This research has developed reproducible methods for the synthesis of Bmim[FeCl4],
Emim[FeCl4], completed the screening for dissolution of switch grass, microcrystalline
cellulose, and fibrous cellulose. It was demonstrated that Bmim[FeCl4] and Emim[FeCl4] can
both facilitate the depolymerization of select biomass. A comparison of the dissolution effects of Bmim[FeCl4], Emim[FeCl4], and FeCl3 indicates that the activity by Bmim[FeCl4] is the
highest. However, it must be noted that the solubility of the magnetic iron complex of [Bmim]Cl- is significantly less than that of the non-magnetic complex which have up to 25
w/w%10. Investigation also shows that a biomass/magnetic ionic liquid reaction needs
sufficiently higher temperatures when compared to [Bmim]Cl- to achieve slight dissolution.
To further this research, results indicate that it might be of worthy investigation to perform many other studies. Determine the water content of Bmim[FeCl4] and Emim[FeCl4] to see if
moisture is preventing greater dissolution. To test other ionic liquids with paramagnetic
properties and adjust the pH to see if dissolution rates and solubility would increase. To develop inexpensive methods of separation intensification for the recovery of the magnetic ionic liquids (i.e. increasing the magnetic field strength induced on the MIL/biomass slurry during
50
use of ultrasound or microwave technology or varying the pressures and temperatures. Lastly, to understand the interaction mechanism between magnetic ionic liquids and cellulose,
51
52
1. Mantz, R. A. et al. Dissolution of Biopolymers Using Ionic Liquids. (2007).
2. Breaking the Biological Barriers to Cellulosic Ethanol: A Joint Research Agenda. Dep. Engergy SC–0095 (2005).
3. Demirbas, A. Biofuels sources, biofuel policy, biofuel economy and global biofuel projections. Energy Convers. Manag. 49, 2106–2116 (2008).
4. Galbe, M. & Zacchi, G. in 41–65 (Springer, 2007).
5. Himmel, M. E. et al. Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315, 804–7 (2007).
6. Fu, D., Mazza, G. & Tamaki, Y. Lignin extraction from straw by ionic liquids and
enzymatic hydrolysis of the cellulosic residues. J. Agric. Food Chem. 58, 2915–22 (2010). 7. Brodeur, G. et al. Chemical and physicochemical pretreatment of lignocellulosic biomass:
a review. Enzyme Res. 2011, 787532 (2011).
8. Zhu, S. et al. Dissolution of cellulose with ionic liquids and its application: a mini-review. Green Chem. 8, 325 (2006).
9. Fredlake, C. P., Crosthwaite, J. M., Hert, D. G., Aki, S. N. V. K. & Brennecke, J. F. Thermophysical Properties of Imidazolium-Based Ionic Liquids. 954–964 (2004).
10. Swatloski, R. P., Spear, S. K., Holbrey, J. D. & Rogers, R. D. Dissolution of Cellose with Ionic Liquids. 4974–4975 (2002).
11. Zheng, Y., Zhang, S. & Wang, J. The application of ionic liquids in dissolution and separation of lignocellulose. 71–84 (2007).
12. Mahalaxmi, S. PREPROCESSING OF BIOMASS USING MECHANICAL CHEMICAL AND MICROBIAL TECHNIQUES. (2012).
13. Giampietro, M., Ulgiati, S. & Pimentel, D. Feasibility of Large-Scale Biofuel Production Does an enlargement of scale change the picture ? 47, 587–600 (2008).
14. Housing Energy Advisor. Installing a Biomass Boiler at Your Home. (2010). at <http://www.housingenergyadvisor.com/blog/install-biomass-boiler-123/>
15. Fengel, D. G. Wood: Chemistry, Ultrastructure, Reactions. (Walter de Gruyte, 1984). 16. McKendry, P. Energy production from biomass (Part 1): Overview of biomass. Bioresour.
53
17. Champagne, P. & Li, C. Enzymatic hydrolysis of cellulosic municipal wastewater treatment process residuals as feedstocks for the recovery of simple sugars. Bioresour. Technol. 100, 5700–6 (2009).
18. Vegas, R., Alonso, J. L., Domínguez, H. & Parajó, J. C. Processing of rice husk
autohydrolysis liquors for obtaining food ingredients. J. Agric. Food Chem. 52, 7311–7 (2004).
19. Hendriks, A. T. W. M. & Zeeman, G. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour. Technol. 100, 10–8 (2009).
20. HAYATSU, R. et al. Lignin-like polymers in coals. Nature 278, 41–43 (1979). 21. SciDAC Review 15 - Overcoming Plant Recalcitrance. at
<http://www.scidacreview.org/0905/html/biofuel.html>
22. Dependence, R. & Oil, F. Bringing Biological Solutions to Energy Challenges. 1–4 (2007).
23. Chang, V. & Holtzapple, M. Fundamental factors affecting biomass enzymatic reactivity. Appl. Biochem. Biotechnol. 5–37 (2000). at
<http://link.springer.com/chapter/10.1007/978-1-4612-1392-5_1>
24. Harmsen, P., Huijgen, W., Bermúdez López, L. M. & Bakker, R. R. C. Literature Review of Physical and Chemical Pretreatment Processes for Lignocellulosic Biomass. 1–49 (2010).
25. Hayashi, S., Saha, S. & Hamaguchi, H. A New Class of Magnetic Fluids: bmim[FeCl4] and nbmim[FeCl4] Ionic Liquids. IEEE Trans. Magn. 42, 12–14 (2006).
26. Fort, D., Remsing, R. & Swatloski, R. Can ionic liquids dissolve wood? Processing and analysis of lignocellulosic materials with 1-n-butyl-3-methylimidazolium chloride. Green … 9, 63 (2007).
27. Graenacher, C. Cellulose Solution and Cellulose Derivative and Process of Making Same. 1–4 (1933).
28. Vitz, J., Erdmenger, T., Haensch, C. & Schubert, U. S. Extended dissolution studies of cellulose in imidazolium based ionic liquids. Green Chem. 11, 417 (2009).
29. Zhang, Y., Du, H., Qian, X. & Chen, E. Y.-X. Ionic Liquid−Water Mixtures: Enhanced K w for Efficient Cellulosic Biomass Conversion. Energy & Fuels 24, 2410–2417 (2010).
54
30. Fukaya, Y., Sugimoto, A. & Ohno, H. Superior solubility of polysaccharides in low viscosity, polar, and halogen-free 1,3-dialkylimidazolium formates. Biomacromolecules 7, 3295–7 (2006).
31. Mikkola, J.-P. et al. Ultrasound enhancement of cellulose processing in ionic liquids: from dissolution towards functionalization. Green Chem. 9, 1229 (2007).
32. Kilpeläinen, I., Xie, H. & King, A. Dissolution of wood in ionic liquids. J. Agric. … 9142–9148 (2007). at <http://pubs.acs.org/doi/abs/10.1021/jf071692e>
33. Sun, N., Rodríguez, H., Rahman, M. & Rogers, R. D. Where are ionic liquid strategies most suited in the pursuit of chemicals and energy from lignocellulosic biomass? Chem. Commun. (Camb). 47, 1405–1421 (2011).
34. Remsing, R. C., Swatloski, R. P., Rogers, R. D. & Moyna, G. Mechanism of cellulose dissolution in the ionic liquid 1-n-butyl-3-methylimidazolium chloride: a 13C and 35/37Cl NMR relaxation study on model systems. Chem. Commun. (Camb). 1271–3 (2006). doi:10.1039/b600586c
35. Turner, M. B., Spear, S. K., Huddleston, J. G., Holbrey, J. D. & Rogers, R. D. Ionic liquid salt-induced inactivation and unfolding of cellulase from Trichoderma reesei. Green Chem. 5, 443 (2003).
36. Jiang, Y., Guo, C. & Liu, H. Magnetically rotational reactor for absorbing benzene emissions by ionic liquids. China Particuology 5, 130–133 (2007).
37. Hayashi, S. & Hamaguchi, H. Discovery of a Magnetic Ionic Liquid [bmim]FeCl4. Chem. Lett. 33, 1590–1591 (2004).
38. Misuk, V., Breuch, D. & Löwe, H. Paramagnetic ionic liquids as “liquid fixed-bed” catalysts in flow application. Chem. Eng. J. 173, 536–540 (2011).
39. Lee, S. H., Sung, H. H., You, C.-Y. & Koo, Y.-M. Recovery of magnetic ionic liquid [bmim] FeCl4 using electromagnet. Korean J. Chem. Eng. 24, 436–437 (2007).
40. Wang, H., Yan, R., Li, Z., Zhang, X. & Zhang, S. Fe-containing magnetic ionic liquid as an effective catalyst for the glycolysis of poly(ethylene terephthalate). Catal. Commun. 11, 763–767 (2010).
41. Yao, A. & Chu, T. Fe-containing ionic liquids as effective and recoverable oxidants for dissolution of UO2 in the presence of imidazolium chlorides. Dalton Trans. 42, 8413–9 (2013).
55
42. Friedman, H. L. The Visible and Ultraviolet Absorption Spectrum of the
Tetrachloroferrate(III) Ion in Various Media 1. J. Am. Chem. Soc. 74, 5–10 (1952). 43. Liu, H., Sale, K. L., Holmes, B. M., Simmons, B. A. & Singh, S. Understanding the
Interactions of Cellulose with Ionic Liquids : A Molecular Dynamics Study. 4293–4301 (2010).
56 APPENDIX
57 APPENDIX A
58
Table A.1: Effect of various pretreatment methods on the chemical composition and
chemical/physical structure of lignocellulosic biomass. H: high effect, L: low effect, ND: not determined, *Depends on the chemical nature of the solvent.7
Pretreatment
method Increases accessible surface area Decrystalizes cellulose Removes hemicellulose Removes lignin Alters lignin structure
Alkaline H ND L H H Acid H H H Green Solvent H H L H or L* L Steam Explosion H H L LHW H ND H L AFEX H H L H H ARP H H L H H SCF H H H L
59
Table A.2 Advantages and disadvantages of different pretreatment methods of lignocellulosic biomass.7
60 APPENDIX B
61
B1: Physical and Chemical properties of 1-ethyl-3-methylimidazolium chloride
Figure B.1: [EMIM]Cl-
Name: 1-ethyl-3-methylimidazolium chloride CAS: 65039-09-0
Empirical Formula: C6H11ClN2
Molecular Weight: 146.62 g/mol pH: 7.7 at 100 g/l
Melting point: 77-79oC
Flash Point: 186.00 oC
62
B2: Physical and Chemical properties of 1-butyl-3-methylimidazolium chloride
Figure B.2: [BMIM]Cl-
Name: 1-butyl-3-methylimidazolium chloride CAS: 79917-90-1
Empirical Formula: C8H15ClN2
Molecular Weight: 174.67 g/mol pH: 7.9 at 100 g/l
Melting point: 70oC
Flash Point: 192 oC
63 APPENDIX C
64
C: Procedure for the Filter Paper Assay for Saccharifying Cellulase
Authors: Y.H. Percival Zhang, Jiong Hong, and Xinhao Ye, Cellulase Assays, Biofuels: Methods and Protocols, Methods in Molecular Biology, Jonathan R. Mielenz (ed.), vol. 581; 2009 Chapter 14 C1: Assay description
FPA is the most common total cellulase activity assay recommended by the International Union of Pure and Applied Chemistry (IUPAC)1. IUPAC recommends a filter paper activity
(FPA) assay that differs from most enzyme assays based on soluble substrate for initial reaction rates. This assay is based on a fixed degree of conversion of substrate, i.e. a fixed amount (2 mg) of glucose (based on reducing sugars measured by the DNS assay) released from 50 mg of filter paper (i.e., both amorphous and crystalline fractions of the substrate are hydrolyzed) within a fixed time (i.e., 60 min). In part due to the solid heterogeneous substrate, reducing sugar yield during hydrolysis is not a linear function of the quantity of cellulase enzyme in the assay
mixture. That is, twice the amount of enzyme does not yield two times the reducing sugar within equal time. Total cellulase activity is described in terms of “filter-paper units” (FPU) per
milliliter of original (undiluted) enzyme solution. The strengths of this assay are that (1) the substrate is widely available and (2) the substrate is reasonably susceptible to cellulase activity. However, the FPA has long been recognized for its complexity and susceptibility to operator errors.2
1Ghose TK (1987) Measurement of cellulase activities. Pure Appl. Chem. 59:257–268.
2 Coward-Kelly G, Aiello-Mazzari C, Kim S, Granda C, and Holtzapple M (2003) Suggested improvements to the standard filter
65 C2: Reagents and Materials
Supplies:
13x100 mm test tubes/caps 30 x 115 mm conical tubes Whatman No. 1 paper strips Phenol
Sodium metabisulfite
Citric acid monohydrate Distilled Water
Sodium Hydroxide 3,5 Dinitrosalicylic acid Sodium Potassium Tartrate Dilute Enzyme Solution (DES)-
Dilute commercial cellulase solution 20 fold using 0.05M Na-citrate buffer (1 part solute, 19 part solvent, CV=CV). Use conical tube.
Enzyme Stock (ES)- Use conical tube.
ES1: 0.10 ml of DES + 1.90 ml of citrate buffer (dilute rate = 0.0250). ES2: 0.15 ml of DES + 1.85 ml of citrate buffer (dilute rate = 0.0375). ES3: 0.20 ml of DES + 1.80 ml of citrate buffer (dilute rate= 0.0500). ES4: 0.30 ml of DES + 1.70 ml of citrate buffer (dilute rate = 0.0750). ES5: 0.35 ml of DES + 1.65 ml of citrate buffer (dilute rate = 0.0850).
Glucose Standards Stocks (GSS)-
A working stock solution of anhydrous glucose (10 mg/mL) should be made up. Use conical tube
GSS1: 1.0 ml of glucose stock + 4.0 ml buffer = 2 mg/ml (1.0 mg/0.5 ml). GSS2: 1.0 ml of glucose stock + 2.0 ml buffer = 3.3 mg/ml (1.65 mg/0.5 ml). GSS3: 1.0 ml of glucose stock + 1.0 ml buffer = 5 mg/ml (2.5 mg/0.5 ml). GSS4: 1.0 ml of glucose stock + 0.5 ml buffer = 6.7 mg/ml (3.35 mg/0.5 ml).
Glucose Standards Tubes (GSs)- Use test tube
GS1: 0.5 ml of GSS1 + 1.0 ml of .05M citrate buffer GS2: 0.5 ml of GSS2 + 1.0 ml of .05M citrate buffer GS3: 0.5 ml of GSS3 + 1.0 ml of .05M citrate buffer GS4: 0.5 ml of GSS4 + 1.0 ml of .05M citrate buffer
Enzyme Controls Tubes (EC)- Use test tube
EC1: 1.0 ml of .05M citrate buffer + 0.5 ml ES1 EC2: 1.0 ml of .05M citrate buffer + 0.5 ml ES2 EC3: 1.0 ml of .05M citrate buffer + 0.5 ml ES3 EC4: 1.0 ml of .05M citrate buffer + 0.5 ml ES4 EC5: 1.0 ml of .05M citrate buffer + 0.5 ml ES5
Substrate Control Tube (SC)- Use test tube
SC: 1.5 ml of 0.05M citrate buffer + filter paper strip
Reagent Blank Tube (RB)- Use test tube
RB: 1.5 ml of 0.05M citrate buffer
Enzyme Assay Tubes (E)- Use test tube
E1: 0.50 ml ES1 + filter paper strip + 1.0 ml buffer E2: 0.50 ml ES2 + filter paper strip + 1.0 ml buffer E3: 0.50 ml ES3 + filter paper strip + 1.0 ml buffer E4: 0.50 ml ES4 + filter paper strip + 1.0 ml buffer E5: 0.50 ml ES5 + filter paper strip + 1.0 ml buffer
66
A. Citrate Buffer: For this procedure, cellulase assays are carried out in 0.05 M citrate buffer pH
4.8. The assay conditions must be defined when reporting results.
1. Mix and dissolve:
Citric acid monohydrate 210 g
Distilled Water 750 mL
NaOH - add until pH equals 4.3 50 to 60 g
2. Dilute to 1 L and check pH. If necessary, add NaOH until the pH is 4.5. This is 1M
stock citrate buffer
3. Citrate buffer (50 mM, pH 4.8):
a. Dilute 1M stock citrate buffer solution 20 fold using distilled water (1 part solute, 19 part solvent, C1V1=C2V2)
b. After diluting the citrate buffer stock check and adjust the pH if necessary to pH 4.8.
4. Store at room temperature (up to 3 months) or at 4oC for longer storage.
B. DNS Reagent
1. Mix and Dissolve on stir plate:
Distilled water 1416 mL 3,5 Dinitrosalicylic acid 10.6 g Sodium hydroxide 19.8 g
2. Add:
Rochelle salts (sodium potassium tartrate) 306 g
Phenol (melt at 50oC) 7.6 mL
Sodium metabisulfite 8.3 g
3. Titrate 3 ml of the DNS reagent using 0.1 M HCl using the phenolphthalein endpoint pH
check. It should take 5–6 ml of HCl for a transition from red to colorless. Add NaOH if required (2 g of NaOH added = 1 ml of 0.1 M HCl used for 3 ml of the DNS reagent) 4. Store in darkness at 4°C for at least 1 month. It could lose its reducing ability after long
67 C3: Procedure
1. Place a rolled filter paper strip into each 13 × 100 test tube.
2. Add 1.0 ml of 50 mM citrate buffer (pH 4.8) to the tubes; the paper strip should be submerged in the buffer.3
3. Prepare the enzyme dilution series, of which at least two dilutions must be made of each enzyme sample, with one dilution releasing slightly more than 2.0 mg of glucose (~2.1 mg) and one slightly less than 2.0 mg of glucose (1.9 mg)3.
4. Prepare the dilute glucose standards (GSs). 5. Prepare the blank and controls.
6. Pre-warm the enzyme solutions, blank, and controls until equilibrium.
7. Add 0.5 ml of the enzyme dilution series to the tubes with filter paper substrate (E1–5); add 0.5 ml of the enzyme dilution series to the tubes without filter paper substrate (EC1–5).
8. Incubate the tubes of E1–5, GSs, RB, EC1–5, and SC in a 50°C water bath (or shaker incubator) for exactly 60 min.
9. Add 3.0 ml of the DNS reagent to stop the reaction, and mix well.3 10. Boil all tubes for exactly 5.0 min.4
11. Transfer the tubes to an ice-cold water bath.
12. Withdraw ~0.5 ml of the colored solutions into 1.5-ml microcentrifuge tubes and centrifuge at ~10,000 g for 3 min.
13. Add 0.200 ml of the supernatant into 3-ml spectrophotometer cuvette tubes, add 2.5 ml of water, and mix well by using a pipette or by inversion several times.
14. Measure absorbance at 540 nm, where the absorbance of RB is used as the blank.
3 See Reagents and Materials sheet for procedure.
4 The boiling condition should be severe, and the volume of the boiling water bath should be maintained above the
68 C4: Calculations
1. Draw a standard sugar curve (sugar along the x-axis vs. absorbance at 540 along the y-axis), as shown in Fig. 1.
2. Calculate the delta absorbance of dilute enzyme solutions (DE1–4) for E1–5 by subtraction of the sum of the absorbance of EC1–5 and SC.
3. Calculate the real glucose concentrations released by E1–5 according to a standard sugar curve.
4. Draw the relationship between the real glucose concentrations and their respective enzyme dilution rates (EDRs) (Fig. 1).
5. Link the points less than 2 mg and greater than 2 mg by a line, and identify the EDR by using the point for 2-mg glucose based on the line (Fig. 1).
6. Calculate the FPA of the original concentrated enzyme solution in terms of FPU/ml: 0.37 FPA= 0.37/EDR where 2 mg glucose = 2 mg/(0.18 mg/mmol) × 0.5 ml × 60 min = 0.37 mmol/min/ml.
69 APPENDIX D
70 D1: Sulfuric Acid Pretreatment
Author: Mahalaxmi, Swetha PREPROCESSING OF BIOMASS USING MECHANICAL
CHEMICAL AND MICROBIAL TECHNIQUES. (2012).
In a set of experiments, >2 mm, 1-2 mm, <1 mm and Un-partitioned samples were subjected to 0.69 %, 2 %, 5 % and 10 % concentrations of sulfuric acid pretreatment for 30 minutes in 20 mL tightly capped hungate tubes at 260 °F. The reaction mixture comprised 1 g biomass and 10 mL of H2SO4 (0.097 mL, 0.28 mL, 0.69 mL and 1.39 mL of 72 % H2SO4 made
up to 10 mL with water, to make 0.69 %, 2 %, 5 % and 10 % H2SO4 respectively). The tubes
were allowed to cool to room temperature; the reaction mixture was filtered and washed to obtain filtrate and residue. The filtrate was analyzed for sugars, furfural, hydroxyl methyl furfural (HMF) and polyphenols using HPLC.
71 VITA
I, Christopher R. Riley, was born in a small southern town- Cordele, Georgia. He spent the early part of his academic career as a homeschool student. He attended such universities as the University of Virginia, Harvard University, and Darton College to obtain college credit before his graduation in May 2005 with his high school diploma with an emphasis in pre-medicine. In the summer of 2005, he attended an intensive pre-medicine summer program at the Medical College of Georgia where he was awarded first place in Medical Library Research. Christopher attended Georgia Southern University where he graduated with Honors in Analytical Chemistry with a minor in Biology, in 2009. His honors research was published in a journal and presented at several conferences.
In 2011, Christopher started the masters program in Chemical Engineering at the University of Mississippi. During his tenure at the university, he was an active member of several campus organizations and gave back to his community via a variety of volunteering activities. Additionally, he continued his relationship within the field of Chemistry and is an active member of the American Chemical Society. During his spare time, he is an avid cyclist, gym enthusiast and thoroughly enjoys cooking, playing video games, and reading about the latest advancements in technology and science. After graduation, he plans to develop his engineering career via a full-time position as a process engineer and looks forward to traveling the world.