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

Filter microalgae in liquid media onto precombusted GFF filters, using gentle vacuum pressure (130 mm Hg).

Equipment

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

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 -80°C

Freeze-dry before processed.

Prepare water bath 95°C

Primary standard

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

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 (6h 0m 0s 500°C ) centrifuge tubes, label tubes from SD1 to SD8.

Equipment

Value	Label
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

Value	Label
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.

A	B	C
SD1	0	500
SD2	25	475
SD3	50	450
SD4	100	400
SD5	150	350
SD6	250	250
SD7	350	150
SD8	450	50

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

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

Transfer each filter into its centrifuge tube, starting from blank.

Add 500µL Milli-Q into each tube, vortex.

Transfer 18 M H₂SO₄ into a 30 mL precombusted glassware (scint vial, beaker... etc)

Vortex sample.

Use reverse pipetting technique, add 500µL 18 M H₂SO₄ into the suspension instead of onto the filter, immediately vortex for 0h 0m 15s (Critical step: monitored by timer or stopwatch)

Add 4.5 mL MilliQ, tightly cap the centrifuge tube, and vortex for 0h 0m 5s .

Place tube into water bath, log the time for each tube.

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

Value	Label
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.

Add 2 mL chloroform into hydrolysate. Vortex.

Centrifuge 3200rpm

Transfer supernatant to 12 mLamber vial by avoiding disturbing organic layer. Keep all hydrolysate in a dark cabinet at Room temperature .

Equipment

Value	Label
Disposable Soda-Lime Glass Pasteur Pipets	NAME
5 3/4"	TYPE
Fisherbrand	BRAND
13-678-6A	SKU
http://www.fishersci.com/us/en/home.html	LINK

Add 1 mL MeOH into the organic layer, mix well, freeze at -80°C until lipids extraction.

Estimate carbohydrate content on the filter for each sample:

[Carbohydrate]_ug/filter= [Chl-a]_ug/L X (15/1.1) X Volume_L

C_assay= 0.4 * Chl * (15/1.1) * V * (Hy/1000)/5.5

Where C_assay 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 1Mass / % volume

A	B	C	D	E	F	G	H
500	0.5	18.00	4500	90	880	30	0.82
500	0.5	18.00	4500	180	760	60	0.82
500	0.5	18.00	4500	270	640	90	0.82
500	0.5	18.00	4500	360	520	120	0.82
500	0.5	18.00	4500	450	400	150	0.82
500	0.5	18.00	4500	540	280	180	0.82
500	0.5	18.00	4500	630	160	210	0.82
500	0.5	18.00	4500	720	40	240	0.82
500	0.5	18.00	4500	750	0	250	0.82

12 M NaOH

Add 15mL 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.

Boiling bath

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.

Transfer 270µLof hydrolysate of standard working solution to amber vial.

Equipment

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

Add 640µL Milli-Q and vortex.

Add 90µL 12 M NaOH and vortex.

Based on the estimation at , transfer a certain volume of hydrolysate to a 12 mL amber vial.

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

In a room with dim light, add 1mL Reagent A into blanks, standards and samples.

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	SD1	SD1
B	SD2	SD2
C	SD3	SD3
D	SD4	SD4
E	SD5	SD5
F	SD6	SD6
G	SD7	SD7
H	SD8	SD8

Microplate layout

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

Load 250 ul hydrolysate into microplate.

Scan UV/VIS spectra from 200 to 850 nm at a step of 2 nm.

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.