Biomass compositional analysis

Introduction

This method is to determine water extractives, ethanol extractives, structural carbohydrates and lignin composition of biomass samples. First extractives are removed from the biomass sample to prevent interference with structural carbohydrate measurement. Then, in quantitative saccharification, the extractives-free biomass samples are subjected to hydrolysis with 72% (w/w) H2SO4 at 30ºC for one hour and subsequently with 4% (w/w) H2SO4 at 121ºC for one hour in an autoclave. The remaining insoluble portion is defined as Klason lignin with ash. These steps are shown in the flow chart below.

Equipment

• ASE 350
• IC
• Furnace
• Oven at 105ºC
• Vacuum oven
• Water bath
• Autoclave
• Filtration set-up
• Multivapor P-12

Supplies

• Filtering Crucibles
• ASE collection vials
• Glass fiber filters for ASE (27mm)
• Glass fiber filters for crucibles (934-AH, 25mm)
• 72% sulfuric acid
• Pressure tubes
• Tube rack

Protocol

Sample Preparation

1. Dry biomass samples at 55ºC in ASI Oven.
2. Grind in Wiley mill using 2 mm screen.
3. Dry ASE 60 mL collection tubes (without lid) in 105ºC oven for at least 12 hours. Cool tubes in dessicator and label according to ASE cell number (e.g. 76 W1, 76W2, 76 E1, 76 E2). You will need 4 tubes for each sample (2 for water extract and 2 for ethanol extract). Record weight with analytical balance to nearest 0.1mg.
4. Ash small crucibles (number labels) with glass fiber filters (934-AH, 25mm) and filtering crucibles (letter labels). Also cash crucible covers. Cool in desiccator.

ASE Water and Ethanol Extractions:

1. Fill 10 mL stainless steel ASE extraction cells.
   1. Place 2 glass fiber filters (27mm) in bottom of cell.
   2. Record cell number.
   3. Tare out cell weight on blue 2-decimal scale.
   4. Pour sample into glass mason jar. Using funnel, add sample to ASE cell, slowly turning the jar to ensure uniform particle distribution.
   5. Clean biomass from cell threads using brush.
   6. Record weight of sample. Typical sample weight is ~1-2 g.
2. Measure moisture and ash content of biomass samples being loaded into ASE cells.
   1. Record empty weight of crucible without the cover (use analytical balance). Record all weights to nearest 0.1 mg.
   2. Tare crucible.
   3. Weigh out ~ 1 g sample and record weight of sample.
   4. Dry samples for 24 h at 105ºC. Cool in desiccator, wigh, and then ash with crucible cover.
3. Weigh collection tubes on analytical balance to nearest 0.1 mg. Place ASE cells and collection tubes in appropriate corresponding positions. For instance, if you are running 12 samples using a sequence, the cell in position 1 has water collection tube in position 1 and ethanol collection tube in position 13. ASE cell should be placed filter side down.
4. All water extractions (method 16) are completed and then follwed by ethanol extractions (method 17). Extraction conditions are:

• Temperature: 100ºC
• Static Time: 7 min
• Flush/rinse volume: 150%
• Purge: 120 s
• Static cycles: 3
• Solvent saver is OFF.
• Both methods can be set up to run automatically in a sequence.

1. Prepare ASE.
   1. Check nitrogen cylinder. Tank pressure should be atleast 400 psi; outlet pressure should be 170 psi.
   2. Fill out log book.
   3. Empty rinse and waste bottles
   4. Fill solvent C reservoir with nanopure water and solvent B reservoir with 95% ethanol (190 proof).
   5. Hit 'rinse' to prime system.
   6. Load sequence.
2. Each extraction takes about 35 mintures. After ASE extractions are finished, hit 'rinse' to clean out ASE tubing. If you run your samples overnight, include a final ASE cell with sand to serve as a blank and rinse out the system.
3. Collect sold samples.
   1. Empty contents of ASE extraction cell into labeled aluminum pan.
   2. Avoid contaminating sample with filter.
   3. Leave solitds in fume hood to air dry (or dry in oven < 50ºC). These will then be used in quantitative saccharification procedure.

Extractives Determination

1. Water and ethanol extractive samples in ASE collection vials will be dried using Buchi Multivapor P-12. Prepare Multivapor:
   1. Turn on cooling water until gentle, steady flow is achieved (about 1/2 turn).
   2. Add nanopure water to each reservoir until it reaches 2 lines about the minimum.
   3. Set hot plate to 70ºC
   4. Fill unused positions with blank adapters.
2. Dry ethanol extractive samples
   1. Split sample between 2 preweighed oven dryed tubes.
   2. Top tube with adapter and seal (use red silicone seal). Use teflon tape on threads if needed. Tape should be removed when tubes are taken out of Multivapor.
   3. Turn on vacuum pump and agitation. Pressure should be gradually decreased to prevent foaming and agitation gradually increased.
   4. Final values are pressure = 175 mbar and agitation = 7.
   5. Samples will dry in ~1 hour. Final pressure may need to be decreased to ~ 100 mbar to dry water in sample (remember solvent was 95% ethanol).
3. Dry water extractive samples.
   1. Split sample between 2 preweighed oven dry tubes.
   2. Top Tube with adapter and seal (use red silicone seal). Use teflon tape on threads if needed. Tape should be removed when tubes are taken out of Multivapor.
   3. Turn on vacuum pump and agitation. Pressure should be gradually decreased to prevent foaming and agitation gradually increased.
   4. Final values are pressure = 72 mbar and agitation = 7.
   5. If you want to collect a sample of the water extract for sucrose determination, this can be done before drying. In order for it to be quantitative, bring total volume of extract to 50 mL with nanopure water suing volumetric cylinder. Return extract to ASE collection tube. Invert to mix. Pipet 10 mL into labeled scintillation vial (this sample will be stored in freezer for future sucrose measurement). Then proceed with steps 1-4 above.
4. Extracts will not be completely evaporated in Multivapor. There will be small amount of liquid remaining. Dry both water and ethanol extractive tubes overnight in vacuum oven at ~45ºC and 25 in. Hg vacuum (include tray of recharged molecular sieve in oven). Then record weight of the tube with extractives to nearest 0.1 mg.
5. Notes on use of the Multivapor:

• If you can not acheive steady vacuum, you should check seals in between the tubes and the adapters. The silicone (red) seals seem to work best (~ $0.06/each) and are compatible with water and 95% ethanol. There are also PEEK seals available (~ $10/each).
• After you are finished with the Mulitvapor, you need to remover the condensation from the pump. Bring the pump back to atmostpheric pressure. Remove the cover above your samples. Then, run the pump in continuous mode until the condenation is removed.

Quantitative Saccharification:

1. Heat large water bath to 30ºC
2. Get sugar recovery standard (SRS) out to thaw.
3. Weigh out 0.29-0.31 g of extratives-free, air-dry sample onto folded weigh paper. Record weiht (use analytical balance). Transfer sample carefully into labeled pressure tube.
4. Determine moisture and ash contents of remaining extractive-free sample (using numbered crucibles), as before (see step 2 under ASE extractions).
5. Add 3 mL 72% (w/w) H2SO$ to each test tube. Use ANKOM pre-made 72% H2SO4
6. Use a teflon stir rod to mix acid and sample thoroughly. Leave teflon stir rod in each test tube.
7. Place test tubes in 30ºC water bath for 1 hour. Every 10 minutes, stir the sample/acid mixture without removing the tube from the water bath.
8. Remove tubes from water bath when 1 hour is complete.
9. Weigh out 84.00 +/- 0.04 g nanopure water fro each sample using blue top-loading scale.
10. Add nanopure water to each pressure tube, usin gthe water to rinse any solids off teflon stir rod (new acid concentration is 4%)
11. Seal pressure tubes with caps. Mix several times by inverting.
12. Autoclave for 1 hour on fluid cycle (250F, 20psi). Also include a sugar recovery standard (10mL standard + 348 uL 72% sulfuric acid).
13. Remove samples from autoclave and allow them to cool.
14. Set up filtration system. EAch sample requires filtering crucible, 500 mL sidearm flask, and adapter.
15. Vacuum filter the sample through previously weighed filtering crucible.
16. Collect a sample of the filtrate in a 20 mL scintillation vial. ASL must be determined immediately. Vial can be stored in refrigerator until sugar/acetyl measurement (see IC sample analysis)
17. Use at least 50 mL nanopure water to transfer all solids from tube into crucible and to rinse solids in crucible (hot water can be used to speed up filtration).
18. Perform acid soluble lignin analysis, using LIGNIN STOEVR smart start method on spectrophotometer. Analyze each sample in duplicate.
    1. Due to small wavelength, use UV cuvettes.
    2. Blank is nanopure water.
    3. Dilute samples so that absorbance is in range of 0.7 - 1.0. Possible dilutions: 250 uL sample + 750 uL water, or 500 uL water + 500uL sample
19. Dry crucible at 105C overnight. Cool and record weight. THese solids are the Klason lignin (acid insoluble) and ash.
20. Place crucibles in furnace overnight to setermine ash content, suing the program specified below.
    1. Ramp to 105ºC. Hold for 12 min.
    2. Ramp to 250ºC at 10ºC/min. Hold for 30 min.
    3. Ramp to 575ºC at 20ºC/min. Hold for 3 hr.
    4. Ramp down to 105ºC. Hold until samples are removed. Cool crucibles and record weight.