The Effect of Free Radical Precursors on the Radiolysis of Cellulose

1. The Effect of Free Radical Precursors on the Radiolysis of Cellulose Matthew O’Reilly Senior Comprehensive Exam Catholic University Chemistry Dept.

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

3. Cellulosic Ethanol • 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

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

5. Cellulose Structure 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.

6. 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.

7. 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.

8. Pre-treatments on Cellulose • 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.

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

10. Cellulose Strucuture • 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/images/benzophenone.png. (accessed Jan. 5, 2010).

11. Breakdown of Cellulose using Gamma Radiation • 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, CO2, CO, etc… • The optimal yield obtained using this method was 50:1 radical formation. This ratio is critical in determing the economic feasibility of this process. Ershov, B.G. Radiation-Chemical Degradation of cellulose and other polysaccharides. Russian Chemical Reviews. 1998,67, 314-334.

12. Radiation Pretreatment • 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?

13. 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/benzophenone.png. (accessed Jan. 5, 2010).

14. Determination of Glucose Yield using Benedict’s Reagent • 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/benzophenone.png. (accessed Jan. 5, 2010).

15. Effect of H2O2 on Glucose Production • 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 H2O2 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 Cu2+ respectively.

16. Determination of Glucose Yields by the use of Benedict’s Reagent • 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+2Cu2++H2O → -COOH+2Cu++2H+

17. Problems • Cannot use Benedict’s method in presence of H2O2 H2O2 + Cu+→ H2O + Cu2+ Thus it is not possible to observe reduction of Cu2+ by glucose. 1st solution: Boil sample to decompose H2O2 before adding Benedict’s solution 2nd solution: Add catalase enzyme before adding Benedict’s solution.

18. Catalase Enzyme • Since residual H2O2 interferes with the Benedict method the analysis of the samples was done by decomposing the excess H2O2 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: 2H2O2 → 2H2O + O2

19. Sample with Catalase enzyme

20. UV Spectrophotometer Glucose Determination • 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 H2O2 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 H2O2 showing the smallest amount of glucose production and the sample irradiated with the largest concentration of H2O2 showing the highest glucose formation. • The method has low sensitivity to low concentrations of glucose

21. ESR • 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/studntres/chem435/Lab7/. (accessed Jan.5 2010).

22. Experiments with Additives • 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

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

24. ESR of Pure Irradiated Cellulose 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

25. Cellulose with 0.2g benzophenone 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.

26. Cellulose with 0.5g benzophenone 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.

27. Discussion • 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.

28. Reasons for Change in Spectra • 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•+O2→RO2• • 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.

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

30. Minimizing Undesirable HMF Formation in Chemical Pre-treatment • HMF (Hydroxymethyl furfural) which leads to the formation of levulinic acid and formic acid • Inhibitory to microbial fermentation

31. AFEX (Ammonia Fiber Explosion) • 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 lignin-carbohydrate linkages. • Good method, but need to increase the extent of delignification to achieve better fractionation of biomass and hence higher yields of glucose.

32. Glucose→Ethanol • Fermentation C6H12O6 → 2 C2H5OH + 2 CO2 • Combustion C2H5OH + 3 O2 → 2 CO2 + 3 H2O

33. Lignin • Lignin structure made up primarily of methoxylated polyphenol rings