Though mass spectrometry has earned a central role in the field of proteomics due to its versatility in a wide range of experiments, challenges and complications are still encountered when using mass spectrometry to characterize protein structures, post-translational modifications (PTMs), and abundances. In this dissertation, analytical methods utilizing mass spectrometry have been developed to address challenges associated with both qualitative and quantitative protein characterization. The effectiveness of using multiple pepsin-like proteases, both separately and in mixtures, combined with online proteolysis using a special triaxial probe has been demonstrated on an amyloid beta peptide related to the onset of Alzheimer’s disease. These findings have broad implications in protein structural characterization studies using hydrogen-deuterium exchange mass spectrometry. A wider range of proteases (Lys-C, Glu-C, and trypsin) and multiple fragmentation methods (collisionally activated dissociation, electron transfer dissociation, and decision tree) have been utilized in the discovery-based PTM characterization of extracellular cellulosome proteins of the bioenergy-relevent organism Clostridium thermocellum, resulting in the identification of 85 previously unknown modification sites in 28 cellulosome proteins. These modifications may contribute to the structure and/or function of the cellulosome protein complex. By using peptide internal standards and a triple quadrupole mass spectrometer operating in selected reaction monitoring mode, a method has been developed for the absolute quantitation of the Clostridium thermocellum cellulosome protein machine in samples ranging in complexity from purified cellulosome samples to whole cell lysates as an alternative to a previously-developed enzyme-linked immunosorbent assay method of cellulosome quantitation. The precision of the cellulosome mass concentration in technical replicates is better than 5% relative standard deviation for all samples, indicating high precision of cellulosome mass concentration for this method. Though methods and results presented in this dissertation have implications in the study of Alzheimer’s disease and bioenergy research, more broadly this dissertation focuses on development of methods to contend with some of the more complex challenges associated with protein characterization currently presented to the field of proteomics.

The focus of this study was to determine the effects of pretreatment of wheat straw by superheated steam (SS) alone or in combination with boiling water (BW) on biomass structure and yields of fermentation products (cell mass and fermentation end-products) by Clostridium thermocellum. Different cultivars of wheat straw were ground to a particle size less than 355 µm, and exposed to the following methods of pretreatment: i) 15 min soaking in 119 °C boiling water under absolute pressure of 193 kPa, followed by processing with SS at atmospheric pressure at different temperatures and retention times; ii) 15 min processing with SS at atmospheric pressure; and iii) 15 min soaking in 119 °C boiling water under absolute pressure of 193 kPa. Processing with SS was conducted at a variety of temperatures in the range of 180-220 °C. The severity of pretreatment was expressed through a treatment severity factor as a measure of harshness of treatment. Pretreatment combinations of boiling water with superheated steam at different retention times inside the SS chamber were also investigated. Wheat straw samples were then used as substrates in fermentation reactions with C. thermocellum. The most noticeable effects on biomass structure and fermentation were observed at the highest severity factor of 6.5, corresponding to 15 min pretreatment with boiling water followed by 15 min treatment with SS at 220˚C. This pretreatment provided the maximum increase in percentage of contribution of amorphous cellulose (% CAC), and the highest fermentation yield in terms of hydrogen, carbon dioxide, and ethanol production. / October 2015

In recent years, fungal and bacterial cellulases have gained popularity for the conversion of lignocellulosic material to biofuels and biochemicals. This study investigated properties of fungal (Trichoderma. reesei) and bacterial (Clostridium thermocellum) cellulases. Enzymatic hydrolysis was carried out with T. reesei using nine enzyme concentration and substrate combinations. Initial rates and extents of hydrolysis were determined from the progress curve of each combination. Inhibition occurred at the higher enzyme concentrations and higher solids concentrations. Mechanisms to explain the observed inhibition are discussed. Samples of C. thermocellum purified free cellulase after 98% hydrolysis were assayed to determine the total protein content (0.15 ± 0.08 mg/mL), the enzymatic activity (0.306 ± 0.173 IU/mL) and the cellulosome mass using the Peterson method for protein determination, the cellulase activity assay with phenol-sulfuric acid assay, and the indirect ELISA adapted for C. thermocellum cellulosomes, respectively. Issues regarding reproducibility and validity of these assays are discussed.

Pretreatment is an essential step in biofuel production from lignocellulose. Disruption of the lignin structure gives enzymes and fermentation organisms access to long chains of cellulose and hemicellulose. For this project’s purposes, the pretreatment must work within the framework of an on-farm butanol bioprocessing facility. Alkaline hydrogen peroxide (AHP) is a delignification method that potentially provides several advantages. At the alkaline pH, powerful hydroxyl radicals are formed; which attack lignin. The objectives of this study were to determine if AHP removes substantial lignin for the feedstocks, corn stover, wheat straw, switchgrass and miscanthus, and to determine if AHP acts as a biocide? Compositional analysis determined if lignin was removed and HPLC data were used to determine whether or not Clostridium thermocellum hydrolyzed the pretreated material. Sterility was determined by plating the AHP material. All materials showed approximately 10% lignin removal with AHP. AHP increased structural carbohydrate concentrations for wheat straw, switchgrass and miscanthus. Corn stover showed no benefit from adding peroxide to a traditional alkaline pretreatment. AHP appears to suppress visible microbial growth for the first 24 hours after pretreatment. If AHP does not provide the additional hygienic effects, AHP does not provide a significant advantage over sodium hydroxide pretreatment.

Current research efforts are directed at developing competitive processes that can utilize lignocellulose as a feedstock for biorefineries. The purpose of this study was to investigate methods of processing lignocellulosic material so that its monosacharides can be more easily accessed for fermentation, the lack of which is hindering the economics and widescale adoption of lignocellulosic biorefining. The monosaccharides are of interest because they can be used by Clostridium beijerinckii downstream of P. chrysosporium and C. thermocellum in a sequential bioprocess to produce butanol. Butanol is an attractive biofuel because it can be utilized without modifying current transportation infrastructure. Butanol is also used as a starting material in organic synthesis. In the first study, the potential for C. thermocellum' s (ATCC 27405) cellulase system to operate outside its optimal temperature range in a high-solids environments was assessed by quantification of the fermentation products lactate, acetate, and ethanol and by quantification of xylose, glucose, and cellobiose remaining. Additionally, the lignin degrading white-rot fungus Phanerochaete chrysosporium RP 78 was investigated as a potential pretreatment for lignocellulose. Elevated temperatures required for Clostridium thermocellum fermentation were examined as a means to improve poor competiveness that is characteristic of P. chrysosporium on unsterile corn stover substrate.

The organism Clostridium thermocellum grows on cellulosic substrates and produces ethanol, acetate, lactate, formic acid, and CO2. The organic acids produced alter the growth environment in which the bacteria grows and ultimately inhibit bacterial growth. One method which has been used successfully to maintain the system at acceptable growth conditions is to intermittently flush out the spent media and metabolic products and replace with new fermentation media. Our goal was to design and build an automated system that will automatically flush the spent media from the growing culture and resupply new media without manual intervention. An automated control system was designed and built to control growth parameters. Heated water was pumped through the jacket of each culture vessel and used to regulate the reactor temperature. Sensors for pH and temperature were connected to a central data acquisition system and NI LabVIEW software was used to control each of the components through the signals provided by the data acquisition system. Peristaltic and vacuum pumps were used to supply growth media and acquire reproducible samples for HPLC analysis with limited contamination. In a series of trials, targeted temperature and moisture conditions were achieved and new media was passed through each reactor using a time trigger. More product was produced in manual and automatically flushed cultures than in batch.

Clostridium thermocellum is a thermophilic anaerobe that is capable of producing ethanol directly from lignocellulosic compounds, however this organism suffers from low ethanol tolerance and low ethanol yields. In vivo mathematical modeling studies based on steady state traditional metabolic flux analysis, metabolic control analysis, transient and steady states’ flux spectrum analysis (FSA) were conducted on C. thermocellum’s central metabolism. The models were developed in Matrix Laboratory software ( MATLAB® (The Language of Technical Computing), R2008b, Version 7.7.0.471)) based on known stoichiometry from C. thermocellum pathway and known physical constraints. Growth on cellobiose from Metabolic flux analysis (MFA) and Metabolic control analysis (MCA) of wild type (WT) and ethanol adapted (EA) cells showed that, at lower than optimum exogenous ethanol levels, ethanol to acetate (E/A) ratios increased by approximately 29% in WT cells and 7% in EA cells. Sensitivity analyses of the MFA and MCA models indicated that the effects of variability in experimental data on model predictions were minimal (within ±5% differences in predictions if the experimental data varied up to ±20%). Steady state FSA model predictions showed that, an optimum hydrogen flux of ~5mM/hr in the presence of pressure equal to or above 7MPa inhibits ferrodoxin hydrogenase which causes NAD re-oxidation in the system to increase ethanol yields to about 3.5 mol ethanol/mol cellobiose.

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