Richard Lab:Dilute-acid pretreatment

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Dilute-Acid Pretreatment of Biomass
Procedure for 1.5 g of dry mass


Dilute-acid pretreatment is a commonly used method of increasing the digestibility of lignocellulosic biomass. While it is difficult for the average lab to achieve the prescibed conditions for standard pretreatment, this protocol is a reduced severity treatment which is achievable using a standard autoclave.



  • Drying Oven
  • Autoclave
  • Filtration aparatus
  • Air lock (optional)


  • Aluminum weigh trays
  • Biomass
  • 0.8% Sulfuric Acid (w/w)
  • Fiber Filters


  1. Prepare biomass samples as necessary (grinding, blending, etc.)
  2. Weigh out ~1g of biomass into a preweighed aluminum tray (do replicates of this).
    1. Be sure to record the weight of the biomass as well as the weight of the tray.
  3. Place the trays in a 105°C oven overnight.
  4. Prepare 0.8% (w/w) sulfuric acid by adding 89 parts water and 1 part 72%.
  5. Once the moisture content is determined, weigh the amount of wet biomass correlating to 1.5g dry mass to a 125ml flask.
    1. Record this weight and repeat for all samples.
  6. Apply 45ml of 0.8% sulfuric acid to each sample.
  7. Put the flasks (with biomass and acid inside) in the airlock and apply vacuum until bubbles stop rising. Repeat 2X.
    1. If an airlock is unavailable just let the flasks (with biomass) sit at on the bench for 1-2 hours.
  8. Cap all flasks with foil.
  9. Autoclave at 121°C for 30min on the liquid cycle.
  10. Immediately after the cycle is finished, remove the samples and filter using a sidearm flask and a fiber filter
  11. Wash the biomass with 200ml water using the filter.