This work integrates perennial feedstock yield modeling using climate model data from current and future climate scenarios, land use datasets, transportation network data sets, Geographic Information Systems (GIS) tools, and Mixed integer linear programming (MILP) optimization models to examine biorefinery network designs in the southeastern United States from an overall systems perspective. Both deterministic and stochastic cases are modeled. Findings indicate that the high transportation costs incurred by biorefinery networks resulting from the need to transport harvested biomass from harvest location to processing facilities can be mitigated by performing initial processing steps in small scale mobile units at the cost of increased unit production costs associated with operating at smaller scales. Indeed, it can be financially advantageous to move the processing units instead of the harvested biomass, particularly when considering a 10-year planning period (typical switchgrass stand life). In this case, the mobile processing supply chain configuration provides added flexibility to respond to year-to-year variation in the geographic distribution of switchgrass yields. In order to capture the effects of variation in switchgrass yields and incorporate it in optimization models, yield modeling was conducted for both current and future climate scenarios. (In general profits are lower in future climate scenarios). Thus, both the effects of annual variation in weather patterns and varying climate scenarios on optimization model decisions can be observed.

Bioenergy has the potential to reduce the world's dependence on fossil fuels, and to decrease the CO2 emissions due to fossil combustion. Lignocellulosic and algae biomass have been presented as promising feedstocks for bioenergy production. In this study, a comparative Life Cycle Assessment (LCA) has been developed to evaluate the environmental impacts associated with different energy products via different routes across the whole life of algal and lignocellulosic bioenergy. Results were compared per energy basis, the production of 1 million BTU of energy products. For the development of the comparative algae biomass conversion LCA, algal biomass was converted to liquid biofuels via a thermochemical gasification and Fisher-Tropsch Synthesis (FTS) process; and to electricity and heat via anaerobic digestion and combined heat and power (CHP) process. Overall results from the algae biomass conversion LCA showed that the process that converts algae biomass through anaerobic digestion and CHP process to electricity and heat had the highest overall environmental impact. Results also showed that the impact categories that appear to contribute the most to the overall impacts are ecotoxicity, human health non-cancer, and human health cancer. For the development of the comparative lignocellulosic biomass conversion LCA, lignocellulosic biomass was converted to ethanol and higher alcohols through thermochemical gasification and alcohol synthesis process, to liquid biofuels via thermochemical gasification and FTS process, and to liquid biofuels via a thermochemical gasification and FTS process that uses methane. Overall results from the lignocellulosic biomass conversion LCA showed that the process that converts lignocellulosic biomass into alcohols has the highest overall environmental impact. Results also showed that the impact categories that appear to contribute the most to the overall impacts are ecotoxicity, human health non-cancer, human health cancer, and global warming. This study determined that cultivated algae biomass feedstock has much higher environmental impacts compared with lignocellulosic biomass feedstock from forestation and agriculture byproducts. It was also concluded that thermochemical gasification and FTS process showed higher efficiency when converting biomass to bioenergy. In addition, the five biomass to bioenergy conversion pathways used in the development of this LCA study were compared. Results showed that the pathway with lignocellulosic biomass (feedstock), thermochemical gasification and alcohol synthesis process (conversion process), and ethanol and higher alcohols (energy products) has the largest environmental impact.

The main approach to studying the proteome is a technique called data dependent acquisition (DDA). In DDA, peptides are analyzed by mass spectrometry to determine the protein composition of a biological isolate. However, DDA is limited in its ability to analyze the proteome, in that it only selects the most abundant ions for analysis, and different protein identifications can result even if the same sample is analyzed multiple times in succession. Data independent acquisition (DIA) is a newly developed method that should be able to solve these limitations and improve our ability to analyze the proteome. We used an implementation of DIA (SWATH) to perform relative protein quantitation in the model bacterial system, Clostridium stercorarium, using two different carbohydrate sources, and found that it was able to provide precise quantitation of proteins and was overall more consistent in its ability to identify components of the proteome than DDA. Relative quantitation of proteins is an important method that can determine which proteins are important to a biochemical process of interest. How we determine which proteins are differentially regulated between different conditions is an important question in proteomic analysis. We developed a new approach to analyzing differential protein expression using variation between biological replicates to determine which proteins are being differentially regulated between two conditions. This analysis showed that a large proportion of proteins identified by quantitative proteomic analysis can be differentially regulated and that these proteins are in fact related to biological processes. Analyzing changes in protein expression is a useful tool that can pinpoint many key processes in biological systems. However, these techniques fail to take into account that enzyme activity is regulated by other factors than controlling their level of expression. Activity based protein profiling (ABPP) is a method that can determine the activity state of an enzyme in whole cell proteomes. We found that enzyme activity can change in response to a number of different conditions and that these changes do not always correspond with compositional changes. Mass spectrometry techniques were also used to identify serine hydrolases and characterize their expression in this organism. / February 2016

Inexpensive petroleum is the cornerstone of the modern global economy despite its huge environmental costs and its nature as a non-renewable resource. While ninety percent of petroleum is ultimately used as fuel and can in principle be replaced by sources of renewable electricity, ten percent is used as a feedstock to produce societally important chemicals that cannot currently be made at a reasonable cost through alternative processes. In this dissertation, I will discuss my efforts, together with several colleagues, to apply synthetic biology approaches to the challenge of producing renewable petrochemical replacements. In Chapter 2, I discuss our efforts to engineer E. coli to produce fatty acids with a wide range of chain lengths at high yield, thereby providing an alternative platform for the production of diverse petrochemicals. In Chapter 3, I describe a novel method of DNA assembly that we developed to facilitate synthetic biology efforts such as those in Chapter 2. This method is capable of simultaneously assembling multiple DNA pieces with substantial sequence homology, a common challenge in synthetic biology. In Chapter 4, I discuss the development of a "bionic leaf": a hybrid microbial-inorganic catalyst that marries the advantages of photovoltaic-based light capture and microbial carbon fixation to achieve solar biomass yields greater than those observed in terrestrial plants. This technology offers a potentially low-cost alternative to photosynthesis as a source of biomass and derived chemicals and fuels. The work described in this dissertation demonstrates the capacity of synthetic biology to address the problem of renewable chemical production, and offers proof of principle demonstrations that both the scope and efficiency of biological chemical production may be improved.

Στην παρούσα διδακτορική διατριβή μελετήθηκε η συνδυασμένη παραγωγή υδρογόνου και μεθανίου από την ενεργειακή καλλιέργεια του γλυκού σόργου. Το γλυκό σόργο είναι ένα μονοετές ενεργειακό φυτό, μεγάλης φωτοσυνθετικής ικανότητας, πλούσιο σε υδατάνθρακες, το οποίο θεωρείται ιδανικό για την παραγωγή βιοκαυσίμων. Η παραγωγή του υδρογόνου από τα σάκχαρα του σόργου, πραγματοποιήθηκε μέσω της ενδογενούς βακτηριακής καλλιέργειας του φυτού, γεγονός που καθιστά τη διεργασία όχι μόνο τεχνικά αλλά και οικονομικά ελκυστική. Σε πρώτο στάδιο, μελετήθηκε η επίδραση των λειτουργικών συνθηκών στη ζυμωτική παραγωγή του υδρογόνου από τα διαλυτά σάκχαρα του γλυκού σόργου, μέσω μικτής μικροβιακής καλλιέργειας, σε συνεχή μεσόφιλο, βιοαντιδραστήρα. Στη συνέχεια, η πλούσια σε οργανικό φορτίο απορροή του ζυμωτικού υδρογονοπαραγωγού βιοαντιδραστήρα, υπέστη περαιτέρω επεξεργασία σε συνεχή μεσόφιλο αναερόβιο χωνευτήρα, με ταυτόχρονη παραγωγή μεθανίου. Το μοντέλο ADM1 (Anaerobic Digestion Model No 1), χρησιμοποιήθηκε για τη μαθηματική προσομοίωση και των δύο βιοδιεργασιών. Η δομή του μοντέλου τροποποιήθηκε προκειμένου να βελτιωθούν οι προβλέψεις για τη διεργασία παραγωγής υδρογόνου. Τέλος, πραγματοποιήθηκε οικονομική αποτίμηση της βιωσιμότητας της συνολικής διεργασίας παραγωγής υδρογόνου και μεθανίου, από το γλυκό σόργο. Η παραγωγή βιοκαυσίμων, με τον τρόπο που έχει σχεδιαστεί, αποδείχτηκε οικονομικά μη συμφέρουσα, αλλά με κάποιες βελτιώσεις πιθανό να αποτελέσει ανταγωνιστική τεχνολογία, στο κοντινό μέλλον. / In the present study we investigated the hydrogen and methane production from sweet sorghum biomass. Sweet sorghum is an annual plant, characterized by high photosynthetic efficiency. Sweet sorghum biomass is rich in readily fermentable sugars and thus it can be considered as an excellent raw material for biofuels generation. It is the first time that this plant is used for the production of hydrogen, although ethanol and methane are among the best-known microbial products produced from sweet sorghum. Τhe fermentative production of hydrogen was achieved using an indigenous mixed microbial culture. The present study concerns the fermentative production of hydrogen from the sugars contained in sorghum extract. The process took place in a mesophilic continuous stirred tank type bioreactor, by an indigenous mixed microbial culture and it was studied at various conditions. Τhe subsequent anaerobic treatment of the effluent of the fermentative hydrogenogenic reactor with the simultaneous production of methane was investigated in a continuous stirred tank type reactor operated at three hydraulic retention times. The recently developed anaerobic model ADM1 was used to simulate the anaerobic digestion process and the fermentative hydrogen production process. However the structure of the model was modified, in order to improve the predictions for biohydrogen production. Finally, technoeconomic analysis was performed to determine the potential economic viability of the process. Biohydrogen and methane production from sweet sorghum biomass was not economic feasible; therefore improvement of the process design is necessary.

Diesel particulate matter (PM) produced from Ultra Low Sulfur Diesel (ULSD), Soy-derived B20 and Animal fat-derived B20 was collected at two engine operating conditions. Gravimetric analysis showed the PM emissions to depend on both fuel and engine operating condition. B20 fuels exhibited higher PM emissions at low load conditions, but lower PM emissions under high load conditions. The differences between the operating conditions were less significant for B20 fuels than for ULSD. Elemental Carbon analysis revealed the B20 PM to contain greater proportions of organic carbon, which may improve diesel particulate filter (DPF) regeneration. Finally, TEM images showed considerable differences between ULSD and B20 soot at low engine loads. The B20 soot displayed a more disordered arrangement of graphene layers. At higher loads, the differences are subtle, but the B20 PM still displayed a more amorphous structure. There was little difference between the Soy and Animal fat-derived B20 soot.

Diesel particulate matter (PM) produced from Ultra Low Sulfur Diesel (ULSD), Soy-derived B20 and Animal fat-derived B20 was collected at two engine operating conditions. Gravimetric analysis showed the PM emissions to depend on both fuel and engine operating condition. B20 fuels exhibited higher PM emissions at low load conditions, but lower PM emissions under high load conditions. The differences between the operating conditions were less significant for B20 fuels than for ULSD. Elemental Carbon analysis revealed the B20 PM to contain greater proportions of organic carbon, which may improve diesel particulate filter (DPF) regeneration. Finally, TEM images showed considerable differences between ULSD and B20 soot at low engine loads. The B20 soot displayed a more disordered arrangement of graphene layers. At higher loads, the differences are subtle, but the B20 PM still displayed a more amorphous structure. There was little difference between the Soy and Animal fat-derived B20 soot.

Project I: [Ru(OH2)3(4'-phenyl-2,2':6',2''-terpyridine)](OTf)2 as a Homogeneous Hydrogenation Catalyst for Biomass Derived Substrates. The complex [Ru(OH2)3(4'-phenyl-2,2':6',2''-terpyridine)](OTf)2 has been shown to be an active ionic hydrogenation catalyst for selected carbonyls, diols and glycerol by the Schlaf group. It was postulated to also be active for other biomass derived substrates such as levulinic acid (LA), furfural and 5-hydroxymethyl furfural (HMF). Synthesis of the complex was optimized and full characterization carried out by 1H/13C –NMR. The complex was tested against LA in aqueous sulfolane medium and the furfural/HMF model system 2,5-hexanedione in water. Activity of the complex was compared to the analogous metal-ligand bifunctional (MLB) system described in Project II. The complex exhibited good thermal stability up to 200 oC in 90/10 wt% sulfolane/water mixtures and was capable of hydrogenation of LA to γ-valerolactone in 95% yield. Addition of protic acids to the reaction mixture and increasing proportions of water decreased the activity of the complex towards the hydrogenation of LA. Project II: [Ru(OH2)3(di(picolyl)amine)](OTf)2 as an acid-, water- stable, metal-ligand bifunctional deoxygenation catalyst. The complex [Ru(OH2)3(di(picolyl)amine)](OTf)2 was postulated to be an active MLB ionic hydrogenation catalyst under acidic aqueous conditions. Using the substantially labile [Ru(DMF)6](OTF)3 ruthenium complex as the precursor, the desired complex was prepared insitu by coordination of the DPA ligand and concomitant reduction of Ru3+ to Ru2+. The complex was characterized by 1H/13C-NMR and tested for the hydrogenation of LA, 2,5-hexanedione, furfural and HMF under acidic aqueous conditions. The complex exhibited thermal stability up to 150 oC and was active for the hydrogenation of carbonyls, as demonstrated by the conversion of 2,5-hexanedione to 2,5-hexanediol in 94% yield in water. Addition of H3PO4 as an acid cocatalyst resulted in nearly complete conversion to dimethyltetrahydrofuran (DMTHF) but further deoxygenation could not be achieved. Direct comparision of [Ru(OH2)3(di(picolyl)amine)](OTf)2 and [Ru(OH2)3(4'-phenyl-2,2':6',2''-terpyridine)](OTf)2 under identical conditions against LA and 2,5-hexanedione demonstrated that the[Ru(OH2)3(di(picolyl)amine)](OTf)2 catalyst is more active than the [Ru(OH2)3(4'-phenyl-2,2':6',2''-terpyridine)](OTf)2 complex in all cases, suggesting that the di(picolyl)amine complex operates through a MLB ionic hydrogenation mechanism. / NSERC

This work investigates the extinction limits of laminar diffusion counterflow flames for various gaseous (methane, syngas, biogas) fuels using a high flow rate counterflow burner designed and built for this work. Equal momenta of the fuel and oxidizer streams were not maintained to provide data to check the fidelity of the numerical schemes and their chemical mechanisms at "non-standard" conditions. Strain rate values at extinction were obtained as a function of fuel mole fraction. Preliminary work with the new burner found that the methane extinction limit results were consistent with results from literature. The results provide insight into the extinction limit conditions of the aforementioned fuels. The strain rate was found to increase with increasing fuel mole fraction. Extinction limit results indicated that fuels with the highest concentration of hydrogen have the greatest extinction limit, which is believed to be attributed to the high diffusivity and reactivity of hydrogen.

This work investigates the extinction limits of laminar diffusion counterflow flames for various gaseous (methane, syngas, biogas) fuels using a high flow rate counterflow burner designed and built for this work. Equal momenta of the fuel and oxidizer streams were not maintained to provide data to check the fidelity of the numerical schemes and their chemical mechanisms at "non-standard" conditions. Strain rate values at extinction were obtained as a function of fuel mole fraction. Preliminary work with the new burner found that the methane extinction limit results were consistent with results from literature. The results provide insight into the extinction limit conditions of the aforementioned fuels. The strain rate was found to increase with increasing fuel mole fraction. Extinction limit results indicated that fuels with the highest concentration of hydrogen have the greatest extinction limit, which is believed to be attributed to the high diffusivity and reactivity of hydrogen.