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Exploiting induced genetic diversity through using mutagenesis is particularly important in giant miscanthus (Miscanthus x giganteus; Mxg) due to its restricted genetic variability. Experiments were conducted to develop an efficient in vitro propagation protocol for Mxg, induce mutations in Mxg using a chemical mutagen, and select Mxg in vitro for heat tolerance. To optimize in vitro propagation of Mxg, five explant types [i.e. immature inflorescences, shoot apex (in vitro), shoot apex (greenhouse), leaf explants (in vitro), and leaf explants (greenhouse)] were tested on five media. Shoot forming calli from immature inflorescences, an excellent source of explant in Mxg, grown in media with 13.6 microM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.44 microM 6-benzylaminopurine (BA) resulted in greatest shoot regeneration rate. Optimization of explant and callus type and media resulted in efficient in vitro proliferation of Mxg and the developed protocol was utilized in consecutive experiments of mutation induction and in vitro selection of Mxg for heat tolerance. Immature inflorescence explants (1-2 mm) were treated with 0.6%, 1.2%, and 1.8% of ethyl methanesulfonate (EMS) whereas the calli (1-2 mm3) were treated with 1.2%, 2.4%, and 3.6% of EMS for 90 min. Results of inter simple sequence repeat PCR analysis revealed polymorphisms indicating presence of genetic differences in Mxg putative mutants. In vitro callus cultures (mutagen treated and non-treated) of Mxg subjected to temperature treatments of 45±2°C for 12 hrs or 40±2°C for 7 days were selected for heat tolerance. Assessment results of electrolyte leakage and photosystem II (PS II) efficiency tests indicated a significant difference in percent membrane damage among Mxg clonal lines whereas PSII was weakly affected by the heat stress. The results suggest that in vitro derived Mxg clonal lines may be utilized for further studies of Mxg heat tolerance in developing potential Mxg ecotypes to adapt to different thermal environments. These studies provided the first investigation of in vitro induced mutagenesis in Mxg using a chemical mutagen. Genetic analysis results presented in this study indicates the potential use of developed Mxg putative mutants in future research programs, although significant morphological alterations were not observed during preliminary screening in the greenhouse.

Alternative fuels are necessary to meet the increasing demands for fuels. Alternative fuels such as biodiesel are produced using vegetable oils, which are prominentt in the food industry. An alternate feedstock could be oil-producing microorganisms. These oleaginous microorganisms are defined as accumulating more than 20% of their weight in oil as lipids. Cultivating these microorganisms for oil production is not economical due to the high production costs from the sugars in the culture medium. Municipal wastewater could be a potential growth medium that has not previously been considered for cultivating oleaginous microorganisms. However, municipal wastewater contains a low concentration of carbon, which does not promote oil accumulation in the oleaginous microorganisms. To increase the carbon concentration in the wastewater, lignocellulosic sugars could be added to the municipal wastewater. These sugars are a potential alternative to sugars that are in the food industry. The goal of this research is to determine the efficacy of using municipal wastewater to cultivate a consortium of oleaginous microorganisms, thus, producing oil for biodiesel production. First, a consortium of oleaginous microorganisms was cultivated on autoclaved wastewater to determine if the wastewater contains any inhibiting substances for the microorganisms. In addition to the substances in the wastewater, indigenous microorganisms are possible inhibitors to the consortium. Therefore, to determine the effect these indigenous microorganisms have on the oleaginous microorganisms, the consortium was cultivated on raw municipal wastewater amended with varying amounts of sugar. Since the municipal wastewater can be used as a cultivation medium, the effect of the addition of lignocellulosic sugars was determined. During the production of lignocellulosic sugars, furfural and acetic acid, known microbial inhibitors, are formed. The effect of these inhibitors on the consortium’s growth and oil accumulation ability was ascertained, and inhibition models were developed to describe their impact. With these results, SuperPro Designer v6.0 was used to perform simulations and economic analyses to determine the efficacy of incorporating an oleaginous microorganism consortium in a wastewater treatment facility.

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Microalgae biomass has potential as a feedstock for various bioproducts, including biofuel. Algae can be cultivated on treated wastewater or on untreated wastewater, accomplishing treatment as a co-benefit. Greater understanding of algal productivity is needed. This study compared the net productivity of naturally forming algae polycultures, and monocultures of Scenedesmus obliquus (DOE0152Z) and Tribonema minus cultivated on treated municipal wastewater or primary clarifier effluent. The experiments were conducted in outdoor, 1350-L and 1000-L, raceway tanks in coastal central California during a multi-year period. A linear regression model of net productivity (i.e., based on the difference of biomass in the influent and effluent of the raceways) was developed. The highest productivity culture was a polyculture grown on primary clarifier effluent at 20.0 +/- 3.8 g/m2-day (ash-free dry weight, AFDW over 12 months of continuous cultivation). The monoculture with the highest productivity was Tribonema minus at 16.1 +/- 0.8 g/m2-day (summer through winter). In the various strain and wastewater type combinations tested, solar radiation was the most statistically significant predictor of net productivity (p

The need for a deeper understanding of public policy instruments is well established in public administration literature. Growth in ethanol and alternative fuel policy instruments across the country and the importance of these policies to national energy security only adds urgency to this need. Policy instruments are defined as tools governments use to address public policy problems. Public policy scholars traditionally focus on processes of policy making or the policies, with little attention paid to how governments accomplish policy goals. This dissertation shifts the focus to policy instruments to fill this void in public administration scholarship. It examines factors that influence policy instruments chosen by policy makers in the fifty states. Using the lens of biofuel policies, it links three diverse public policy theories: Policy Instrument Theory, New Public Management (NPM) Theory, and Political Culture Theory, into a single model of policy instrument choice. The dependent variable is derived using cluster analysis methods and results in four distinct groups of states based on state level biofuel policy instrument characteristics. These groups are used to test proposed hypotheses regarding state level characteristics including levels of NPM reform, individual state political culture and elite political ideology as well as fundamental measures of state policy capacity of state wealth, impacts of economic sectors, and political interests. Multinomial logistic regression analysis is used to establish the likelihood of membership in one group of states versus other groups with specific instrument characteristics. The results conclude that policy makers in states make different instrument choices based on state level characteristics. Wealthy states choose policy instruments that rely upon changing citizen behavior rather than direct government intervention. The levels of agricultural and manufacturing employment influence instrument choice. Agricultural employment was the most influential variable introduced to the model. These economic sectors did not appear to receive favorable treatment as policy instrument theorists contend. Strong evidence was found for a connection between political ideology and policy instrument choice. States with liberal elite ideology choose different biofuel policy instruments than states with conservative elite ideology. The research offered initial evidence that NPM philosophies translate to policy instrument adoption.

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Levoglucosan is the major anhydrosugar component of bio-oil produced by fast pyrolysis. Previous research has shown that levoglucosan yield can be greatly increased if a mild acid pretreatment is applied to demineralize the feedstock prior to pyrolysis. The interest in levoglucosan production is that it provides a route to production of monomeric sugars, primarily glucose, which can be utilized to produce biochemically derived fuels (ethanol, butanol, etc.) In one study, four different lignocellulosic biomass were subjected to pyrolysis as feedstocks to produce bio-oils via fast pyrolysis in a 7 kg/h feed rate auger reactor. Feedstocks were pretreated with dilute phosphoric acid and bio-oils were produced and analyzed to compare the bio-oil characteristics from both untreated and treated feedstocks. The results shown in this study strongly indicate that the ash content and alkali metal content are very important parameters which can greatly affect the yield and many properties of bio-oils produced during fast pyrolysis. The dilute acid pretreatment performed in this study significantly reduced the total ash content and alkali metal content in the feedstocks, resulting in a great increase in the bio-oil and levoglucosan yields. It was also shown that dilute acid pretreatment was more effective in treating herbaceous feedstocks than woody biomass in terms of increasing bio-oil production yield and improving bio-oil properties. In one study, bio-oil composed of high levoglucosan concentration was produced via fast pyrolysis of dilute acid pretreated loblolly pine wood in an auger reactor. Water-to-bio-oil ratio, temperature, and time were selected as the three parameters to investigate the optimal condition for extracting the maximum amount of levoglucosan from the bio-oil. The optimal condition for levoglucosan extraction determined was 1.3 : 1 (water-to-bio-oil ratio), 25 oC, and 20 min, producing a levoglucosan yield of 12.7 wt %. The final study developed a new method based on pyrolysis of dilute acid pretreated loblolly pine wood and modification of the pyrolysis process. This new method resulted in a significant 30.7 wt % increase in levoglucosan concentration in the bio-oil organic portion. The results indicated that this method successfully suppressed the levoglucosan decomposition during fast pyrolysis.

The 2007 Energy Independence and Security Act mandates the production of 135 billion liters of transportation fuel achieved through the use of alternative energy sources. The most economically and sustainably competitive bioenergy crops will need to be high yielding, perennial species which require minimal inputs coupled with the ability to grow on marginal land. Grower adoption will be imperative to the success of this industry. Our results indicate that after three years of growth, several species including Arundo donax and Miscanthus × giganteus are able to produce up to 48.2 and 61.4 Mg ha⁻¹ dry weight of feedstock respectively. We also found that weed management may not be necessary under ideal growing conditions, but a group of herbicides appear promising for preemergence and postemergence application timings, tested under greenhouse conditions. The herbicides pyroxasulfone, sethoxydim and nicosulfuron may prove to be useful tools for the control of bioenergy crops if they escape cultivation. The potential for many of these exotic and highly competitive species to become invasive has hindered advancement of this industry. We develop a beginning framework to evaluate the potential for seeded Miscanthus × giganteus to become invasive, as no proven formula currently exists. We developed a novel methodology to assess the invasive potential of bioenergy crops across a continuum of invasiveness, which proved to be critical to the interpretation of our results. This methodology allows us to make relative comparisons of risk, evaluating our otherwise isolated results in broader context. We chose to evaluate M. × giganteus in a direct comparison with known exotic invasives, as well as with species that are known not to be invasive. We use a tiered approach, combining qualitative risk assessment models with quantitative field trials. Results from two weed risk assessment models indicate that many bioenergy feedstocks are of high risk, but agronomic crops such as Oryza sativa were also found to be high risk. Subsequent field trials indicate that seedling establishment for M. × giganteus in perennial systems may be low as only 0.1% of emerged seedlings survived after six months, while up to 10% of the invasive Microstegium vimineum survived. Spread of fertile M. × giganteus seedlings into annual systems with low competition had greater success; we observed 316 culms m⁻², at a distance of 1.5 m from the planted plot, after three years of growth. The observed spread of fertile M. × giganteus, in the low competition environment, did not differ from that of our positive control species; however, spread of fertile M. × giganteus seedlings into areas of high competition was significantly lower than our positive controls, up to 3 m from the cultivated plot. Importantly, A. donax and sterile M. × giganteus never spread from the cultivated plot. Results from our study and application of this novel methodology will help to prepare for the expansion of this burgeoning industry while providing useful information for the implementation of best management practices and improved stewardship. / Ph. D.