These commercial vines were inoculated early in the growing season, which has been shown to be most effective inoculation timing to result in chronic infection in Napa Valley . Therefore, the diseased plants that we observe under commercial growing conditions may represent a fraction of plants that are exposed to X. fastidiosa. Overwinter curing is still poorly understood, but temperature adaptation in PD strains across California might contribute to the pathogen’s tolerance of colder winters . Typical infections averaged 105 CFU/g of plant petiole tissue; however, population size was not found to impact symptoms. Only binary infection status significantly influenced symptoms. Plants that tested positive were above the threshold of population size needed for transmission by the local vector, Graphocephala atropunctata, measured as populations above 104 CFU/g of stem tissue . In May, qPCR detection was rare, even for plants that tested positive later in the growing season. Given the complications with detection via qPCR that vary seasonally and throughout a plant, this study supports symptom identification as the best way to detect infected grapevines in Napa Valley early in the growing season.

These data also indicate that even in plants that are chronically infected and those with severe PD symptoms, plastic pot populations will still be undetectable if measured even a few months too early. This project provides new insight into the characteristics of early PD infection, which should improve disease detection in vineyards. The long-term goal of elucidating the development of this infectious disease in a high-value agricultural system is vital to the California agricultural economy. As PD has recently expanded into global grape growing regions in Europe and Asia , there has never been a more urgent time to understand the infection process of X. fastidiosa in Vitis vinifera.Free resveratrol is a naturally occurring plant-made stilbene compound produced in over 70 plant species1 , including but not limited to grapes, peanuts , and blueberries. Resveratrol, an isomer where the trans-form of the double bond separates two phenyl groups , is a phytoalexin capable of both antifungal and antimicrobial properties. This defensive property has prompted researchers to investigate the compound further in search for any potential benefits. In recent years, over 1,900 studies have been published reporting the numerous therapeutic benefits and applications which resveratrol contains . In particular, resveratrol is widely recognized for its beneficial pharmacological effects which include anti-tumor, antiinflammatory, prevention of cardiovascular disorders, and anti-aging. For this reason, the utilization of resveratrol is predominantly seen as a supplement in food or in the form of pill capsules in the nutraceutical industry . However, the use of resveratrol is also seen present within the pharmaceutical and cosmetic industries.

Resveratrol has been gaining a lot of attention recently since chemical research literature has demonstrated that it can be utilized as an active ingredient in synthesizing bio-based polymers and resins. Notably, using biorenewable compounds like resveratrol to produce biopolymers is not a novel subject but interest in using bio-based compounds for polymer development is growing. In fact, an emerging topic in the field of green chemistry is the investigation of biobased compounds for polymer development. An example of a bio-based compound used for biopolymer applications is lactic acid. Lactic acid, which is mainly derived from corn, has been shown to form polylactic acid typically used in 3D printing applications. Another example of a biobased compound used for polymer applications is vanillin. Vanillin, which can be derived from vanilla orchid extract, has been shown to be a precursor for a variety of polymers ranging from polyethylene Therephthalate to polybenzoxazines. The attractiveness of these compounds emerges from a variety of reasons, ranging from their unique chemical structure to their biodegradability. One reason resveratrol is highly sought after in the development of thermoset polymers is the number of hydroxyl groups located on the backbone of the aryl compound. It is extrapolated that these functional groups act as proton donors which can catalyze the curing reaction, allowing for curing to occur at low temperatures while also increasing reactivity of the polymer. Another reason why using resveratrol as a raw material for polymer synthesis is of inTherest is its sustainability. Typical epoxy resins are designed using environmentally unfriendly petrochemicals which must be chemically synthesized, such as bisphenol A , which is harmful towards the human immune system20. Using alternative, naturally produced, less toxic compounds like resveratrol offers scientists the ability to use renewable materials at large quantity in a safe environmental workplace. An example of a resveratrol-based biopolymer resin and its chemical synthesis pathway can be seen in Figure 1.2. InTherestingly, literature focused on resveratrol-based biopolymers have demonstrated that resveratrol epoxy resins have been characterized to hold high flame resistance properties. Specifically, these resins can hold high thermal stability at temperatures of 350°C and higher, causing them to become a viable alternative to environmentally harmful petroleum-based epoxy resins currently being used. Additionally, researchers from Hubei University reports epoxyresins fabricated using bio-based resveratrol can achieve char yields up to 62% at 800°C. Such thermal stability performance makes using resveratrol as a raw material desirable. However, the utilization of resveratrol as a precursor for biopolymers may require a much higher purity than what is currently existing for nutraceutical applications. Unlike the stringent regulations placed on the pharmaceutical industry, the nutraceutical industry remains an unregulated market which receives little to no oversight from the U.S. Food and Drug Administration . Thus, domestically produced resveratrol capsules can be expected to vary in resveratrol concentrations. A study conducted by Rossi et al., demonstrated that only 5 out of 14 nutraceutical resveratrol products tested contained concentrations of resveratrol in similar values to that branded on the bottle’s labels. Further investigation from the same authors allowed them to discover that 2 out of the 14 nutraceutical resveratrol products sampled had resveratrol content below the limit of detection. Such variation prompts the need for an efficient manufacturing process which can produce concentrated amounts of resveratrol reliably.Literature featuring different production processes capable of producing resveratrol at 98% purity and higher is available but is often only described for small quantities in laboratory-based environments, limiting its potential for large scale production. It has also been demonstrated that highly pure resveratrol can be produced via different expression systems, grow bag either through a plant-based, microbial, or chemical synthesis approach. The most common methodutilized for commercial production of resveratrol is the plant-based approach, demonstrated by the dozen companies offering natural plants as their source for production. While alternative production routes such as microbially and chemical synthesis are commercialized, two individual companies, Evolva and DSM, dominate the manufacturing of resveratrol using those methods globally.

Japanese knotweed is typically used as the plant source since it naturally produces resveratrol at high concentrations within its rhizomes, concentrations ranging from 0.5 – 12 mg/g fresh weight. Other plant sources such as grapes, peanuts, and seeds of melinjo endosperms only reach a fraction of the resveratrol present in knotweed, yielding values as high as 0.01 to 84.63 µg resveratrol/ g grape leaves FW, 0.03 to 0.14 μg of resveratrol/g peanuts Dry weight and .223 mg resveratrol/g dried melinjo endosperms. Ironically, an additional advantage to using Japanese knotweed is that resveratrol is not the leading compound found within the plant. The resveratrol precursor polydatin, a resveratrol glucoside found in Japanese knotweed, is found in higher concentrations, even up to 7-fold higher compared to resveratrol under certain growing conditions. A deglycosylation and hydrolysis step can convert polydatin to resveratrol, thus increasing the total concentration of resveratrol present in knotweed. The chemical structure of polydatin is shown in Figure 1.3.The global market for resveratrol is increasing, with current analysts estimating that the global resveratrol market to be within the range of $71.9 to $97.7 million in 2020 alone withanticipated growth in the coming years14,30. A discussion with a resveratrol manufacturer in China led to the claim that the global production volume was roughly 300 MT per year. Despite being able to recover resveratrol from a variety of expression systems, no biotechnological large-scale production facility has been established. It is to the best of our knowledge, the production capacity for a single manufacturer producing 98% pure resveratrol is well below 100 MT per year. Implementation of such bio-manufacturing facility presents a viable solution to provide a reliable supply of resveratrol in high purity suitable for biopolymer applications domestically. Nonetheless, launching a new facility for such production scale requires extensive planning, market analysis, process development, and product development. Certain tools such as process simulation tools are beneficial in providing decision making guidance for the design of a new facility as it can provide clarity on unknown processes or technologies. In fact, the utilization of PSTs during different stages of the product life cycle can be used to assess and eliminate impractical methods before the actual design and implementation. PSTs are a feasible tool used extensively when designing new manufacturing facilities as estimates for capital investment, equipment size, costs and scheduling, annual operating costs, and cost of goods can be determined. This information is invaluable as it can guide investors and companies on the economics earlier in the design process. In the following chapters, a techno-economic analysis , including process design, scale-up simulation, and scenario and sensitivity analyses, is presented for the large-scale plant based production of resveratrol. A base case production facility will be modeled for an annual production target of 100 MT of 98% pure resveratrol. In Chapter 2, the economic analysis performed for the upstream processing of the field-grown and field harvested Japanese knotweed plants will be discussed. Next, Chapter 3 will address the downstream bio-processing steps needed for extraction and purification of resveratrol from the cultivated Japanese knotweed roots. In Chapter 4, a series of sensitivity and scenario analysis will be performed to assess the relationship between certain bio-processing parameters to economic factors such as capital expenditures and cost of goods sold, along with discussion of certain advantages and disadvantage of the different scenarios performed. Chapter 5 summarizes the environmental impact of the base case model. Chapter 6 will summarize the work performed within this analysis and offer further work and modifications which can be applied to the model.Large scale, open-field agriculture is common practice utilized for the production and cultivation of several thousand metric tons of crops in the United States . In a report distributed by the United States’ Department of Agriculture , it was announced that in 2019 the U.S. harvested over 284 million acres of farmland for principal crops including but not limited to corn, oats, wheat, and potatoes1 . Outdoor agriculture is a technique which has been gaining traction for molecular farming applications, or the production of recombinant proteins or metabolites in plants. Transgenic, or genetically modified, plants have been shown to grow at agricultural scale, reaching protein yields as high as 1000 kg per year . While literature has demonstrated that transgenic plants can produce resveratrol , the use of modified plants are not needed for large scale production of resveratrol since it can already be produced in native plants at high concentrations. An additional advantage of using a natural plant source for resveratrol production outdoors over transgenic plants is the exclusion of adhering to regulations for field grown, genetically modified crops. Furthermore, no transformation step is needed when establishing the plant sources, thus reducing upstream costs. For outdoor production, Japanese knotweed is chosen as the host best suitable to reach the market’s need for resveratrol. Japanese knotweed has been shown to grow in open-field plantations by numerous oversea companies, thus demonstrating its usefulness for bulk resveratrol production. In addition to the high concentration of resveratrol found within Japanese knotweed rhizomes and its ability to grow at large scale, there are several other advantages to utilizing it over other natural plant sources. First, and most importantly, Japanese knotweed grows rapidly in a variety of habitats and environments; it classified is an invasive species. In fact, Japanese knotweed has been namedone of the 100 worst invasive species in the world by the Invasive Species Specialist Group and World Conservation Union 5 .