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As a warm season grass native to the North American continent, switchgrass is considered as one of the most promising biofuel crops in the USA. It is a C4 plant that makes it energy efficient. Switchgrass has a deep root system that allows it to grow on marginal land with low water and nutrient input. Switchgrass has been used as a forage crop and its use for biofuel will not affect food security. Biofuels are more environment-friendly than fossil fuels as they do not produce net greenhouse gases. However, the problem of high cost of production per unit for biofuel has to be overcome if we want to replace fossil fuels with biofuels. One of the major factors related to the high cost of biofuel are the expensive cellulase enzymes used in the pretreatment of feedstock. Endoglucanase is the key enzyme used for breaking down cellulose before fermentation. Currently, endoglucanase is produced from engineered E. coli or yeast strains, which is still expensive for enzyme production and purification of industrial scales. Expression of endoglucanase in plants has been previously reported. However, there are no reports of transgenic switchgrass producing cellulase enzyme. In this study, the catalytic domain of beta-endoglucanase gene was codon-optimized and synthesized based on the cDNA cloned from Hypocrea jecorina. Rice RuBisCO small subunit targeting signal peptide was fused to the N-terminus of the beta-endoglucanase gene, which was expected to target the fusion protein to chloroplast. This subcellular compartment targeting could minimize negative effects on cell function and plant development. The endoglucanase gene was cloned with maize ubiquitin promoter in a modified binary vector pCambia 1305-2 and transformed into switchgrass genotype HR8 by using Agrobacterium tumefaciens. In this study, I generated five independent transgenic switchgrass lines and they were confirmed by growing on the selection agent hygromycin, GUS assay, PCR amplification, southern blotting hybridization, for the presence of hygromycin and endoglucanase genes. However, based on RT-PCR analysis, only two transgenic lines were confirmed to produce mRNAs of the endoglucanase gene. These two transgenic lines were further characterized for their agronomic traits and the chlorophyll contents. Our results suggested that expression of endoglucanase in switchgrass could reduce chlorophyll content and affect plant development. Nevertheless, in this study, we demonstrated that a fungal endoglucanase gene could be expressed in switchgrass transgenic plants, though the gene expression level and the subcellular localization need to be carefully regulated in order to minimize the toxic effect of endoglucanase on plant cells. / Master of Science

Aphthona spp. flea beetles were released in the Little Missouri National Grasslands (LMNG) in western North Dakota in 1999 to control leafy spurge (Euphorbia esula L.). The changes in soil seed bank composition and leafy spurge density were evaluated on two ecological sites five (2004) and ten years (2009) after Aphthona spp. release to monitor the effectiveness of the insects on weed control and associated change in plant communities. In 2009, leafy spurge stem density averaged 2 and 9 stems m-2 in the loamy overflow and loamy sites, respectively, compared to 110 and 78 stems m-2, respectively, in 1999 and 7 and 10 stems m-2, respectively, in 2004. Leafy spurge constituted nearly 67% of the loamy overflow seed bank in 1999 compared to 17% in 2004 and 2% in 2009. In the loamy seedbank, the weed represented nearly 70% in 1999 compared to approximately 11% in 2004 and 15% in 2009. As leafy spurge was reduced, native species diversity and seed count increased ten years following Aphthona spp. release. High-seral species represented 17% of the loamy overflow seedbank in 2009, an increase from 5% in 1999. However, Kentucky bluegrass, a non-target weedy species, increased over 250% in the loamy overflow seedbank from 2004 to 2009. The reestablishment of native plant species has often been slow in areas where leafy spurge was controlled using Aphthona spp. A bioassay was completed to evaluate native grass establishment when grown in soil from Aphthona spp. release and non-release sites throughout North Dakota. Native grass production was not affected when grown in soil collected from established Aphthona spp. sites (1.5 g per pot) compared to soil without insects (1.6 g per pot). The cause of reduced native grass production in sites with Aphthono spp. previously observed is unknown but may have been due to a chemical inhibition caused by the insects within the soil that no longer exists. The native warm-season switchgrass (Ponicum virgotum L.) may be an alternative to corn for efficient biofuel production; however, control of cool-season grassy weeds has been a problem in switchgrass production. Various herbicides were evaluated for smooth bromegrass (Bromus inermis Leyss.) and quackgrass [Elymus repens (L.) Gould] control in an established switchgrass stand near Streeter, ND and a weed-infested field in Fargo, ND. Switchgrass yield was higher than the control 14 mo after treatment (MAT) when aminocyclopyrachlor or sulfometuron were applied early in the growing season, but no treatment provided satisfactory long-term grassy weed control. Herbicides were reevaluated at increased rates for smooth bromegrass or quackgrass control in Fargo. Sulfometuron provided 99% smooth bromegrass control when applied at 280 g ha-1 in the fall but injured other grass and forb species as well. Sulfometuron would likely be injurious to switchgrass and could not be used for biofuel production. Aminocyclopyrachlor did not injure other grass species but only reduced smooth bromegrass control by 76% when applied at 280 g ha-1 in the fall. No treatment provided satisfactory long-term quackgrass control.

La sfida nel 21esimo secolo è quella di fornire cibo e energia ad un mondo in continua crescita demografica e allo stesso tempo conservare l’ambiente. In questa tesi uno scenario alternativo di uso del suolo per la produzione di bioenergia è stato testato: le fasce tampone bioenergetiche. Considerate le problematiche ambientali legate al trilemma “cibo-energia-ambiente”, la struttura del Millennium Ecosystem Assessment sui servizi ecosistemici (SE) fornisce l’opportunità di esaminare l’impatto ambientale di questo nuovo scenario bioenergetico. In questa tesi ho mirato a determinare in che misura le colture bioenergetiche poliennali influenzino la fornitura multipla di SE quando coltivate come fasce tampone. Per raggiungere questo obiettivo, ho combinato una revisione sistematica della letteratura sui SE forniti da colture energetiche poliennali (CEP) con una prova sperimentale su fasce tampone bioenergetiche. Applicando una metodologia di attribuzione di punteggi agli impatti sui SE estratti dal materiale bibliografico raccolto, ho mostrato come coltivando le CEP lungo i margini dei campi coltivati esista una grande opportunità per sostenere la fornitura multipla di SE. La coltivazione delle CEP come fasce tampone adiacenti a campi agricoli può migliorare i SE di regolazione del clima, dell’acqua e della biodiversità, sostenere la salute del suolo e fornire biomassa dedicata alla produzione di bioenergia. Al contrario, la conversione di margini di campo di prati stabili ha mostrato un impatto netto negativo sulla fornitura multipla di SE. Tuttavia, due sono i principali svantaggi che sono stati individuati relativamente alla creazione e alla gestione delle fasce tampone bioenergetiche. Primo, diversi sono i fattori sito-specifici di tipo idro-pedologico lungo i margini dei campi che devono essere tenuti in considerazione poiché possono avere un impatto negativo sull’affrancamento delle colture e la loro produttività a medio-lungo termine. Secondo, riguardo la catena di approvvigionamento della biomassa, uno spazio di lavoro limitato per le macchine agricole è stato riconosciuto come principale inconveniente per le fasce tampone bioenergetiche rispetto alle CEP coltivate in pieno campo. Questo limite logistico di natura spaziale può inevitabilmente incrementare i tempi e le operazioni di taglio e raccolta della biomassa e quindi in ultima il consumo di combustili fossili. Grazie ad una prova sperimentale su fasce tampone bioenergetiche condotta in un terreno sabbioso-limoso con falda acquifera poco profonda contaminata da nitrati di origine agricola, si è dimostrato come fasce tampone coltivate con miscanto e salice siano in grado di intercettare e rimuovere i nitrati in falda (>60%) tanto quanto fasce tampone con specie avventizie. CEP come miscanto e salice, grazie ai loro apparati radicali profondi, hanno mostrato essere in grado di promuovere delle relazioni pianta-suolo-microorganismi lungo l’intero profilo del suolo utili ai fini ambientali delle fasce tampone bioenergetiche. Infatti, negli strati più profondi, una maggiore biomassa radicale ha portato le CEP a superare le specie avventizie in termini di rimozione biologica dei nitrati dal suolo e mitigazione potenziale dei gas serra. Inoltre, i risultati relativi alla produzione di biomassa e le asportazioni di N legata alla fase di raccolta hanno confermato ulteriormente come la coltivazione di CEP lungo i corsi d’acqua sia una strategia win-win: produzione di biomassa e protezione dell’ambiente. In conclusione, il potenziale rivelato dalle CEP in termini di fornitura multipla di SE suggerisce che la loro coltivazione, come elementi paesaggistici perenni in posizioni strategiche all'interno di paesaggio agricolo, è un'opzione promettente per promuovere l'intensificazione ecologicamente sostenibile degli agroecosistemi. / The 21st century will challenge agriculture to feed and fuel a growing world while conserving the environment. In this thesis an alternative bioenergy land use scenario to the conversion of marginal land has been tested: the bioenergy buffers. Given the environmental issues related to “food-energy-environment” trilemma, the Millennium Ecosystem Assessment framework on ES provides an opportunity to examine the environmental impacts of this new bioenergy land use scenario. In this thesis I aimed to determine to what extent do the perennial bioenergy crops affect the delivery of multiple ES when cultivated as bioenergy buffers. To reach this aim, I combined a systematic revision of literature on ES provided by perennial bioenergy crops with a field experiment on bioenergy buffers. Applying an impact scoring methodology to the effects on ES extracted from literature, I showed that, cultivating perennial bioenergy crops along field margins of former croplands offer a great opportunity to sustain the provision of multiple ES. The cultivation of perennial bioenergy crops on field margins can improve climate, biodiversity and water regulation services, sustain soil health and provide biomass for energetic purposes. On the contrary, grassland conversion showed a net negative impact on multiple ES provision. Nevertheless, I found two main shortcomings related to bioenergy buffers establishment and management. First, several site-specific factors along field margins must be taken into account, because they can affect crop establishment and buffers long-term productivity. Second, regarding to biomass supply chain, a limited working space for the farm machinery operations has been recognized as the main disadvantages of bioenergy buffers compared to large-scale bioenergy plantations. This spatial logistics constraint may inevitably increase harvest and collection operation times and fossil fuel consumption. Conducting a field experiment with bioenergy buffers in a nitrate-enriched shallow groundwater, I showed that miscanthus and willow buffers are able to efficiently intercept and remove from groundwater the incoming NO3-N as much as buffer strips with spontaneous species. Yet, due to their deep rooting systems, bioenergy buffers promote significant plant-microbial linkages along the soil profile. At deeper soil layers, a higher fine root biomass led perennial bioenergy crops to outperform patches of adventitious vegetation in terms of biological N removal from soil and belowground GHG mitigation potential. The results on biomass production and N removal via harvesting further confirmed that the cultivation of perennial bioenergy crops along watercourses is an effective win-win strategy: biomass production and protection of the environment. In conclusion, the revealed potential of perennial bioenergy crops on multiple ES provision implies that their cultivation as perennial landscape elements in strategic locations within landscape is a promising option to promote the ecological sustainable intensification of agroecosystems.

Recently, there has been a surge in the research and application of "Green energy" in the United States. This has been driven by the following three objectives: (1) to reduce the nation's reliance on foreign oil, (2) to mitigate emission of greenhouse gas, and (3) to create an economic stimulus within the United States. Switchgrass is the biomass of choice for the Southeastern United States. In this dissertation, we address a feedstock logistics problem associated with the delivery of switchgrass for conversion into biofuel. In order to satisfy the continual demand of biomass at a bioenergy plant, production fields within a 48-km radius of its location are assumed to be attracted into production. The bioenergy plant is expected to receive as many as 50-400 loads of biomass per day. As a result, an industrialized transportation system must be introduced as early as possible in order to remove bottlenecks and reduce the total system cost. Additionally, we assume locating multiple bioenergy plants within a given region for the production of biofuel. We develop mixed integer programming formulations for the feedstock logistics problem that we address and for some related problems, and we solve them either through the use of decomposition-based methods or directly through the use of CPLEX 12.1.0. The feedstock logistics problem that we address spans the entire system-from the growing of switchgrass to the transporting of bio-crude oil, a high energy density intermediate product, to a refinery for conversion into a final product. To facilitate understanding, we present the reader with a case study that includes a preliminary cost analysis of a real-life-based instance in order to provide the reader appropriate insights of the logistics system before applying optimization techniques for its solution. First, we consider the benefits of active versus passive ownership of the production fields. This is followed by a discussion on the selection of baler type, and then, a discussion of contracts between various business entities. The advantages of storing biomass at a satellite storage location (SSL) and interactions between the operations performed at the production field with those performed at the storage locations are then established. We also provide a detailed description of the operations performed at a SSL. Three potential equipment options are presented for transporting biomass from the SSLs to a utilization point, defined in this study as a Bio-crude Plant (BcP). The details of the entire logistics chain are presented in order to highlight the need for making decisions in view of the entire chain rather than basing them on its segments. We model the feedstock logistics problem as a combination of a 2-level facility location-allocation problem and a multiple traveling salesmen problem (mATSP). The 2-level facility location-allocation problem pertains to the allocation of production fields to SSLs and SSLs to one of the multiple bioenergy plants. The mATSP arises because of the need for scheduling unloading operations at the SSLs. To this end, we provide a detailed study of 13 formulations of the mATSP and their reformulations as ATSPs. First, we assume that the SSLs are always full, regardless of when they are scheduled to be unloaded. We, then, relax this assumption by providing precedence constraints on the availability of the SSLs. This precedence is defined in two different ways and, is then, effectively modeled utilizing all the formulations for the mATSP and ATSP. Given the location of a BcP for the conversion of biomass to bio-crude oil, we develop a feedstock logistics system that relies on the use of SSLs for temporary storage and loading of round bales. Three equipment systems are considered for handling biomass at the SSLs, and they are either placed permanently or are mobile, and thereby, travel from one SSL to another. We use a mathematical programming-based approach to determine SSLs and equipment routes in order to minimize the total cost incurred. The mathematical program is applied to a real-life production region in South-central Virginia (Gretna, VA), and it clearly reveals the benefits of using SSLs as a part of the logistics system. Finally, we provide a sensitivity analysis on the input parameters that we used. This analysis highlights the key cost factors in the model, and it emphasizes areas where biggest gains can be achieved for further cost reduction. For a more general scenario, where multiple BcPs have to be located, we use a nested Benders' decomposition-based method. First, we prove the validity of using this method. We, then, employ this method for the solution of a potential real-life instance. Moreover, we successfully solve problems that are more than an order of magnitude larger than those solved directly by CPLEX 12.1.0. Finally, we develop a Benders' decomposition-based method for the solution of a problem that gives rise to a binary sub-problem. The difficulty arises because of the sub-problem being an integer program for which the dual solution is not readily available. Our approach consists of first solving the integer sub-problem, and then, generating the convex hull at the optimal integer point. We illustrate this approach for an instance for which such a convex hull is readily available, but otherwise, it is too expensive to generate for the entire problem. This special instance is the solution of the mATSP (using Benders' decomposition) for which each of the sub-problems is an ATSP. The convex hull for the ATSP is given by the Dantzig, Fulkerson, and Johnson constraints. These constraints at a given integer solution point are only polynomial in number. With the inclusion of these constraints, a linear programming solution and its corresponding dual solution can now be obtained at the optimal integer points. We have proven the validity of using this method. However, the success of our algorithm is limited because of a large number of integer problems that must be solved at every iteration. While the algorithm is theoretically promising, the advantages of the decomposition do not seem to outweigh the additional cost resulting from solving a larger number of decomposed problems. / Ph. D.