Richard Lab:Enzymatic Hydrolysis of Biomass

Back to Protocols Enzymatic Saccharification of Lignocellulosic Biomass Laboratory Analytical Procedure for 0.15 g of dry mass

Introduction
This procedure describes the enzymatic saccharification of cellulose from native or pretreated lignocellulosic biomass to glucose in order to determine the maximum extent of digestibility possible. A saturating level of a commercially available cellulase preparation and hydrolysis times up to one week are used. This procedure is appropriate for lignocellulosic biomass. This procedure can also be used to measure the efficacy of a given pretreatment based on a maximum enzyme loading.

Interferences
Unwashed acid- and alkaline-pretreated biomass samples may change solution pH to values outside the range of enzymatic activity; and the unwashed glucose in the biomass may influence the final result.

Equipment

 * 1) A suitable shaking or static incubator set at 50°C ± 1°C
 * 2) A fixed speed rotator that can hold vials and operate in a static incubator.
 * 3) 5ml or 30ml centrifuge tubes
 * 4) pH meter
 * 5) Analytical balance, accurate to 1 mg or 0.1 mg
 * 6) YSI analyzer with appropriate membranes or equivalent glucose quantification method such as HPLC or IC
 * 7) pipet with tips

Reagents

 * 1) Antibiotics
 * 2) Tetracycline stock solution (10 mg/mL in 70% ethanol). (light sensitive, wrap in foil)
 * 3) Cycloheximide stock solution (1 mg/mL in distilled water).
 * 4) Alternate antibiotic – Sodium Azide (20 mg/ml in distilled water)
 * 5) Sodium citrate buffer (1 M, pH 4.80).
 * 6) Cellulase enzyme of known activity, FPU/mL. (we use spezyme)
 * 7) Beta-glucosidase enzyme of known activity, pNPGU/mL (we use novozme)


 * (If necessary) Xylanase enzyme of known protein concentration, mg/ml

Procedure
Alternatively, instead of adding each reagent singly, a mixture can be made containing citrate buffer, tetracycline, cycloheximide, water, Spezyme and Novozyme. The mixture is added to the biomass and then water to total 10mL.
 * 1) Determine the solids content for all cellulose containing samples to be digested.
 * 2) label metal trays
 * 3) mass of metal tray
 * 4) mass of tray plus about 1 gram of material
 * 5) oven dry at 105 C for 24 hours
 * 6) mass of tray and material
 * 7) calculate percent solids
 * 8) Weigh out a biomass sample equal to the equivalent of 0.15 g dry biomass (calculated based on the results of the previous step) and add to a 15mL centrifuge tube. It is assumed that this is roughly equivalent to 0.1g cellulose.
 * 9) To each vial, add:
 * 10) 500μL sodium citrate buffer (1 M, pH 4.8)
 * 11) 40μL tetracycline solution (400 μg
 * 12) 30μL cycloheximide solution (300 μg)
 * 13) Add the appropriate calculated volume of water to each tube to bring the total volume to 10.00 mL after addition of the enzymes specified in the following step.
 * 14) All solutions and the biomass are assumed to have a specific gravity of 1.000 g/mL. Thus, if 0.200 g of biomass was added to the vial, it is assumed to occupy 0.200 mL and 9.733 mL of liquid is to be added.
 * 15) Add the following to each tube:
 * 16) an appropriate volume of the cellulase enzyme preparation to equal approximately 5 FPU/dry g cellulose (low enzyme loading) or 15 FPU/dry g cellulose (high enzyme loading) calculations below
 * 17) the appropriate volume of β-glucosidase enzyme to equal 20 CBU/dry g cellulose or 60 CBU/dry g cellulose. calculations below
 * 18) Xylanase may be added at the same time (if desired).
 * 19) Prepare control blanks
 * 20) The substrate blank contains buffer, water, and the identical amount of substrate in 10.00 mL volume.
 * 21) Enzyme blanks for cellulase, β-glucosidase, and xylanase with buffer, water, and the identical amount of the enzyme.
 * 1) Close the tubes tightly and place them in a shaking incubator or fixed speed rotator that has been placed in the incubator at 50°C.
 * 2) Shake or rotate sufficiently enough to keep solids in constant suspension for a period of 72 to 168 hours or until the release of soluble sugars from the sample(s) becomes negligible when measured by YSI, as described in the next step.
 * 3) If the progress of the reaction is to be measured:
 * 4) A 0.3-0.5 mL aliquot is removed at each predetermined time interval after the tube contents have been well mixed by shaking.
 * 5) Use a 1-mL plastic syringe or 1ml pipette with tip cut off to draw a representative sample while constantly suspending the contents of the vial.
 * 6) This will allow solids, as well as liquid, to be withdrawn into the orifice.
 * 7) The sample is filtered through a 0.45 μm filter
 * 8) Analyze the glucose concentration using the YSI glucose analyzer or appropriate HPLC method.

Calculations
0.0099 g/mL x 10 mL x 0.9 = 0.0891 g
 * 1) To calculate the percent digestibility of the cellulose
 * 2) Determine glucose concentration in the centrifuged supernatant by YSI.
 * 3) Subtract the glucose concentrations, if any, from the substrates and enzyme blanks.
 * 4) Correct for hydration (multiply the glucose reading by 0.9 to correct for the water molecule added upon hydrolysis of the cellulose polymer)
 * 5) Multiply by 10mL total volume of assay.
 * 6) Example: If the glucose analyzer reading (corrected with blanks) is 9.9 mg/mL, then the amount of cellulose digested is:
 * 1) Calculate percent digestion:
 * 2) %digestion= 100 X (grams cellulose digested/grams cellulose added)

To calculate Low enzyme loading


 * 1) Spezyme 5 FPU/ g of Dry Matter (DM)
 * 2) Novozyme 20 IU/ g of Dry Matter (DM)  note FPU and IU are units specific to the enzymes we purchase.
 * 3) 5x0.15 g DM= 0.75 units of Spezyme needed.  Our bottle is 110 units/ul  so add 6.8 ul
 * 4) 20x 0.15 g DM= 3 units of Novozyme needed.  Our bottle is 288 units /ul so add 10.4 ul

To calculate High enzyme loading


 * 1) Spezyme 15 FPU/ g of Dry Matter (DM)
 * 2) Novozyme 60 IU/ g of Dry Matter (DM)
 * 3) Using the same calculations as above the results will be 20.5 uL Spezyme and 31.3 uL Novozyme