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 0.18 to 10 µg C/mL. 








[STEPS]
SECTION: Day 1 - Samples
12. Considering the working hours from 9 am to 4 pm, suggested sample number is:
# blank + # samples = 24
SECTION: Day 1- Hydrolysis
17. Transfer 18 M H2SO4 into a 30 mL precombusted glassware (scint vial, beaker... etc)
SECTION: Day 1- Hydrolysis
20. Add 4.5 mL MilliQ, tightly cap the centrifuge tube, and vortex for 5 s . 

SECTION: Day 1- Hydrolysis
25. 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.
SECTION: Day 2- Alkalinization of samples
46. Add MilliQ and 12 M NaOH based on the sheet   , 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
47. In a room with dim light, add 1 mL Reagent A into blanks, standards and samples.
SECTION: TPTZ method
49. Keep in a boiling water bath for 10 min 
SECTION: TPTZ method
51. Add 1 mL Reagent B and 2 mL Reagent C into the vial and vortex.
SECTION: TPTZ method
52. Shake at Room temperature for 30 min .
SECTION: TPTZ method
53. 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
54. 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: Standard curve
59. Subtract the average absorbance of blank (0 ug glucose) from the absorbance of each standard
SECTION: Total particulate carbohydrate in the samples
62. Subtract the average absorbance of blank filter from the absorbance of each sample. 
SECTION: Standard curve
60. Plot the standard curve as blank subtracted absorbance versus carbon (ug/mL)
  
SECTION: Standard curve
61. Calculate slope and intercept of the standard curve
SECTION: Total particulate carbohydrate in the samples
63. Use the blank subtracted absorbance of sample, the slope and intercept of the standard curve, dilution factor (DF) in neutralization and total volume of hydrolysis (5.5 mL) to calculate the amount of carbon in total particulate carbohydrate per filter.

C_ug/filter = {[Abs(595)-Abs(blank)] - Intercept}/slope*5.5/DF
SECTION: Sample collection
2. Filter microalgae in liquid media onto precombusted GFF filters, using gentle vacuum pressure (130 mm Hg).
 

SECTION: Sample collection
1. Combust GFF filter for 240 min at 450 °C 
SECTION: Sample collection
3. Rinse filtration funnel with filtered seawater to avoid sample loss.
SECTION: Sample collection
4. Place sample filters in 2 mL Cryogenic Vials.
SECTION: Sample collection
6. Flash freeze filters and store at -80 °C
SECTION: Sample collection
5. Filter blank media (without cells) through filter as blank. 
SECTION: Day 1- Glucose standard solutions
9. Primary standard
SECTION: Day 1- Glucose standard solutions
9.1. In a 2 mL microtube, weigh 1 ~ 2 mg D-glucose  
SECTION: Day 1- Glucose standard solutions
9.2. Add Milli-Q for a final concentration of 1 mg/mL (Volume requirement for preparing standard working solutions: >1800 µL).
SECTION: Day 1- Glucose standard solutions
10. Prepare eight 10 mL precombusted (360 min 500 °C ) centrifuge tubes, label tubes from SD1 to SD8.
 

Caps for the standard working solutions are acid-washed.
 


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

 

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

SECTION: Day 1 - Samples
15. Transfer each filter into its centrifuge tube, starting from blank.
SECTION: Day 1 - Samples
16. Add 500 µL Milli-Q into each tube, vortex.

SECTION: Sample collection
7. Freeze-dry before processed.
SECTION: Day 1- Preparation
8. Prepare water bath 95 °C 
SECTION: Day 1- Hydrolysis
19. Use reverse pipetting technique, add 500 µL 18 M H2SO4 into the suspension instead of onto the filter, immediately vortex for 15 s (Critical step: monitored by timer or stopwatch) 
 

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

 

SECTION: Day 1- Hydrolysis
22. After all samples are placed in the water bath, reduce temperature to 90 °C .
SECTION: Day 2- Alkalinization of standards
42. Transfer 270 µLof hydrolysate of standard working solution to amber vial.
 

SECTION: Estimation
32. 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
SECTION: Estimation
33. 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 1 Mass / % volume

 


      MQ (ul)  H2SO4 (mL)  H2SO4 (M)  MQ (ul)  ug/mL  hydrolysate (ul)  MQ (ul)  12M NaOH (ul)  [H+]/[OH-]    500  0.5  18.00  4500  12.36  90  880  30  0.82    500  0.5  18.00  4500  12.36  180  760  60  0.82    500  0.5  18.00  4500  12.36  270  640  90  0.82    500  0.5  18.00  4500  12.36  360  520  120  0.82    500  0.5  18.00  4500  12.36  450  400  150  0.82    500  0.5  18.00  4500  12.36  540  280  180  0.82    500  0.5  18.00  4500  12.36  630  160  210  0.82    500  0.5  18.00  4500  12.36  720  40  240  0.82    500  0.5  18.00  4500  12.36  750  0  250  0.82  

SECTION: Day 2- Alkalinization of samples
45. Based on the estimation at   , transfer a certain volume of hydrolysate to a 12 mL amber vial.

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

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

SECTION: Spectra of hydrolysate (optional step)
55. Load 250 ul hydrolysate into microplate.
SECTION: TPTZ method
48. Tightly cap the vial and vortex.
SECTION: TPTZ method
50. 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)
56. Scan UV/VIS spectra from 200 to 850 nm at a step of 2 nm.
SECTION: Waste disposal
57. All hydrolysate and TPTZ reagents need to be neutralized by soda before disposed into the sink.
SECTION: Day 1- Hydrolysis
18. Vortex sample.
SECTION: Day 2 Preparation
38. Boiling bath
SECTION: Estimation
31. Estimate carbohydrate content on the filter for each sample:

 [Carbohydrate]ug/filter= [Chl-a]ug/L X (15/1.1) X VolumeL
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
23. Label pre-combusted 5 mL centrifuge tubes for supernatant.

# of vials = # of samples + # of blanks
SECTION: Day 1- Hydrolysis
24. Label 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
26. 
 

SECTION: Day 1 - Samples
13. Label 10 mL centrifuge tubes, log sample information. 
SECTION: Day 1- Prepare for lipids extraction
27. Add 2 mL chloroform into hydrolysate. Vortex. 
 

 

SECTION: Day 1- Prepare for lipids extraction
28. Centrifuge 3200 rpm, 5 min 
SECTION: Day 1- Prepare for lipids extraction
29. Transfer supernatant to 12 mLamber vial by avoiding disturbing organic layer. Keep all hydrolysate in a dark cabinet at Room temperature . 
 

 

SECTION: Day 1- Prepare for lipids extraction
30. Add 1 mL MeOH into the organic layer, mix well, freeze at -80 °C  until lipids extraction.
SECTION: Waste disposal
58. TPTZ reagent B is collected in trace metal waste container.
SECTION: Waste disposal
58.