TITLE: Total particulate carbohydrate from microalgae
AUTHORS: Ying-Yu Hu, Zoe V. Finkel

[DESCRIPTION]
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. 








[BEFORE_START]
Combust pasteur pipets at 500 °C 120 min 
Combust vials at 500 °C 360 min 
Combust glassware to hold 18 M H2SO4 at 500 °C 360 min 
Combust glass centrifuge tubes at 500 °C 360 min 
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

[STEPS]
SECTION: Day 1 - Samples
15. Considering the working hours from 9 am to 4 pm, suggested sample number is:
# of blank + # of samples = 24
SECTION: Day 1- Hydrolysis
20. Transfer 18 M H2SO4 into a precombusted glassware (such as, scintillation vial, beaker... etc)
SECTION: Day 1- Hydrolysis
22. Add 4.5 mL MilliQ, tightly cap the centrifuge tube, and vortex for 5 s . 
SECTION: Day 1- Hydrolysis
27. 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.
SECTION: Day 2- Alkalinization of samples
47. Add 250 µL 12 M NaOH , vortex.
 


SECTION: Prepare reagents
34. 12 M NaOH
SECTION: Prepare reagents
34.1. Add 15 mL Milli-Q water into a 50 mL Falcon tube.
SECTION: Prepare reagents
34.2. Add 12 g NaOH pellet into the water, swirl and have the pellets completely dissolved, let it cool down to Room temperature .
SECTION: Prepare reagents
34.3. 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.
SECTION: Prepare reagents
35. Alkaline solution for potassium ferricyanide
Dissolve 400 mg NaOH and 20 g Na2CO3 in volumetric flask and top to 1 L by Milli-Q. Store at room temperature.
  
 
SECTION: Prepare reagents
36. Sodium acetate solution
SECTION: Prepare reagents
37. 3 M acetic acid
Weigh 180 g acetic acid in fumehood, transfer the acid into volumetric flask, top to 1 L with Milli-Q water. Store at room temperature.

SECTION: Day 2 TPTZ reagents
39. Potassium ferricyanide (Reagent A)
Weigh 23 mg potassium ferricyanide and transfer into a 100 mL amber reagent bottle. Add 100 mL alkaline solution, vortex until powder is completely dissolved. It is stable for two weeks at room temperature.
  
 

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

SECTION: Prepare reagents
36.2. Store at room temperature.
SECTION: Prepare reagents
36.3. Dispense solution by serological pipet to avoid having salt precipitated around sealing surface of the bottle.
SECTION: TPTZ method
48. In a room with dim light, add 1 mL Reagent A into blanks, standards and samples.
SECTION: TPTZ method
50. Keep in a boiling water bath for 10 min 
SECTION: TPTZ method
52. Add 1 mL Reagent B and 2 mL Reagent C into the vial and vortex.
SECTION: TPTZ method
53. Shake at Room temperature for 30 min .
SECTION: TPTZ method
54. 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.

SECTION: TPTZ method
55. 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
SECTION: Sample collection
2. Filter microalgae in liquid media onto precombusted GF/F filters, using gentle vacuum pressure (130 mm Hg).
 

SECTION: Sample collection
1. Combust GF/F filter for 240 min at 450 °C 
SECTION: Sample collection
3. Rinse filtration funnel with filtered saline to avoid sample loss.
SECTION: Sample collection
5. Place sample filters in Cryogenic Vials.
SECTION: Sample collection
9. Flash freeze filters and store at -80 °C
SECTION: Sample collection
6. Filter same volume of blank media (without cells) through filter as blank. 
SECTION: Day 1- Glucose standard solutions
12. Primary standard
SECTION: Day 1- Glucose standard solutions
12.1. In a 2 mL microtube, weigh ~ 2 mg D-glucose  
SECTION: Day 1- Glucose standard solutions
12.2. Add Milli-Q for a final concentration of 1 mg/mL 
 

SECTION: Day 1- Glucose standard solutions
13. Prepare eight 10 mL precombusted centrifuge tubes, label tubes from SD1 to SD8.
Caps for the standard solutions: acid-washed and dried
 

 

SECTION: Day 1- Glucose standard solutions
14. Follow the sheet to add primary standard and Milli-Q into the tube for working standard solutions.
 

SECTION: Day 1 - Samples
17. Rinse forceps with 95% ethanol and air dry.
 

SECTION: Day 1 - Samples
18. Transfer each filter strip into its centrifuge tube, starting from blank.
SECTION: Day 1 - Samples
19. Add 500 µL Milli-Q directly to the bottom of the tube, , avoiding contact with the filter.
 

SECTION: Sample collection
10. Freeze-dry before processed.
SECTION: Day 1- Preparation
11. Prepare water bath 95 °C 
 

SECTION: Day 1- Hydrolysis
21. Using the reverse pipetting technique, carefully dispense 500 µL 18 M H2SO4 into the MilliQ solution, ensuring it does not touch the filter. Immediately vortex the mixture for 15 s using a timer or stopwatch.
 


SECTION: Day 1- Hydrolysis
23. Place tube into water bath, log the time for each tube. 
 

SECTION: Day 1- Hydrolysis
24. After all samples are placed in the water bath, reduce temperature to 90 °C .
SECTION: Day 2- Alkalinization of standard solutions
42. Forward pipetting: transfer 270 µLof hydrolysate of standard working solutions to amber vial.
 

SECTION: Day 2- Alkalinization of samples
46. Use reverse pipetting to transfer 750 µL hydrolysate to a 12 mL amber vial.

 

SECTION: Day 2- Alkalinization of standard solutions
43. Add 640 µL Milli-Q and vortex.

SECTION: Day 2- Alkalinization of standard solutions
44. Add 90 µL 12 M NaOH and vortex. 
 

SECTION: Spectra of hydrolysate (optional step)
61. Load 250 ul hydrolysate into microplate.
SECTION: TPTZ method
49. Tightly cap the vial and vortex.
SECTION: TPTZ method
51. Remove boiling bath from the heat, keep all vials in the hot water and move them into the room with dim light.
SECTION: Spectra of hydrolysate (optional step)
62. Scan UV/VIS spectra from 200 to 850 nm at a step of 2 nm.
SECTION: Waste disposal
63. All hydrolysate and TPTZ reagents need to be neutralized by soda before disposed into the sink.
SECTION: Day 2 Preparation
38. Boiling bath
SECTION: Day 2 TPTZ reagents
40. 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
 

SECTION: Day 2 TPTZ reagents
41. 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. 
 
 

SECTION: Day 1- Hydrolysis
25. Label pre-combusted 12 mL clear vials for supernatant.

# of vials = # of samples + # of blanks
SECTION: Day 1- Hydrolysis
26. Label pre-combusted 12 mL amber vials for TPTZ measurement with white oil based sharpie.

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

SECTION: Day 1- Prepare for lipids extraction
28. 
 

SECTION: Day 1 - Samples
16. Label 10 mL centrifuge tubes, log sample information. 

 

SECTION: Day 1- Prepare for lipids extraction
29. Use glass serological pipet, add 2 mL chloroform into the hydrolysate of the samples (not the standard solutions). Vortex well. 

 
 

 

SECTION: Day 1- Prepare for lipids extraction
30. Centrifuge 3200 rpm, 5 min 
SECTION: Day 1- Prepare for lipids extraction
31. 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. 
 

 

SECTION: Day 1- Prepare for lipids extraction
33. Use glass serological pipet, add 1 mL methanol into the organic layer, vortex well, freeze at -80 °C  until lipids extraction. 
SECTION: Waste disposal
64. TPTZ reagent B is collected in trace metal waste container.
SECTION: Sample collection
4. 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
SECTION: Sample collection
7. 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
SECTION: Sample collection
8. Place filter in Cryogenic Vials.
SECTION: Day 2- Alkalinization of samples
45. Transfer 2 mL of hydrolysate into 2 mL microtube, centrifuge 13000 rpm, 5 min 
SECTION: TPTZ method
56. 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.

SECTION: TPTZ method
57. Transfer    of sample into microplate in duplicate

SECTION: TPTZ method
58. Transfer    of sample blank into microplate in duplicate
SECTION: TPTZ method
59. Add   into the sample
SECTION: TPTZ method
60. 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
SECTION: Day 1- Prepare for lipids extraction
32. Keep all hydrolysate (standards, blank and samples) in a dark cabinet at Room temperature .