Matthew O’Reilly
Senior Comprehensive Exam
Catholic University Chemistry Dept.

• Cellulose and its degradation
• Methods of pre-treatment
• Experimental Approaches
• Acknowledgements

• Cellulosic ethanol is the preferred biofuel
• Cellulose is one of the most widespread natural polymers and is a key component of plant’s cell walls
• The production of cellulosic ethanol biofuel is based on fermentation of glucose derived from plant cellulose.
• Producing cellulose (and subsequently ethanol) from cellulose is difficult because of the complex and rigid structure of the lignocellulosic materials of the cell walls in plant materials
• Pre-treatment with chemicals or radiation is necessary in order to break down the lignocellulosic structure and produce cellulose fragments

Lignocellulosic Plant Material
↓ Pre-treatment (e.g. AFEX, other acids or bases)
Cellulose and large fragments
↓ Hydrolysis (enzymatic or acidic)
Glucose ( and xylose etc.)
↓
Ethanol
Fermentation

Mosier, N.; Wyman, C.; Dale,B.;
Elander,R.; Lee,Y.;
Holtzapple,M; Ladisch,M.
Features of promising technologies for pretreatment of lignocellulosic biomass .
BioResource Technology 2005 ,
96, 673-686.

Mosier, N.; Wyman, C.;
Dale,B.; Elander,R.;
Lee,Y.; Holtzapple,M;
Ladisch,M. Features of promising technologies for pretreatment of lignocellulosic biomass .
BioResource Technology.
2005 , 96, 673-686.

Mosier, N.; Wyman, C.; Dale,B.; Elander,R.;
Lee,Y.; Holtzapple,M; Ladisch,M. Features of promising technologies for pretreatment of lignocellulosic biomass . BioResource
Technology. 2005 , 96, 673-686.

• Requirements for effective pre-treatment:
1) Avoids need for reducing the size of biomass particles.
2)Limits formation of degradation products that inhibit growth of fermentative microorganisms
3) Minimizes energy demands and limits cost.

The University of Edinburgh, Institute of Cell &
Molecular Biology: The Microbial World. http://www.biology.ed.ac.uk/research/groups/jde acon/microbes/armill.htm (accessed Jan 3,
2010).

• The structure of cellulose is very rigid and strong, which is why radiation or strong acids or enzymes must be used to facilitate its break-down. The monomer units of cellulose consist of pyranose rings in the chair conformation with axial hydrogen atoms. This structure is characterized by high rigidity and restricts the conformational transitions.
MIT. Chemistry- A Way of Life. web.mit.edu/clubchem/i mages/benzophenone.p
ng. (accessed Jan. 5,
2010).

Ershov, B.G. Radiation-Chemical Degradation of cellulose and other polysaccharides. Russian
Chemical Reviews . 1998, 67, 314-334.
• The propagation of the free radical with scission of the polymeric chain.
• First, cellulose decomposes into large fragments and then to the ulitimate radiolysis products which include RCHO, RCOOH, CO
CO, etc…
2
,
• The optimal yield obtained using this method was 50:1 radical formation. This ratio is critical in determing the economic feasibility of this process.

• Radiation pre-treatment helps in accomplishing two purposes:
1) Break-up of the lignocellulosic cell wall structure to allow access to the cellulose fibers during subsequent treatment
2) Degradation of cellulose into smaller polymeric fragments
• The economic feasibility of using radiation as a pretreatment depends on the yield of free radicals formed upon radiolytic scission of bonds.
• The focus of the research conducted dealt with the following question: Is it possible to enhance the yield of radicals through the use of free radical initiators?

•Adsorbing free radical initiators onto cellulose and exposing them to radiation may enhance the number of free radicals that are formed upon irradiation and thus enhance the degradation of cellulose. The different species used as intiators in the present study have been hydrogen peroxide, benzoyl peroxide, bromine water and benzophenone.
MIT. Chemistry- A Way of Life. web.mit.edu/clubchem/images/b enzophenone.png. (accessed
Jan. 5, 2010).

• Method: Soaking cotton balls with differing concentrations of 30% hydrogen peroxide (a source of OH radicals upon irradiation)
• OH radicals are very powerful oxidizing agents and they produce radicals capable of initiating chain reactions.
MIT. Chemistry- A Way of Life. web.mit.edu/clubchem/images/ben zophenone.png. (accessed Jan. 5,
2010).

2
2
• The degradation of cellulose was by measured by glucose yields using Benedict’s reagent.
• Cotton balls were soaked in different concentrations of hydrogen peroxide were exposed to gamma radiation.
• Samples of differing concentrations of H
2
O
2 were made and a cotton ball was placed in each respective solution.
The samples were then exposed to gamma radiation.
• The samples were analyzed using a GenTech UV/Vis
Spectrophotometer. The two wavelengths obtained for each sample were at 700nm and 515nm which corresponded to the Cu + and Cu 2+ respectively.

• The Benedict’s Reagent test is used to analyze the content of reducing sugars in solutions, which in this case would be glucose. The reaction for the Benedict’s reagent is shown below.
• If glucose is present the solution will turn from green
(representing the Cu2+ in the reagent) to a brick red color (representing the Cu+ product).
-CHO+2Cu 2+ +H
2
O → -COOH+2Cu + +2H +

• Cannot use Benedict’s method in presence of H
2
O
2
H
2
O
2
+ Cu + → H
2
O + Cu 2+
Thus it is not possible to observe reduction of Cu 2+ by glucose.
1 st solution: Boil sample to decompose H
2 adding Benedict’s solution
O
2 before
2 nd solution: Add catalase enzyme before adding
Benedict’s solution.

• Since residual H
2
O
2 interferes with the Benedict method the analysis of the samples was done by decomposing the excess H
2
O
2 with catalase and then allowing the glucose product to react with Benedict’s solution
• Catalase is a common enzyme that catalyzes the breakdown of hydrogen peroxide into water and oxygen.
The decomposition of hydrogen peroxide with catalase is as follows:
2H
2
O
2
→ 2H
2
O + O
2

• The data obtained from the first trials showed a consistent error that was believed to be caused by the presence of the hydrogen peroxide. In order to try and remove the residual H
2
O
2 interferences the samples were boiled first for 5 minutes and then for 20. The results were inconsistent.
-The sample with the catalase enzyme showed good results, with the sample irradiated with the smallest concentration of H
2
O
2 showing the smallest amount of glucose production and the sample irradiated with the largest concentration of H
2
O
2 showing the highest glucose formation.
• The method has low sensitivity to low concentrations of glucose

• ESR: Detects substances with unpaired electrons such as free radicals.
• In the ESR technique, a magnetic field is applied allowing the unpaired electron spins to form two distinct energy levels.
•
Microwaves are applied at a frequency of 9 GHz in the presence of a magnetic field to allow resonance transition between the two levels.
New Mexico State University: Dept. of Chemistry.
http://www.chemistry.nmsu.edu/stud ntres/chem435/Lab7/. (accessed
Jan.5 2010).

• 1g of cellulose powder
• 10 mL of 1% solution of each additive
• Stirring together for 1 day
• Decantation and air-drying
• Irradiation of part of the sample with 100 kGy Co-60 gamma source
• ESR spectroscopy performed

Pure irradiated benzophenone ESR spectra of pure irradiated benzophenone with a peak at 3350 gauss.
Pure unirradiated benzophenone

Pure irradiated celluloseESR spectra of pure irradiated cellulose with a maximum peak around 3345G and some splitting peaks at 3320G and
3335G.
Pure unirraditated cellulose

Irradiated cellulose with 0.2g benzophenone ESR shows a peak at
3345 with a larger magnitude than that of cellulose and benzophenone as well as a peak at 3320G.
Irradiated cellulose with 0.2g benzophenone after 2 months shows a splitting of the large peak at 3347G with another smaller one at 3320G.

Irradiated cellulose with 0.5g benzophenone ESR shows a split peak at 3340G with smaller peak at
3320G.
Irradiated cellulose with 0.5g benzophenone after 2 months ESR shows a peak at 3345G and another at 3320G.

• The ESR technique showed free radicals that were present in the structure.
The samples that were not exposed to radiation showed no results on the
ESR spectra because no free radicals were present. The samples that were exposed to radiation each showed unique spectra caused by the free radicals present in that structure.
• The spectra of the pure irradiated benzophenone and cellulose each had distinctive features and when benzophenone was added, elements of each ones spectra can be seen in the spectra of the mixture. This mixture also showed unique elements of its own that were not present in either the benzophenone or cellulose ESR. This supports the idea that perhaps the benzophenone incorporated itself into the structure of cellulose.
• The spectra after two months from when the original samples were analyzed, the spectra of the 0.2g of benzophenone showed interesting changes in the shape of the spectra. The 0.5g of benzophenone added did not show distinct changes.

• The spectrum of the irradiated benzophenone-treated cellulose is different from that of the separate ingredients because the free radical may have incorporated itself into the cellulose structure.
• The later change in the spectrum (of the 0.2g benzophenone-doped sample) can be attributed to the reaction of the original radical with atmospheric oxygen.
R •+O
2
→RO
2
•
• This hypothesis could have been tested by storing the sample under an inert gas such as argon or nitrogen instead of air.
• Further experiments were planned but could not be carried out due to the failure of the ESR system.

• Dr. Barkatt
• Dr. Al-Sheikhly of University of Maryland
• Ms. Marina Chumakov

• HMF (Hydroxymethyl furfural) which leads to the formation of levulinic acid and formic acid
• Inhibitory to microbial fermentation

• Flow aqueous ammonia through a column reactor packed with biomass at elevated temperatures.
• Aqueous ammonia reacts with lignin and cause depolymerization of lignin and cleavage of lignincarbohydrate linkages.
• Good method, but need to increase the extent of delignification to achieve better fractionation of biomass and hence higher yields of glucose.

• Fermentation
C
6
H
12
O
6
→ 2 C
2
H
5
OH + 2 CO
2
• Combustion
C
2
H
5
OH + 3 O
2
→ 2 CO
2
+ 3 H
2
O

• Lignin structure made up primarily of methoxylated polyphenol rings