Hydrolysis of Cellulose

Stephanie Henscheid
Intensive Research J-term Final Paper
Dr. Speckhard
24 January 2014

Table of Contents
Introduction and Background . . . . . . . . . . . . . . . . . . . . . . . . . . . .p. 2-5
Materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 5-6
Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .p. 6-9
Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .p. 9-10
Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p. 10-12
Reflection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .p.13
Works Cited. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .p. 14

Henscheid 2
Introduction and Background
In todays society many people are trying to become more green and when I say that I
mean that people are trying to be more conscience about the effects of their actions. One way
that people like to think about being more green is they like to think that they are being
sustainable to their actions. According to the article written by Alan D. Hecht called
Perspectives on Achieving Sustainable Energy Production, he say that, the traditional
denition of sustainability calls for policies and strategies that meet societys present needs
without compromising the ability of future generations to meet their own needs (Hecht). In a
world where there is a dependence on oil is about to outgrow the supply, we have begun the
search to find alternative fuel sources that help us be less dependent on these fossil fuels.
People have studied new ways of making fuels out of biomass like corn ethanol. The
biomass that people look at in order to make biofuels is generally plant biomass; biomass is
defined as natural material that is derived from recently living organisms. The reason why
biomass is so important is because it is renewable (Welcome). When using biomass, we can
use any carbon based materials, which is all plants; there is a large amount of plant waste that is
accumulated every year then disposed of in wasteful manners because no one knows what to do
with it. With further study on this topic to find the most cost efficient process, this plant waste
can be turned into fuel to decrease our dependency of fossil fuels.
People have realized the need to find renewable energy sources dating back to before
1979, during the second oil crisis. During this time the US government decided to promote the
use of fuel ethanol, which was derived from corn. They did this by making an incentive for
people to use ethanol, which was 54 cents per gallon of fuel ethanol used. Since then the
government has reduced the incentive to 51 cents per gallon. Then in 2005 the government

Henscheid 3
created the Energy Policy Act in which it established a renewable fuel standard. This mandate
required the use of renewable fuels to increase per year from 4 billion to 7.5 billion gallons from
2005 to 2012. In 2006, the amount of ethanol used was 4.9 billion gallons which exceeded the
mandated 4.2 billion gallons (Wang). The use of ethanol has continued to exceed the mandates
limits throughout the years. The graph below shows the use of ethanol and how much is
mandated by the Energy Policy Act of 2005.

Henscheid 4
While the use of corn ethanol is wonderful there are a few issues that come along when
using corn to make ethanol. The first problem is that corn is a food source and by using it to
make ethanol is draining a food source that is vital to many lives. A second problem to using
corn is that it depletes the soil of it nutrients very quickly therefore it requires many chemical
inputs in order for it to grow properly, including potash fertilizer, phosphate fertilizer, nitrogen
fertilizer, and lime. It also requires the use of fossil energy to operate farming machinery, to
pump water for irrigation, and to dry corn kernels (Wang). The third major problem using corn it
that the gas is that is produced does not provide a lot of energy, so we have to use more of it.
This is a table comparing fossil energy ratios of how much energy is delivered to the customers
per unit of fossil energy used. As you can see the ratios for using gasoline and electricity are
really low with their values significantly lower than 1. The value for corn ethanol is a little bit
higher coming in at 1.36 so still low but about twice as much as the others. But when you look
at the predicted value of the ratio for cellulosic ethanol coming in at 10.3, we can see that this
type of ethanol will provide us with a lot more energy while using less of our fossil fuels

Henscheid 5

(Carbon). Because of the following problems that occur when using corn as the main source
for biofuels, I decided to study the different ways to use cellulosic biomass to make ethanol.
The most common process to convert cellulosic biomass to ethanol is through hydrolysis.
There are two main types of hydrolysis that would be considered when thinking about trying to
break down cellulose, acid catalyzed hydrolysis and enzymatic hydrolysis. Acid catalyzed
hydrolysis is the use of acid to break down cellulose polymers into monomeric sugars. The
chemical catalytic hydrolysis is a faster acting reaction that requires much less residence time in
the reactor, but has a lot of waste products that are expensive to dispose of (Taherzadeh). In
enzymatic hydrolysis, enzymes are used to break down the cellulose polymers into monomeric
sugars. This involves multiple steps to fully break down the cellulose and these include,
pretreating the cellulosic source to remove the lignins which block the enzymes from being able
to fully break down the cellulose into glucose, once this is done the cellulose broken down into 5
or 6-carbon sugars for conversion into the fuel source, by fermentation and distillation

Henscheid 6
(Chandra). Enzymatic hydrolysis can be run at much lower
temperatures which bring utility costs of a process down.
I will be using the process of enzymatic hydrolysis to break
down the cellulose despite it taking much more time than acid
catalyzed hydrolysis because it takes a lot of acid to break down the cellulose and there would be
much waste from the process. The sources of cellulose that I am using are spinach, to act as
grass which was unavailable at the time, corn husks, and wood shavings. I have chosen to use
the sources of cellulose that I have because it reduces strain on to food industry from other forms
of ethanol production and because cellulosic ethanol is also a better choice than corn ethanol.
There is a greater accessibility and abundance of forest, agricultural, and other cellulosic
resources which do not compete with existing food reserves. Also more energy is produced from
cellulosic sources of ethanol. I believe that out of the three sources the spinach sample will have
the highest glucose concentrations, followed by the corn husks, and then finally the wood
shavings.
Materials:
Materials for Enzymatic Hydrolysis
Spinach: Marketside: Fresh Spinach bought from Walmart Dubuque, IA
Corn Husks: Obtained from Dr. Maslowskys personal garden Dubuque, IA
Aspen Wood Shavings: AlfaPet Aspen Bedding bought from Walmart Dubuque, IA
Cellulase: Fischer Scientific Cellulase from Aspergillus Niger Pittsburg, PA
Materials for Buffer Solution
Sodium acetate
Acetic acid
Distilled water
Materials for Benedicts Reagent
Sodium citrate
Sodium carbonate
Potassium thiocyanate
Copper sulfate
Potassium hexacyanoferrate

Henscheid 7
Distilled water
Dextrose (for test samples)
Materials for Acid Hydrolysis
Cellulose
5% Sulfuric acid
5% Hydrochloric acid
6M Potassium hydroxide
Methods:
Pretreatment of Cellulose
Before any type of hydrolysis was preformed simple pretreatment on the forms of
cellulose were necessary. The spinach was baked in a 450F (approx. 232C) oven for 7 minutes
or until completely dried out. Once it was dry the spinach was ground into a powder to
maximize surface area. The corn husks were already dried out and were ground into a powder.
The wood shavings had inconsistent sizes of pieces, a strainer was used to allow only the small
pieces to be collected while the larger pieces were discarded.
Enzymatic Hydrolysis
To preform enzymatic hydrolysis, 0.100g of each type of cellulose was measured out and
place in a 25mL vial and 10 mL of buffered cellulase solution was added to each vial (except for
the blank vials in which only the buffer was added and no cellulase). The sealed vials were
placed into a 40C oven for 24 hours. Quantitative Benedicts reagent was used to measure
glucose concentrations. The reaction was stopped when the enzymes were placed in boiling
water as a part of using Benedicts reagent (Wang, Nam).
http://www.eng.umd.edu/~nsw/ench485/lab4.htm#Procedures
Buffer Solution
The buffer solution used was had a pH 4.76 and was 50mM. It was made by dissolving
0.286g sodium acetate in 50mL 0.1M acetic acid. Water was added to make it 100mL. When

Henscheid 8
used with the enzymes the solution was diluted to be 5mM by mixing 1mL buffer to 9 mL H2O.
The buffered cellulase solution contained 0.100g celluase per 10mL buffer solution.
Acid Catalyzed Hydrolysis
0.200g of cellulose was added to 10mL of 5% H2SO4 solution in a capped 25mL vial.
Blanks were prepared with 0.200g cellulose and 10mL distilled water. All vials were placed in a
90C oven for 2 hours. The reaction was stopped by neutralizing the sample with 6M potassium
hydroxide. This whole process was repeated only the second time 5% HCl was used. To
neutralize the samples 1.7mL KOH was added to the solutions with H2SO4 and 8.3mL KOH was
added to the solutions with HCl (Wang, Nam).
http://www.eng.umd.edu/~nsw/ench485/lab4.htm#Procedures
Pretreatment with Acid
When pretreatment with acid was completed, 0.100g cellulose was added to 5mL 5%
H2SO4 in a 25mL vial and capped, and placed in an oven at 90C for 1 hr. Then the acid was
neutralized with 83.3L 6M potassium hydroxide. After that was completed then 10mL of
buffered cellulase solution was added to each vial (except for the blank vials in which only the
buffer was added and no cellulase). The vials were placed into a 40C oven for 24 hours.
Quantitative Benedicts reagent was used to measure glucose concentrations.
Quantitative Benedicts Reagent
Solution 1
Sodium citrate
Sodium carbonate (anhydrous)
Potassium thiocyanate
Dissolve in 400mL H2O
Solution 2
Copper sulfate .5 H2O
Dissolve in 50mL H2O

100g
32.5g
62.5g

9g

Henscheid 9
All chemicals were measured out and combined into the 2 different solutions. Solution 2
was added to solution 1 while it was rapidly stirred. After the solutions were combined 0.13g
potassium hexacyanoferrate (II) was added, then whole solution was diluted to 500mL.
When used to measure the amount of glucose in a sample the solution was diluted with
35mL of this solution to 100mL with water. 2mL of Benedicts reagent was added to 4mL of the
glucose sample in a test tube. The test tube was placed in boiling water for 5 minutes. The tubes
were allowed to cool and to stand until the precipitate settled.
The solution was then pipetted into micro-centrifuge tubes to make sure that all of the
precipitate was not in solution. The solution was poured into a cuvette and the spectrometer
measured the absorbance of the solution.
http://www.mystrica.com/Applications/Benedicts
Test Samples of Glucose
To practice using Benedicts reagent test samples with known concentrations of glucose
were prepared. For one set of known concentrations 0.180g dextrose was dissolved in 10 mL of
water to create a solution that was 0.1M. 5mL of the solution was used and then 5mL of water
was added back causing the concentration to be cut in half. This process was repeated so that the
samples that were tested were 0.1M, 0.05M, 0.025M, 0.0125M, and 0.00625.
A similar process was repeated with 2.882g of dextrose and 20mL water to start with a
solution that had a concentration of .8M. 5mL of the solution was used and then 5mL of water
was added back causing the concentration to be decreased by one-fifth. This was repeated to get
solutions with concentrations of 0.8M, 0.64M, 0.48M, 0.32M, and 0.16M.
Each sample was reacted with Benedicts reagent and their absorbance was tested. The
absorbances, at 398nm, were plotted versus the known molarities of glucose on a graph in

Henscheid 10
Logger Pro 3.8.6.1. A best fit line was tried out and it was the quantic line, this graph is shown
below. Once the graph of the known concentrations of glucose was obtained, the absorbances of
the samples with unknown molarities glucose were able to be plotted on the graph to be able to
see the amount of glucose produced in the reaction.

Results:
Based on the comparison of molarities in the chart below, in general the spinach samples
got the highest concentrations of glucose. There were higher concentrations of glucose
measured from the samples taken after 18 hours of heating. In general the enzymes worked
better than the acids.
On the table below in the set column, when it says 1st, 2nd, or 3rd, that refers to the
trial of regular enzymatic hydrolysis. The 18 and 24 refer to what hour the samples were
taken from, either after 18 hours of being in the oven or 24 hours after. Acids means when I

Henscheid 11
did the acid hydrolysis of the cellulose. Both refers to the method of pretreating the cellulose
by running it with acid and then adding the enzyme to finish the reaction.

Set
1st 18

1st 24

2nd 18

2nd 24

Type
ASpinach
BSpinach

Absorban
ce

Molarit
y

Absorbanc
e

Molarit
y

A- Spinach

1.579

0.154

B- Spinach
C- Spinach
(Blank)
D- Corn
E- Corn
F- Corn (Blank)

1.748

0.79

1.614
1.546
1.534
0.852

0.483
0.146
0.14
0.051

G- Wood

1.148

0.077

1.053

0.069

0.115
0.12 Acids
0.078
0.068

H- Wood
I- Wood
(Blank)
Spinach H2SO4
Spinach HCl
Spinach Blank

0.295
1.302
1.354
1.852

0.012
0.098
0.104
0.84

1.597

0.161

Corn H2SO4

0.916

0.053

1.659
1.609
1.606
1.482
1.398

0.682
0.479
0.478
0.131
0.113

Corn HCl
Corn Blank
Wood H2SO4
Wood HCl
Wood Blank

0.634
1.169
0.55
0.326
0.784

0.032
0.077
0.028
0.011
0.044

1.665

0.709 Both

A- Spinach

1.065

0.0701

1.601

0.163

1.047

0.0678

1.432
1.479
1.085
Broke

0.117
0.129
0.072
Broke

B- Spinach
C- Spinach
(Blank)
D- Corn
E- Corn
F- Corn (Blank)
G- Wood
H- Wood
I- Wood

1.076
0.779
0.739
0.735
0.579
0.517
0.573

0.0724
0.0445
0.043
0.0422
0.0298
0.0259
0.0293

Type

1.741

Set
3rd
0.793 24

1.652

0.662

C- Corn
D- Corn
E- Wood
F- Wood
ASpinach
BSpinach

1.517
1.609
1.075
1.232

0.138
0.436
0.071
0.089

1.528

0.14

1.715

0.772

C- Corn
D- Corn
E- Wood
F- Wood
ASpinach
BSpinach
C- Corn
D- Corn
E- Wood
F- Wood
ASpinach
BSpinach

1.404
1.432
1.145
1.041

C- Corn
D- Corn
E- Wood
F- Wood

Henscheid 12
(Blank)

Discussion:
As suspected the spinach samples had the highest glucose concentrations, followed by the
corn husk samples and lastly the wood shaving sample. This is because of how much lignin is in
each of the types of the samples, what the lignin does is it protects the cellulose from degradation
and has to be removed before the enzymes can work to fullest capacity to breakdown the
cellulose. Wood has a very high amount of lignin compared to other softer sources of cellulose
(Demers).
The relatively high levels of glucose are great results because that means that it is a good
source of ethanol. The more glucose in a sample, the more can be fermented into ethanol. On a
commercial level this process of enzymatic hydrolysis is not to the point where we can produce
ethanol cheap enough where people would actually buy it. With that being said, the technology
is being looked and studied so that one day this can be a process used just like it is for corn
ethanol.
There may be another reason why the spinach absorbance levels and that could be due to
the chlorophyll affecting the absorbance readings. If this were the case one could start the
process with another pretreatment step to get rid of the chlorophyll, this method would include
acetone rinses until all of the chlorophyll (green color) was removed. The reason why this was
not a problem for the wood shavings or the corn husks was because both of them were a light tan
color and they had no chlorophyll left in them.
The first set of samples taken had low concentration levels because the oven that was
used would not go any lower than 55C, optimal temperature for the enzymes is 40C. After that

Henscheid 13
set, a different oven was used to run the samples, that oven was able to stay at approximately
40C, therefore better results were achieved.
When using the acid catalyzed hydrolysis method and the pretreatment with acid method,
the results obtained were subpar. That is believed to be due to the improper neutralization of the
acid before running the Benedicts reagent.
As the absorbance levels grew higher the resulting molarities were not what was
expected. As seen on the graph on page 9 the absorbance levels leveled off even though the
concentration of the glucose continued to grow. This is not what was supposed to happen but it
did. This error can be traced back to 2 things. First off, the spectrometer does not give a very
accurate reading once the absorbance hit a certain point and the levels that were measured could
have hit that point because they were quite high. Secondly, the absorbance could have dropped
off because there was not enough copper from the Benedicts reagent to react with all of the
reducing sugar in the samples causing the absorbance to stay the same. To solve the first
problem, one would need to change the way of testing the levels of glucose. They could either
use a spectrometer that takes more accurate readings or use a whole different method like
fermentation to measure the levels. To solve the second problem, one could react the samples
with more Benedicts reagent so that the copper is not a limiting reagent.
As of right now I do not plan on continuing this project. Although if I were to continue
on this project I would make a few changes, that include taking care of the chlorophyll problem
with the spinach and using a better method for measuring glucose concentrations. I had already
given an idea of how I would take care of the chlorophyll problem but I would consider dropping
the use of the spinach in general. The point of the project was to not use food sources but with
the lovely Iowa winters I had no access to grass or other yard waste. Next time I would collect

Henscheid 14
grass and leaf samples that are dried. To have a better method for measuring glucose
concentrations I would ferment my final samples to test glucose levels because I would get more
accurate results and I would know for sure that what I have is glucose. The reason I would know
that it is only glucose is because the yeast only eat the glucose and not the other byproducts
created from the enzymatic hydrolysis of the cellulose. I did not do this process during J-term
because of the time constraint that I had. Fermentation takes a long time and with only having
three weeks that was not a plausible process.
Reflection:
As a general thought this J-term of research was interesting, but I will not be pursuing
this as a career option. By the end of the J-term I felt more comfortable with the process and was
enjoying myself a little bit more. My opinion of science changed because when you are doing
research you are exploring the unknown which I have not had much experience with that because
it makes me feel very uncomfortable and is not what I like to surround myself with. Coming into
this I thought I had an idea of what research was going to be like, this fulfilled that expectation
and exceeded it by a hundred times. I didnt realize how mentally exhausting this was going to
be. What I hated most about the research is also what I like the most which was the freedom. In
science labs that I have had before it is very structured, you get a procedure, you do it, and the
professor knows what is supposed to happen, simple as that. While that is nice sometimes, it
gets old after a while. During research I was able to find some procedures but they werent
spelled out word by word, there was still things that I needed to find on my own and I couldnt
go to a professor because they would do the same thing I had to do: look up other research on the
internet. I had the freedom to choose how I did a method and make my own solutions. That was

Henscheid 15
fun. I dont plan on doing much research outside of what is required in the future but this was a
good starting experience so I know what I am getting myself into later on in life.

Works Cited
"Benedict's Test." Mystrica. N.p., n.d. Web. 25 Jan. 2014.
"CARBON DIOXIDE/ NET ENERGY." Ceres. Ceres Inc., 2007. Web. 25 Jan. 2014.
Chandra, R. P., R. Bura, W. E. Mabee, A. Berlin, X. Pan, and J. N. Saddler. "Substrate
Pretreatment: The Key to Effective Enzymatic Hydrolysis." Springer-Verlag Berlin
Heidelberg, 2007. Web. 20 Jan. 2014
Demers, Alexander, Richard Doane, Scott Guzman, and Ryan Pagano. "Enzymatic Hydrolysis of
Cellulosic Biomass for the Production of Second Generation Biofuels." Worcester
Polytechnic Institute. N.p., 01 May 2009. Web.
Hecht, Alan D., and C. Andrew Miller. Perspectives On Achieving Sustainable Energy
Production and Use. Journal of Renewable & Sustainable Energy 2.3 (2010) 031002.
Academic Search complete. Web. 25 Jan. 2014.
Taherzadeh, Mohammad J., and Keikhosro Karimi. "Acid-Based Hydrolysis Processes for
Ethanol From Lignocellulosic Materials." NCSU.edu. N.p., 26 Aug. 2007. Web. 5 Jan.
2014.

Henscheid 16
Wang, Michael, May Wu, and Hong Huo. "Life-cycle Energy and Greenhouse Gas Emission
Impacts of Different Corn Ethanol Plant Types." IOP Science. N.p., 22 May 2007. Web.
17 Jan. 2014.
Wang, Nam Sun. "Cellulose Degradation." Cellulose Degradation. N.p., n.d. Web. 25 Jan. 2014.
"Welcome to the Website of the BIOMASS Energy Centre." Welcome to the Website of the
BIOMASS Energy Centre. BIOMASS Energy Centre, n.d. Web. 25 Jan. 2014.