Protocol

 

Please Note

This protocol was contributed by Robin Martin in conjunction with the Carnegie Institution for Science. Please visit the Spectranomics website at https://asnerlab.org/projects/spectranomics/ for more information and spectranomics tools.

Overview

Carbon extractions to determine hemicellulose, cellulose and lignin in leaf tissue.

Materials/Equipment

Equipment

• Ankom fiber analyzer
• Electric tea kettle
• Muffle furnace
• Crucibles and crucible tongs
• 1 L beakers
• 2 L beakers
• Heat sealer

Consumable Materials

• Neutral detergent Solution (1800-1900 ml)
• Acid detergent (5X solution at 1500-1900 ml)
• 72% sulfuric acid (300 ml for 48 sample bags)
• Acid resistant gloves, goggles, and apron
• Filter bags
• Acid resistant Ankom marker
• 3-4 Aluminum Trays
• Deionized water (DI)

Procedure

Sample Preparation

• Label bags with Ankom marker or sharpie and dry in oven at 105 ^∘C for 30 min. Cool in desiccator for 30 min.
• Weigh each bag, record weight and tare bag.
• Weigh 0.5 g (∓ 0.05 g) of ground (40 mm screen) dried sample into bag and record total weight.
• Include a blank bag in each run (each batch of 24) for a blank bag correction.
• Seal the bag within 0.5 cm of open edge using heat sealer. Be sure to also seal the blank bag.
• Spread sample uniformly inside the filter bag by shaking and lightly flicking the bag to eliminate clumping.
• A maximum of 24 bags may be placed in the sample tree. All nine trays are used regardless of the number of bags being processed. Place three bags per tray and then stack trays on center post with each level rotated 120 degrees to sit in the notches of the tray below it. The weight is placed on top of the empty 9^th tray to keep the bag suspender submerged.

Measurement Procedure

• NDF (neutral detergent fiber) Extraction: Fill chamber with 1800-1900 ml neutral detergent solution and agitate with heat for 75 min to extract carbohydrates, lipids, pectin, starch, soluble proteins, and non-protein nitrogen. You will be left with hemicellulose, proteins bound to the cell walls, cellulose, lignin, and recalcitrant materials. Rinse the samples of neutral solution by agitating them 3 times with 1800 ml of boiling DI water for 5 min each agitation. Once rinsing is complete, dry samples overnight on aluminum trays to prepare for ADF extraction.
• ADF (acid detergent fiber) Extraction: First weigh bags once they have cooled in desiccator and record the weights. Add 1900 ml ADF solution to the chamber and agitate sample at ambient temperature for 60 min to extract hemicellulose and bound proteins. After acid solution is removed from the chamber, agitate sample with 1800 ml of boiling DI water 3 times to rinse sample. Dry overnight on aluminum trays at 90-100 ^∘C.
• ADL (acid determined lignin) Extraction: Add ~ 300 ml 72% sulfuric acid to 2L beaker containing 48 completely dried sample bags. Place 1 L beaker on top of samples to keep the bags submerged. Manually agitate the bags every 30 min by lifting 1 L beaker up and down 30 times. Repeat for 3 hours. After sulfuric acid is removed, add 1000 ml boiling DI water to rinse sample about 6-7 times until pH is neutral. Dry samples overnight at 90-100^∘C. This final extraction removes cellulose and leaves lignin and recalcitrant materials.
• Ashing: Rinse crucibles with DI water and dry overnight at 65 ^∘C. Set muffle furnace to 150 ^∘C and equilibrate overnight. The next day, remove crucibles from the oven and place in the desiccator for 1 hour. Remove dried bags from oven and cool in desiccator for 30 min. Weigh bags and record the weight. Place bags in crucibles. Place crucibles in muffle furnace at 500 ^∘C for 5.5 hours. After 5.5 hours, reduce temperature to 150 ^∘C and leave samples in furnace overnight. The next day, remove crucibles from furnace using crucible tongs and cool in desiccator for 2 hours. Turn off muffle furnace. Weigh bags and crucibles and record weight.

Data Preparation and Finalization

Note: blank bag correction (C) = blank bag wt. after extraction / blank bag tare wt.

NDF Calculations

Soluble cell contents =

[((Tare wt. sample bag + sample wt.) – bag wt. after NDF) x 100] / sample wt.

Hemicellulose, bound proteins, cellulose, lignin, recalcitrants =

[(bag wt. after NDF – (tare wt. sample bag x C_NDF)) x 100] / sample wt.

ADF Calculations

Hemicellulose and bound proteins =

[((bag wt after NDF – (tare wt sample bag x C_NDF)) – (bag wt after ADF – (tare wt. sample bag x CADF)) x 100] / sample wt.

Cellulose, lignin, and recalcitrants =

[(bag wt. after ADF – (tare wt. sample bag x C_ADF)) x 100] / sample wt.

ADL Calculations

Cellulose =

[((bag wt after ADF – (tare wt sample bag x C_ADF)) – (bag wt after ADL – (tare wt. sample bag x C_ADL)) x 100] / sample wt.

Lignin and recalcitrants =

[(bag wt. after ADL – (tare wt. sample bag x CADL)) x 100] / sample wt.