MICROSCOPIC AND LABORATORY SCALE CHARACTERIZATION METHODS TO EVALUATE BIOMASS DECONSTRUCTION

Renewable fuels from lignocellulosic biomass are an appealing option because they can seamlessly integrate into the existing fuel distribution infrastructure. Lignocellulosic biomass constitutes nonedible plant material obtained from plant cell walls. The natural recalcitrance of lignocellulosic biomass poses a challenge in accessing the cell wall carbohydrates during biochemical conversion. Despite various approaches, enzymatic hydrolysis of lignocellulosic biomass remains economically impractical due to incomplete knowledge about biomass recalcitrance and the influence of environmental factors on biomass quality. The first goal of this dissertation was to construct a microfluidic imaging reactor to better understand the tissue-specific deconstruction of plant materials. Confocal laser scanning microscopy was conducted on thin sections (60 mm thickness) of corn stems at different time points during dilute acid and dilute alkali pretreatment. The digestibility of the acid and alkali-pretreated biomass sections during enzymatic hydrolysis were evaluated using brightfield imaging. Corn stem parenchyma cells were more susceptible to deconstruction than vascular bundles in both pretreatment and enzymatic hydrolysis. After 48 hours of enzymatic hydrolysis, only the protoxylem remained undegraded. The second goal was to investigate how increasing stem solidness impacts enzymatic digestibility in wheat straw using the microfluidic imaging reactor. This was based on the rationale that the pith parenchyma cells are more digestible than the other vascular cell types. During the pretreatment and enzymatic hydrolysis, the solid stemmed samples showed considerably greater amenability to degradation than the hollow and semisolid cultivars, which based on the imaging was largely due to the greater digestibility of the pith parenchyma cells. The third goal was to develop a high-throughput, moderate-scale enzymatic hydrolysis method at high-solids loading to study the impact of drought and extreme weather conditions on biomass deconstruction. At the laboratory scale, high solids loading results in improper mixing and low saccharification due to low water availability. This was overcome using horizontal mixing on a laboratory scale roller to improve enzyme accessibility and obtain higher sugar yields. The saccharification for the roller bottle method was about 25-50% higher than the traditional shake flask method. This was evaluated for a variety of AFEX-pretreated feedstocks, including corn stover, sorghum, miscanthus, native prairie, and switchgrass

Comparison of the infection biology and transcriptome of wild-type and single gene deletion strains of Fusarium graminearum

Fusarium Ear Blight is a devastating fungal disease of cereals and due to the contamination of the harvested grain with a range of trichothecene mycotoxins presents a risk to human and animal health. The re-emergence of Fusarium graminearum on wheat and maize, the evolution of more aggressive fungal strains and the lack of an effective control strategy, has increased the need for a greater understanding of the disease aetiology. This project aimed to enhance the understanding of the interaction between F. graminearum and wheat (Triticum aestivum), through the utilisation of microscopy and molecular pathogenomics. A detailed investigation of the infection process revealed a prolonged latent period of intercellular infection that preceded host cell death, intracellular colonisation and the onset of disease symptoms. Phenotypic differences in colonisation and mycotoxin gene expression implied that hyphae within the two phases of infection were transcriptionally distinct, while a bioinformatic analysis described the fungal secretome. The two fungal gene-deficient strains assessed, top1 and tri5, were unable to establish symptomless infection or spread throughout the wheat ear, in the presence or absence of mycotoxin production, suggesting the existence of additional virulence factors. Subsequently, a genome wide transcriptome investigation of the two phases of infection, using both Affymetrix and RNA-sequencing technologies, revealed the unique expression profile, and secretome, of the advancing hyphal front of the symptomless infections. This greater understanding of the biphasic interaction will provide a benchmark for comparison with the single gene deficient strains. Finally, a laser capture microdissection procedure was developed to enable future cell-type specific transcriptome experiments. Collectively, I have discovered and developed a model of how F. graminearum establishes symptomless and symptomatic infection. In doing so, this study has enhanced the understanding of this non-biotrophic pathosystem, providing many new lines of investigation, which could greatly improve crop protection strategies

Cellulose Filaments for Responsive and Functional Materials

Cellulosic filaments, found in plants, are crucial structural elements for their survival and are a great source of inspiration to obtain new functional materials. The work performed out in this thesis aimed for the isolation, physical characterization, study of the morphology and shape, as well as the mechanical behaviour of cellulosic filaments obtained from the leaves of Agapanthus africanus and Ornithogalum thyrsoides and from the filaments that form the ribbons existing on the awns of the Erodium plants. These cellulosic filaments and filament networks were chosen due to the fundamental role that they play in the plant kingdom and their ability to change shape in the presence of an external stimuli. The study of the chosen systems served as an inspiration for the preparation and production of new membranes formed by non-woven networks of micro/nano filaments. A prototype, which allows for the selective removal of oil micro droplets from aqueous emulsions, was developed. This dissertation begins with a general introduction, based on two review papers, which the author of this thesis is the first author, in which the main concepts used in the following chapters are addressed and interconnected. The original part of the work is in chapters II, III and IV. In chapter II, the study of microfilaments from the leaves tracheary of two plants, Agapanthus africanus and Ornithogalum thyrsoides, is made. These microfilaments belong to plants of the same order and have identical shapes (left helices), chemical composition and skeletons, but different mechanical properties. For the first time, micrometric droplets of a nematic liquid crystal were used as sensors to reveal the morphology of the filaments. In order to obtain quantitative characteristics on the surface of the microfilaments, photos of the textures of pierced droplets were obtained by Polarized Optical Microscopy (POM). These textures were compared with simulated optical microphotographs obtained by numerical modelling for the nematic droplets. Homeotropic anchoring at the air, and different anchoring conditions, at the interfaces with the filaments were considered for the nematic structure. This study allowed the establishment of relationships between the physical properties/morphology of the filaments and to determine their interactions with other filaments and with the environment. In chapter III, cellulosic networks existing on dead tissues of the Erodium awns were isolated and characterized. An interesting feature of these cellulosic networks is that they form ribbons that change reversibly the shape in the presence of moisture. When dried these ribbons are right-handed helices, that uncoil remaining taut, in the presence of moisture. The work performed allowed the preparation of helical ribbons that can change the shape from right- to lefthanded helices in the presence of moisture. The behaviour observed was explained using computational simulations, considering filaments that contract and expand asymmetrically. Birefringent transparent ribbons were also isolated. The asymmetric arrangement of cellulosic fibres allows the material to be stimuli-responsive without the use of complicated lithography and intricate deposition techniques, making it suitable for a diverse range of applications, such as the production of intelligent textiles and environmental friendly micro components. In chapter IV, non-woven membranes obtained from cellulosic materials and cellulose nanocrystals were prepared and characterized. Different patterns were designed using the screenprinting technique. The adhesion between the different types of cellulosic fibres was promoted through a heat treatment. The non-woven membranes produced allowed the development of a prototype that selectively removes oil droplets from aqueous emulsions with an efficiency of approximately 80%. Throughout this thesis (chapters II and III), the complexity of the systems increases. The work begins by studying the morphology of a filament and its mechanical behaviour in the presence of other filaments. After a much more complex system, in which anisotropic filament networks, produced by the Erodium plant were addressed. The stimuli-responsive behaviour of these anisotropic networks was investigated after being released by the plant. Based on the understanding of the systems formed by cellulosic filaments, studied in chapters II and III, functional non-woven membranes were produced, printed, and characterized. The non-woven membranes led to the development of a prototype, which allows the selective removal of micro droplets of oil form aqueous emulsions. At the end, a summary of the main scientific results and future work including a technological application, which was developed in the framework of this thesis, are presented.Os filamentos celulósicos, existentes nas plantas, são elementos estruturais cruciais para a sua sobrevivência e fontes de inspiração para a obtenção de novos materiais funcionais. O trabalho realizado nesta tese visou o isolamento, caracterização física, estudo da morfologia e forma, assim como do comportamento mecânico de filamentos celulósicos obtidos a partir das folhas das plantas Agapanthus africanus e Ornithogalum thyrsoides e de filamentos que formam fitas existentes nas hastes da planta Erodium. Estes filamentos e redes de filamentos celulósicos foram escolhidos devido ao papel fundamental que desempenham no reino vegetal e à resposta que podem apresentar na presença de estímulos externos. O estudo dos sistemas escolhidos serviu de inspiração para a preparação e fabrico de novas membranas formadas por redes não tecidas de filamentos micro/nanométricos e o desenvolvimento de um protótipo, que permite a remoção seletiva de microgotas de óleo de emulsões aquosas. Inicia-se com uma introdução de caracter geral, baseada em dois artigos de revisão, dos quais a autora desta tese é primeira autora, na qual se abordam e interligam os conceitos principais, que são utilizados nos capítulos seguintes. A parte original do trabalho reparte-se pelos capítulos II, III e IV. No capítulo II é feito o estudo de microfilamentos existentes nos sistemas vasculares das folhas de duas plantas, Agaphantus africanus e Ornithogalum thyrsoides. Estes microfilamentos pertencem a plantas da mesma ordem e possuem formas (hélices esquerdas), composições químicas e esqueletos idênticos, mas apresentam propriedades mecânicas distintas. Pela primeira vez foram utilizadas gotas micrométricas de um cristal líquido nemático como sensores da morfologia dos filamentos isolados. De modo a obter características quantitativas sobre a superfície dos microfilamentos, fotos de texturas obtidas por microscopia com luz polarizada foram comparadas com microfotografias óticas simuladas a partir de modelação numérica de um meio contínuo das estruturas das gotas nemáticas, com ancoragem homeotrópica na superfície com o ar suspensas nos microfilamentos com diferentes ancoragens. O estudo realizado permitiu estabelecer relações entre as propriedades físicas/morfologia dos filamentos e determinar as suas interações com outros filamentos e com o meio ambiente. No capítulo III foram isoladas e caracterizadas redes celulósicas existentes nas hastes de tecidos mortos da planta Erodium. Uma característica interessante destas redes celulósicas é a de formarem fitas que mudam reversivelmente de conformação na presença de humidade. Quando secas estas fitas são hélices direitas, que na presença de humidade desenrolam para fitas esticadas, sem torção nem flexão. Este trabalho permitiu induzir uma inversão de quiralidade nas fitas que foi explicada pelo uso de simulações computacionais considerando que as fitas contraem e esticam de modo assimétrico. Fitas birrefringentes transparentes também foram isoladas. A disposição assimétrica das fibras celulósicas permite a resposta do material não envolvendo o uso de técnicas complicadas de litografia nem de deposição, podendo ser aplicado, por exemplo, no fabrico de têxteis inteligentes e de microcomponentes amigos do ambiente. No capítulo IV foram preparadas e caracterizadas membranas não tecidas obtidas a partir de soluções de derivados celulósicos e de celulose nano cristalina. Foram desenhadas diferentes geometrias pela utilização da técnica de “screenprinting” e promovida a adesão entre os diferentes tipos de fibras celulósicas através de tratamento térmico. As membranas não tecidas produzidas permitiram o desenvolvimento de um protótipo que remove, de forma seletiva, microgotas de óleo de emulsões aquosas com uma eficiência de cerca de 80%. Ao longo desta tese (capítulo II e III), a complexidade dos sistemas estudados aumenta, isto é, começa-se por estudar a morfologia de um filamento e o seu comportamento mecânico na presença de outros filamentos, para depois se passar para um sistema muito mais complexo em que redes anisotrópicas de filamentos, impressas pela planta Erodium, permitem a resposta a estímulos externos da estrutura formada, mesmo após esta ter abandonado a planta. Tendo por base a compreensão dos sistemas formados por filamentos celulósicos, estudados nos capítulos II e III, foram produzidas, impressas e caracterizadas, no laboratório, membranas funcionais tecidas de filamentos micro/nano celulósicos. As membranas não tecidas originaram o desenvolvimento de um protótipo, que permite a remoção seletiva de micro gotas de óleo existentes em emulsões aquosas. No final é apresentado um sumário dos principais resultados científicos e perspetivas de avanço tecnológico alcançadas por este trabalho

“Turning A New Leaf” Development of Spectroscopic Protocols to Study Plant Health

Improved methods to monitor plant health may be pivotal for future protection of crops from disease and environmental stressors. Epicuticular waxes, and macronutrients (K+ and Ca2+) hold important physiological functions within plants, and are therefore, excellent markers of plant health. This thesis has developed new spectroscopic analytical methods to directly image changes in epicuticular waxes and Ca2+ and K+ on plant leaves, for future application to monitor plant health

Developing marker technologies to probe complex polymers and products

Chemistry and Polymer Scienc

Evaluation of parameters influencing plant response to carbon nanotube contamination

Carbon nanotubes (CNTs) are among the most used nanomaterials (NMs) thanks to their excellent physicochemical properties. All along their lifecycle, they may be spread unintentionallyor intentionally in the environment. It is thus essential to assess their behavior and potential impacts on ecosystems and particularly on crop plants. Overall, behaviour and effects of CNTs in plants are not well understood and still very controversial. In this work, we aimed to assess the influence of several parameters on plant response after exposure in a CNT-contaminated soil. We first focused on the analytical challenge of detecting CNT in biological matrices and tested several spectroscopic techniques. Then, we evaluated the response of tomato plants to two different NMs (CNTs and TiO2-NPs). Our results highlight that despite being different for several parameters (i.e. shape, size, surface chemistry), CNT exposure led to a similar response in tomato plants, in particular on the alteration of plant cell wall components. The study of different plant species (tomato, canola, maize and cucumber) exposed to DWCNT contamination highlighted different responses according to plant species, maize (monocot) being the most sensitive. Different types of CNTs are currently available. Five types of CNTs varying in diameter, functionalization and length were used to investigate their impact on canola. Canola was more sensitive to CNTs with the smallest diameters, but it was also observed that the functionalization greatly modulated the plant response. Finally, we tested the impact of a combined stress: canola plants grown in optimal growth conditions were not impacted by CNT exposure at the tested dose, while we observed that plants were more sensitive to CNTs when submitted to a concomitant heat stress

Pyrene availability as determined by the interactions of natural organic matter with the soil mineral matrix

Soil organic matter (SOM), especially humic substances and biological material, profoundly affect polyaromatic hydrocarbon (PAH) movement, toxicity, and degradation when assessing acceptable end points for in situ remediation practices. The complex, inter-dependent relationship between soil chemical and physical attributes makes determining absolute PAH fate, transport, and availability a formidable task. This research attempted to correlate the impact known SOM components have on pyrene sorption as related to sorbent loading and type and mineral phase aggregation. The objective was achieved by generating a synthetic soil, composed of mineral matter, humic acid (HA), and peptidoglycan (PG), and comparing this matrix to freshly contaminated and aged natural soils, using pyrene as the representative hydrophobic organic contaminant. In contrast to HA, a larger concentration of PG sorbed to both mineral phases (hematite and montmorillonite clay); thus, promoting further particle aggregation, and effectually resulting in greater pyrene sorption as evidenced by HPLC and fluorescence microscopy. The greater the concentration and number of constituents introduced into the synthetic matrix, culminating in a HA+PG mixture loaded onto clay, the greater the resulting pyrene sorption and similarity to spiked natural soils, both in chemical and physical attributes. The final result was a compiled fluorescence map visually detailing pyrene location and percentage sorbed in relation to PG, HA, and the mineral phases for natural and synthetic soils, where the biological constituent held a significant influence over pyrene compared to that by the humic substances. In summary, the type and concentration of SOM constituents, specifically humic substances and biological material, sorbed to a mineral phase significantly impact soil physical and chemical characteristics, and therefore, should be accounted for when determining pyrene fate, transport, and availability within contaminated soil systems. Potentially, when these observations are combined with fluorescence microscopy, consultants could determine the most effective remediation technology to target specific locations, such as pore spaces, or strong sorptive entities, such as PG, and achieve acceptable end-points

Evidence of Late Quaternary Fires from Charcoal and Siliceous Aggregates in Lake Sediments in the Eastern U.S.A.

The late-glacial transition to the Holocene, 15,000–11,600 cal yr BP, is an enigmatic period of dynamic global changes and a major extinction event in North America. Fire is an agent of disturbance that transforms the environment physically and chemically, and affects plant community composition. To improve understanding of the linkages between fire, vegetation, and climate over the late glacial and Holocene in the eastern U.S., I analyzed lake-sediment cores for charcoal and indicators of wood ash, and compared results to existing pollen records. A new microscopic charcoal record from Anderson Pond, Tennessee revealed high fire activity from 23,000–15,000 cal yr BP when conifers dominated, and during the Mid-Holocene Warm Period (8000–5200 cal yr BP), when hardwoods dominated. Macroscopic charcoal analysis of sediments from Pigeon Marsh, Georgia showed high fire activity from 16,500–14,500 cal yr BP, below a major hiatus. Jackson Pond, Kentucky and Cahaba Pond, Alabama had low macroscopic charcoal concentrations during the late glacial; largest charcoal peaks occurred around 5000 cal yr BP at Jackson Pond, and from 1370–640 cal yr BP at Cahaba Pond. Thin sections were prepared for cores from the four southeastern U.S. sites and from Swift and Slack Lakes, Michigan, and analyzed together with nitrogen isotopes and element data from XRF. Thin sections showed the presence of siliceous aggregates, a unique grain type, in sediments from five sites. These grains are rare, occurring in only three periods, around 19,250, 14,000 and 12,400 cal yr BP. In laboratory experiments, I produced siliceous aggregates from wood ash with simulated rain, and found their formation requires silt, but not high acidity. On the landscape, siliceous aggregates form after fires in wood ash by the action of water. The alkaline pH of the wet ash dissolves phytoliths, and amorphous silica nucleates around silt-sized quartz grains. Then aggregates are transported into lake sediments. My research demonstrates that siliceous aggregates are a new proxy for wildfires in paleoenvironmental records. The wildfire-derived siliceous aggregates in cores examined from the eastern U.S. are contemporaneous with combustion signals in Greenland ice cores, suggesting widespread late-glacial fire events