TITLE: Measuring leaf carbon fractions with the ANKOM2000 Fiber Analyzer
AUTHORS: Jocelyne Ayotte, Etienne Laliberté

[DESCRIPTION]
Here we describe the standardized protocol used by the Canadian Airborne Biodiversity Observatory (CABO) to measure carbon fractions in leaf samples, using the ANKOM2000 Fiber Analyzer. Prior to the analysis, leaf samples are oven-dried, and then ground with a cyclone mill (2-mm screen). Then, carbon fractions are measured using a sequential digestion procedure to measure neutral detergent fiber (NDF, acid detergent fiber (ADF), acid detergent lignin (ADL). Finally, the residues are ashed in a muffle furnace to determine inorganic recalcitrant materials. This protocol is based on the manufacturer's protocols.

[BEFORE_START]
Leaf samples need the have been dried for 72 h at 65°C and then ground with a cyclone mill (2-mm screen). 

[STEPS]
SECTION: Sample preparation
1. Use a solvent resistant marker to label the filter bags to be used in the analysis.
  

SECTION: Sample preparation
2. Weigh and record the weight of each empty filter bag to the nearest 0.0001 g (in Fulcrum). Zero the balance. 


 


 


SECTION: Sample preparation
3. Place 0.45–0.50 g of prepared sample in up to 23 of the bags and record the weight to the nearest 0.0001 g (in Fulcrum) of each. Avoid placing the sample in the upper 4 mm of the bag.


SECTION: Sample preparation
4. Include at least one empty bag in the run to determine the blank bag correction (C1).


 

SECTION: Sample preparation
5. Using a heat sealer, completely seal each filter bag closed within 4 mm of the top to encapsulate the sample. 


 

 

SECTION: Sample preparation
6. Spread the sample uniformly inside the filter bags by gently shaking the bags to eliminate clumping.
SECTION: Neutral Detergent Fiber
7. Place up to 3 bags on each of eight Bag Suspender Trays (maximum of 24 bags). Stack the trays on the center post of the Bag Suspender with each level rotated 120 degrees in relation to the tray below it. Place the empty 9th tray on top.
  
  

 



SECTION: Neutral Detergent Fiber
8.  Verify that the hot water supply is on (room B-236). 
  



SECTION: Neutral Detergent Fiber
10. Attach the Neutral Detergent (ND) solution hose to the Cubetainer and then to Port A on the instrument.

  

SECTION: Neutral Detergent Fiber
9. Verify that the drain hose is securely positioned in an empty yellow waste bin. 

  

SECTION: Neutral Detergent Fiber
12. Open the Vessel Lid and insert the Bag Suspender with bags into the Vessel and place the Bag Suspender weight on top of the empty 9th tray to keep the Bag Suspender submerged. 
  
  

SECTION: Neutral Detergent Fiber
13. Follow the instructions on the ANKOM2000 display: 
Select NDF. (Wait to close the Vessel Lid.)
  

SECTION: Neutral Detergent Fiber
15. Confirm hot water is on (>70 °C) and press START. 

  



SECTION: Neutral Detergent Fiber
16. After the ND solution has been automatically inserted and agitation begins, manually add 20g of Na2SO3 and 4.0 ml of alpha-amylase.
20 g4.0 mL

  
  
 

 

 



SECTION: Neutral Detergent Fiber
17. Close the Vessel Lid.

 
This step takes about 90 min.

SECTION: Neutral Detergent Fiber
18. When the NDF extraction and rinsing procedures are complete, open the Vessel Lid and remove the filter bags. Gently press out excess water from the bags. 


 

 

SECTION: Neutral Detergent Fiber
19. Completely dry the filter bags in an oven at 102 ± 2°C for 3 h. 
180 min
During that time, go to Step 20 and Flush Procedure (Step 21). 

 

  

  


SECTION: Neutral Detergent Fiber
28. When dry, remove the filter bags from the oven and immediately place them directly into a collapsible desiccant pouch and flatten it to remove any air. Cool to ambient temperature for 30 min. 


 


SECTION: Neutral Detergent Fiber
29. Weigh the filter bags to the nearest 0.0001 g and add record the weight (in Fulcrum). 

 

SECTION: Neutral Detergent Fiber
30. Remove the yellow waste bin and label it properly. 
  
 


SECTION: Acid Detergent Fiber
31. Place up to 3 bags on each of eight Bag Suspender Trays (maximum of 24 bags). Stack the trays on the center post of the Bag Suspender with each level rotated 120 degrees in relation to the tray below it. Place the empty 9th tray on top. 
  
 


SECTION: Acid Detergent Fiber
32. Insert the drain hose securely into an empty white waste bin. 
  

SECTION: Acid Detergent Fiber
33. Read the Temperature Controller on the right side of the instrument. 

 

SECTION: Neutral Detergent Fiber
20. During that time, run a flush procedure if necessary. 

Detach both cubitainers from ports A and B. 
 

 

SECTION: Acid Detergent Fiber
34. Attach the acid detergent (AD) solution hose to the Cubetainer and then to Port B on the instrument.

SECTION: Acid Detergent Fiber
35. Open the Vessel Lid and insert the Bag Suspender with bags into the Vessel and place the Bag Suspender Weight on top of the empty 9th tray to keep the Bag Suspender submerged. 

SECTION: Acid Detergent Fiber
36. Follow the instructions on the ANKOM2000 display: 
a. Select ADF. 
b. Close the Vessel Lid. 
c. Confirm hot water is on (>70°C). 
d. Press START.
60 min

SECTION: Acid Detergent Fiber
37. When the ADF extraction and rinsing procedures are complete, open the Vessel Lid and remove the filter bags. Gently press out excess water from the bags. 

 

SECTION: Acid Detergent Fiber
38. Completely dry the filter bags in an oven at 102 ± 2°C for 3 h. 
180 min

 


SECTION: Acid Detergent Fiber
40. During that time, run a flush procedure if necessary. 

Detach both cubitainers from ports A and B. 
 


SECTION: Acid Detergent Fiber
48. Remove the filter bags from the oven, immediately place them into a collapsible desiccant pouch, and flatten it to remove any air. Cool to ambient temperature.
  
 


SECTION: Acid Detergent Fiber
49. Weigh the filter bags to the nearest 0.0001 g and record the weight (in Fulcrum).

 

 

SECTION: Acid Detergent Fiber
39. During that time, remove the white waste bin and label it properly. 
  

 

SECTION: Lignin method in beakers
50. Prepare a 72% H2SO4 solution or take a 250 ml bottle of 72% H2SO4. 

 

 

. 
SECTION: Lignin method in beakers
51. Place dried filter bags with samples into a 2 L glass beaker and completely cover the bags with 72% H2SO4 (approximately 250 ml). 

 

SECTION: Lignin method in beakers
52. Place a 1 L beaker inside the 2 L beaker to keep the filter bags submerged. 
  

SECTION: Lignin method in beakers
54. After 3 h, pour off the H2SO4 and rinse with tap water to remove all of the acid.


 

 

SECTION: Lignin method in beakers
55. Repeat rinses until the pH paper shows neutral color when touching the bags. Handle the bags gently during rinsing. Fine lignin particles can exit the filter bag if not handled carefully.

 

SECTION: Lignin method in beakers
56. Dry the bags in an oven at 100 °C for 3 h.
180 min
 


SECTION: Lignin method in beakers
57. Remove the filter bags from the oven and place them directly into a Desiccant/Moisture Stop pouch. Flatten the pouch to remove air. Cool to ambient temperature and weigh the bags to the nearest 0.0001 g. Record the weight (in Fulcrum).

  

SECTION: Ashing
58. Rinse 24 crucibles and covers with dH2O. 
SECTION: Ashing
59. Dry the crucibles and covers in an oven at 150 °C for 1 h. 
60 min

 


SECTION: Ashing
60. Cool down crucibles (with lids on) in a metal dessicator for 1h30min.
90 min

 

SECTION: Ashing
61. Weigh empty crucibles, with lids, to the nearest 0.0001 g. Record the weight (in Fulcrum).

 

 

SECTION: Ashing
62. Fold filters bags into four and insert each bag into its crucible, with the corresponding sample number. Cover with the lid. 
SECTION: Ashing
63. Put the crucibles (with lids on) in the muffle furnace at 500 °C for 5 h. 
300 min
 


SECTION: Ashing
64. Change the temperature at 150 °C, O/N.
840 min

 


SECTION: Ashing
65. Turn off the muffle furnace and transfer the crucibles (with lids on) in a metal dessicator for 1h30min. 
90 min

 

SECTION: Ashing
66. Weigh crucibles with lids on to the nearest 0.0001 g. Record the weight (in Fulcrum).

 

SECTION: Ashing
67. Wash crucibles and lids with tap water.


SECTION: Acid Detergent Fiber
41. Flush procedure:
Verify that the drain hose is securely inserted into a waste bin.

  

SECTION: Acid Detergent Fiber
42. Flush procedure: 
Press the arrow keys on the Keypad until you see “Select Flush” on the Display.

  

SECTION: Acid Detergent Fiber
43. Flush procedure: 
Press ENTER on the Keypad.


SECTION: Acid Detergent Fiber
44. Flush procedure:
Attach the Amylase Dispenser Assembly to port B.


SECTION: Acid Detergent Fiber
45. Flush procedure:
Fill the dispenser with 100 ml of hot water.

  

SECTION: Acid Detergent Fiber
46. Flush procedure:
Attach the Amylase Dispenser Assembly to port A.


SECTION: Acid Detergent Fiber
47. Flush procedure:
Fill the dispenser with 100 ml of hot water.


 

SECTION: Neutral Detergent Fiber
11. Add 50 ml of tap water in the alpha-amylase dispenser. Add 8.0 mL of alpha-amylase and enough tap water to fill the dispenser (until the line). Attach the Amylase Dispenser Assembly to Port B on the instrument.

Keep the pipette and alpha-amylase bottle next to the instrument for step 16. 

Weigh 20 gof Sodium sulfite and keep it for step 16. 


  
 

 





SECTION: Neutral Detergent Fiber
14. The screen will show: Insert samples. Press Enter.

  


SECTION: Lignin method in beakers
53. Agitate the bags at the start and at 30-minute intervals by gently pushing and lifting the 1 L beaker up and down approximately 30 times. 

  

SECTION: Neutral Detergent Fiber
21. Flush procedure:
Verify that the drain hose is securely inserted into a waste bin.

  

SECTION: Neutral Detergent Fiber
22. Flush procedure: 
Press the arrow keys on the Keypad until you see “Select Flush” on the Display.

  

SECTION: Neutral Detergent Fiber
23. Flush procedure: 
Press ENTER on the Keypad.


SECTION: Neutral Detergent Fiber
24. Flush procedure:
Attach the Amylase Dispenser Assembly to port B.

SECTION: Neutral Detergent Fiber
25. Flush procedure:
Fill the dispenser with 100 ml of hot water.

  

SECTION: Neutral Detergent Fiber
26. Flush procedure:
Attach the Amylase Dispenser Assembly to port A.


SECTION: Neutral Detergent Fiber
27. Flush procedure:
Fill the dispenser with about 100 ml of hot water.