Measurement of dissolved carbohydrate

GFF filter is combusted for 4h 0m 0s at 450°C

Glass filter holder is combusted for 2h 0m 0s at 500°C

Glass filter funnel, flask and 10 mL centrifuge tubes are combusted for 6h 0m 0s at 500°C

Equipment

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

Tube caps are acid-washed.

Equipment

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

Filter microalgae sample and collect the filtrate, using gentle vacuum pressure (130 mm Hg).

Transfer 5 mL filtrate into centrifuge tube and flash freeze.

Freeze dry samples before measurement.

Primary standard solution

In a 2 mL microtube, weigh 1 ~ 2 mg D-glucose

Add Milli-Q for a final concentration of 1 mg/mL (>600 µL).

Secondary standard for total dissolved carbohydrate

Add 45µL primary solution into a 2 mL microtube

Add 955µL Milli-Q and then vortex for a good mix

In 10 mL centrifuge tubes, prepare the following standard solutions:

A	B	C
TCHO-SD1	0	100
TCHO-SD2	20	80
TCHO-SD3	40	60
TCHO-SD4	60	40
TCHO-SD5	80	20
TCHO-SD6	100	0

Secondary standard for total dissolved monosaccharide

Add 10µL primary solution into a 2 mL microtube

Add 990µL Milli-Q and then vortex for a good mix

In 12 mL amber vials, prepare the following standard solutions:

A	B	C	D
MCHO-SD1	0	984	16
MCHO-SD2	10	974	16
MCHO-SD3	20	964	16
MCHO-SD4	50	934	16
MCHO-SD5	100	884	16
MCHO-SD6	150	834	16

Equipment

Value	Label
Storage Vials and Closures	NAME
12 mL amber	TYPE
Thermo Scientific	BRAND
B7800-12A	SKU
VWR 66030-686	SPECIFICATIONS

Prepare water bath 95°C

Add 100µL Milli-Q to each tube with freeze-dried sample.

Use reverse pipetting technique, add 100µL 18 M H₂SO₄ to standard solution/sample, immediately vortex for 0h 0m 15s (monitored by timer or stopwatch)

Add 900µL Milli-Q, tightly cap the centrifuge tube, and vortex for 0h 0m 5s .

Place tube into water bath, log the time.

After all samples are in the water bath, reduce temperature to 90°C .

Label amber vials for TPTZ measurement with white oil based sharpie.

# of vials = # of samples + # of blanks + # of standards

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.

Keep all hydrolysate in a dark cabinet at Room temperature .

12 M NaOH

Add 15 mL Milli-Q water into a 50 mL Falcon tube.

Add 12g NaOH pellet into the water, swirl and have the pellets completely dissolved, let it cool down to Room 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 400mg NaOH and 20g Na₂CO₃in volumetric flask and top to 1 L by Milli-Q. Store at room temperature.

Sodium acetate solution

Dissolve 164g sodium acetate, 42g citric acid and 300g acetic acid in a 1 L volumetric flask and top to1 L with Mill-Q water.

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 180g acetic acid in fumehood, transfer the acid into volumetric flask, top to 1 L with Milli-Q water. Store at room temperature.

Prepare boiling bath

TPTZ reagents

Potassium ferricyanide (Reagent A)

Weigh 23mg potassium ferricyanide and transfer into a 100 mL amber reagent bottle. Add 100mL alkaline solution, vortex until powder is completely dissolved. It is stable for two weeks at room temperature.

Equipment

Value	Label
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

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.

Note

This solution is stored at room temperature and stable for one week.

Total dissolved carbohydrate samples

Use reverse pipetting technique, transfer 750µL hydrolysate of standard/sample to amber vial.

Add 250µL 12 M NaOH and vortex.

Total dissolved monosaccharide samples

Add 1200µL Milli-Q into the tube with freeze-dried sample

Use reverse pipetting technique, transfer 984µL solution to amber vial.

Add 16µL 12 M NaOH and vortex.

In a room with dim light, add 1mL Reagent A into each amber vial.

Tightly cap the vial and vortex.

Keep in a boiling water bath for 0h 10m 0s

Remove boiling bath from the heat, keep all vials in the hot water and move them into the room with dim light.

Add 1mL Reagent B and 2mL Reagent C into the vial and vortex.

Shake at Room temperature for 0h 30m 0s .

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.

A	B	C	D	E	F	G	H	I	J	K	L	M
A	MCHO-SD1	MCHO-SD1
B	MCHO-SD2	MCHO-SD2
C	MCHO-SD3	MCHO-SD3
D	MCHO-SD4	MCHO-SD4
E	MCHO-SD5	MCHO-SD5
F	MCHO-SD6	MCHO-SD6
G
H

Microplate layout for dissolved monosaccharide samples

A	B	C	D	E	F	G	H	I	J	K	L	M
A	TCHO-SD1	TCHO-SD1
B	TCHO-SD2	TCHO-SD2
C	TCHO-SD3	TCHO-SD3
D	TCHO-SD4	TCHO-SD4
E	TCHO-SD5	TCHO-SD5
F	TCHO-SD6	TCHO-SD6
G
H

    Microplate layout for total dissolved carbohydrate samples

Read in microplate reader:Shake for 5 s at 600 rpm in a continuous and high force modeRead endpoint 595 nm with a measurement time 100 ms

Hydrolysate

Load 200µL hydrolysate into microplate.

Blank:

Milli-Q : H₂SO₄= 10:1

Monosaccharide solutions

Load 200µL solution into microplate.

Blank: Milli-Q

Scan UV/VIS spectra from 200 to 400 nm at a step of 1 nm.

Total dissolved carbohydrate

Subtract the average absorbance of blank (0 ug glucose) from the absorbance of each standard for total dissolved carbohydrate.

Obtain standard curve by plotting blank subtracted absorbance (Abs') versus carbon (uM C)

Subtract the average absorbance of blank (0 ug glucose) from the absorbance of each sample

Total dissolved monosaccharide

Subtract the average absorbance of blank (0 ug glucose) from the absorbance of each standard for total dissolved monosaccharide.

Obtain standard curve by plotting blank subtracted absorbance (Abs') versus carbon (uM C)

Subtract the average absorbance of blank (0 ug glucose) from the absorbance of each sample

Total dissolved polysaccharide

All hydrolysate and TPTZ reagent C need to be neutralized by soda before disposed into the sink.

TPTZ reagent B is collected in trace metal waste container.