How to Load Enzyme Based on Glucan Contents in Raw Biomass Material
2010-09-22
Xiadi Gao
1. Background
Enzymatic hydrolysis allows nearly theoretical yields after appropriate pretreatment. However, enzymes
are expensive and ,therefore, there is an important parameter deserves attention: the ratio of enzymes
to total potential ethanol from cellulose and hemicellulose be the same for different pretreatment
conditions or technologies, which is the most critical factor for economical and fair comparisons. If a
pretreatment (or a particular pretreatment at some conditions) can remove more glucan in that step
with less degradation of glucose and xylose, the technology (or pretreatment conditions) will benefit by
getting better yields with the same amount of enzyme ( consequently, higher enzymes to glucan ratio in
enzymatic hydrolysis step) than the technology ( or conditions) that releases less glucose during
pretreatment and would have a lower enzyme loading per unit glucan in enzymatic hydrolysis step.
This comparison is particularly important at low enzyme loadings that are more commercially affordable.
As a result, it is vital to apply enzymes based on glucan in raw biomass and not on glucan in the
pretreated solids.
2. Calculation Basis
To load enzyme based on glucan content in raw material, there are two calculation methods. One is using
glucan yield and the other one is using solid yield. To make the statement clear, a real case example is
applied as follow:
10 g raw material
4 g glucan
Pretreatment
7 g pretreated material
3.6 g glucan
Glucan content in raw material = 4/10=40%
Glucan content in pretreated material = 3.6/7 = 51%
Glucan yield= 3.6/4 = 90%
Solid yield = 7/10 = 70%
Let us assume enzyme loading is 10 mg enzyme /g glucan in raw material and enzymatic hydrolysis is
performed with 25mL total reaction volume and 1% glucan loading.
So, in each reaction flask, the amount of glucan = 25mL × 1g/mL × 1% = 0.25 g glucan.
2.1 Calculation based on glucan yield
Pretreated material to weigh for each flask = 0.25 g/ glucan contents in pretreated material = 0.25g/
51%= 0.49 g (dry)
Enzyme loading for each flask = 10 (mg/g) × 0.25 g / glucan yield = 10 × 0.25 / 90% = 2.78 mg
Pros.
Each bottle contains exactly the same amount of glucan.
Cons.
It requires composition analysis for pretreated material, which cost more time.
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2.2 Calculation based on solid yield and glucan in raw material
Raw material to weigh for each flask for 1 wt.% glucan in untreated biomass = 0.25 g / glucan
contents in raw material = 0.25 g/40% = 0.625 g of raw material
Pretreated material to weigh if assuming glucan content does not change = 0.625 g × solid yield=
0.625×70% = 0.4375 g of pretreated material (dry)
Amount of glucan in flask = 0.4375*0.51 = 0.2231 g
Enzyme loading for each flask = 10(mg/g) × 0.25 g = 2.5 mg
Pros.
It is more convenient because it does not require composition analysis on pretreated material.
Cons.
Each bottle contains slightly different amount of glucan depending on pretreatment conditions and
would differ by a significant amount for hydrolysis with higher glucan content.
2.3 To better understand if both of the methods reflect the effectiveness of pretreatment, the
actual enzyme loadings to glucan (after pretreatment) are calculated as below:
Method 2.1
Enzyme loading per g glucan = 2.78 mg/0.25 g glucan in flask = 11.12 mg/g
Method 2.2
Enzyme loading per g glucan = 2.5 mg / (0.2231 g glucan in flask) = 11.2 mg/g
The calculations above show that both loading methods are capable of showing the effectiveness of
pretreatment, which means that the more glucan is removed during pretreatment, the higher enzyme
loading per g residual glucan will be loaded actually.
To compare the convenience of the two methods, method 2.1 requires a compositional analysis to obtain
the glucan content in pretreated material, which is not necessary for method 2.2. In terms of error that
may occur, method 2.1 assure the glucan loading in each flask for enzymatic hydrolysis, avoiding the
different inhibition that may happen in method 2.2 in which each flash contains slightly different amount
of glucan. However, compared to method 2.1, method 2.2 is easier due to avoiding an extra
step—compositional analysis.
3.
Method 1: Based on glucan yield calculation
1) Determine the composition of raw biomass and pretreated solids following NREL LAPs. The
glucan contents in raw material is x % and in pretreated material is y%.
2) The total amount of starting dry material is A g and pretreated material is a g
A g raw material
x % glucan
a g pretreated material
y % glucan
Pretreatment
3) Calculate glucan yield GY% as following:
𝐺𝑌% =
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑔𝑙𝑢𝑐𝑎𝑛 𝑙𝑒𝑓𝑡 𝑎𝑓𝑡𝑒𝑟 𝑝𝑟𝑒𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡
𝑇𝑜𝑡𝑎𝑙 𝑔𝑙𝑢𝑐𝑎𝑛 𝑎𝑚𝑜𝑢𝑛𝑡 𝑖𝑛 𝑠𝑡𝑎𝑟𝑡𝑖𝑛𝑔 𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙
2
=
𝐴 ×𝑥%
𝑎 ×𝑦%
4) Measure the moisture z% of pretreated material by using moisture analyzer or 105oC oven.
5) Enzymatic hydrolysis is performed in a 25mL reaction volume with 1% glucan loading and enzyme
loading of w mg/g glucan in raw material
6) The amount of pretreated material to weigh for each flask for enzymatic hydrolysis reaction =
0.25g /[ y%×(1-z%)]
7) Enzyme loading for each bottle, mg = w (mg /g) ×0.25 g/ GY%
4 Method 2: Base on solid yield and glucan in raw material
This method can be applied to Parr reactor as well as tube reactor pretreatment.
4.1Parr reactor
4.1.1 Pretreatment and solid yield
1) Weight A g of raw dry biomass (If contains moisture, convert mass into mass of raw dry material)
and soak in targeted concentration of acid solution. Follow standard pretreatment procedure to
perform pretreatment.
2) Prepare a filter-funnel and 2 pieces of filter paper. Using 110oC oven, dry the filter papers and
then weight the total mass of filter-funnel+ filter papers as m g.
3) Filter the hydrolyzate from pretreatment using the pre-weighed funnel and filter papers. Make
sure to minimize the lost of solids.
4) Keep the vacuum pump on for 30-40 min in order to remove more moisture from pretreated
solid.
5) Use the Ziploc bag to collect solids. So weigh a plastic Ziploc bag as p g.
6) Then Weigh Ziploc bag + pretreated biomass as q g.
7) Determine the moisture of pretreated biomass as z% using moisture analyzer or 105oC oven
(usually around 70%).
8) Because some pretreated solid will be left on the filter paper in funnel, dry the funnel + filter
paper + residual biomass in 110oC oven overnight and weight the total mass as n g.
9) Calculate solid yield as following :
Solid yield S% =
Total mass of dry staring material
Total mass of dry pretreted material
=
(𝐪−𝐩)×(𝟏−𝐳%)+(𝐧−𝐦)
𝐚
× 100%
where, q is biomass weight with bag; p is bag weight; z% is moisture content; n is filter-funnel,
filter paper, and residual biomass weight; m is weight of filter-funner and filter paper
4.1.2 Enzymatic hydrolysis
1) Determine the composition of raw biomass following NREL LAPs. The glucan contents in raw
material is x %
2) Assume that enzymatic hydrolysis is performed in a total volume of 25 mL with 1% glucan in raw
biomass loading.
3) Calculate the amount of pretreated material required b g for each flask for enzymatic hydrolysis as
following :
bg=
25 ×1% × S%
x% ×(1−z%)
Where S% is solid yield, x% is glucan content in raw material, z% is moisture in pretreated material.
Explanation for the Equations:
3
25mL × 1g/mL× 1% = mass of glucan for each flask
25 × 1% / x% = raw material for each flask
(25 × 1%/x% ) × S% = dry pretreated biomass needed for each flask if assuming that the absolute glucan
amount does not change before and after pretreatment
[(25 × 1%/x% ) × y % ] / (1-z%) = amount of pretreated biomass for each flask
4) Enzyme solution concentration is c mg enzyme/mL. If enzyme loading is w mg enzyme / g glucan in
raw material, then the volume v mL of enzyme solution needed for each flask can be determined as
following
v=
25 ×1% ×w
c
4.2 Tube reactor
The major difference between using Parr reactor and tube reactor is how to determine solid yield.
The next part will introduce how to determine solid yields when using tube reactor
1) Use 3 (22 mL internal volume) or 4 (10 mL internal volume) tubes as a group. (Note: The number of
tubes depends on how many pretreated samples are needed) Assume all tubes in the same group
have the same solid yield.
2) Load A’g raw dry biomass to each tube and perform pretreatment.
3) Prepare funnel and dry filter paper (recommending two pieces) and weight it as m’ g.
4) After pretreatment, carefully collect ALL pretreated biomass from one tube to the weighted funnel.
5) Collect the pretreated biomass from the rest tubes in the same group for further use in enzymatic
hydrolysis. Determine moisture as z ‘%.
6) Dry the funnel and biomass from step 4 in 110oC oven overnight and weight (the funnel+ filter
papers+ biomass) as n’ g.
7) Solid yield S% can be calculated as following.
m′ − n′
× 100%
A′
8) Enzyme loading for enzymatic hydrolysis can be performed the same way as described in 4.1.2..
S% =
5 Method 3: Base on all raw material that undergoes pretreatment calculation
There is an alternative method may be applied to tube reactor pretreatment. This method does not
require determining solid yields
1) Load A’’ g raw dry biomass to a tube reactor. The glucan content in raw material is x%
2) Perform pretreatment.
3) After pretreatment, collect ALL hydrolyzate from the tube into a 50mL centrifuge tube.
4) Centrifuge to separate liquid from solid.
5) Remove liquid and using 3× 40mL DI water to wash the solid.
6) The total reaction volume V’ mL of enzymatic hydrolysis (1% glucan loading) for each centrifuge
tube can be determined by equation 5.
V=
A′′ × x%
1%
1
× 1g/mL
(5)
7) The volume of enzyme solution t mL is calculated by equation 6.
4
t mL =
A′′ g × x % × w mg /g
c mg /mL
(6)
Note:
1. Enzyme can also be loaded based on glucan+xylan contents in raw material, depending on the
enzyme species used in enzymatic hydrolysis.
2. 0.05 M, pH= 4.8 citrate buffer which containing 0.1g/L sodium azide is used in enzymatic hydrolysis.
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