Sustainability through replacement of non-renewable fibers with renewable fibers is an ecological need. Impact of transportation costs from South-east Asia on the life cycle analysis of the composite is detrimental. Kenaf is an easily grown crop in America. Farm based processing involves placing the harvested crop in rivers and ponds, where retting of the fibers from the plant (separation into fibers) can take 2 weeks or more. The objective of this thesis is to analyze industrially viable processes for generating fibers and examine their synergistic impact on mechanical performance, surface topography and chemistry for functional composites. Comparison has been made with commercial and conventional retting process, including alkali retting, enzymatic retting, retting in river and pond water (retting occurs by natural microbial population) with controlled microbial retting. The resulting kenaf fibers were characterized by dynamic mechanical analysis (DMA), Raman spectroscopy (FT-Raman), Fourier transform infrared spectroscopy (FT-IR), polarized optical microscopy (POM), X-ray photoelectron spectroscopy (XPS), Scanning electron microscopy (SEM) optical fluorescence microscopy, atomic force microscopy (AFM) and carbohydrate analysis. DMA results showed that pectinase and microbe treated fibers have superior viscoelastic properties compared to alkali retting. XPS, Raman, FT-IR and biochemical analysis indicated that the controlled microbial and pectinase retting was effective in removing pectin, hemicellulose and lignin. SEM, optical microscopy and AFM analysis showed the surface morphology and cross sectional architecture were preserved in pectinase retting. Experimental results showed that enzymatic retting at 48 hours and controlled microbial retting at 72 hours yield uniform and superior quality fibers compared to alkali and natural retting process. Controlled microbial retting is an inexpensive way to produce quality fibers for polymer composite reinforcement.

<p>Pectinases are a group of enzymes produced by bacteria, fungi, higher plants and animals. Pectinases can modify and degrade pectins, a class of heterogeneous and multifunctional polysaccharides present in middle lamellae and primary cell walls of plants. Pectins have been showed to play diverse roles in cell physiology, growth, adhesion and separation. Pectinases are used technically in the processing of fiber production and fruit juice or wine making. We have studied the mechanisms and applications of pectinases, especially in retting, a microbiological process where bast fibers in flax and other bast fiber cultivars are released from each other and from the woody core.</p><p>A strong correlation was found between the ability to perform retting and the degradation of sparsely esterified pectin, a substrate of polygalacturonase. This led to the conclusion that polygalacturonase plays a key role in the enzymatic retting of flax. We purified and characterized an extracellular polygalacturonase produced by Rhizopus oryzae, a very potent retting organism. The purified enzyme which appeared to be the single active component in retting, has non-methylated polygalacturonan as its preferred substrate. Peptide sequences indicate that the enzyme, like another polygalacturonase (EC. 3.2.1.15), belongs to glycosyl hydrolase family 28. It contains, however, an N-terminal sequence absent from other fungal pectinases, but present in an enzyme from the phytopathogenic bacterium, Ralstonia solanacearum.</p><p>Our finding that removal of calcium ions from the plant material by pre-incubation in dilute acid in enzymatic retting could reduce enzyme consumption by several orders of magnitude, improves the economical feasibility of the enzymatic retting process. Comparisons with different acids showed that the action was mainly pH dependent.</p><p>Pectinases were employed as analytical tools in a study of stored wood discoloration and, together with cellulases, in a mechanical process for making pulp from flax and hemp in paper production. </p>

Pectinases are a group of enzymes produced by bacteria, fungi, higher plants and animals. Pectinases can modify and degrade pectins, a class of heterogeneous and multifunctional polysaccharides present in middle lamellae and primary cell walls of plants. Pectins have been showed to play diverse roles in cell physiology, growth, adhesion and separation. Pectinases are used technically in the processing of fiber production and fruit juice or wine making. We have studied the mechanisms and applications of pectinases, especially in retting, a microbiological process where bast fibers in flax and other bast fiber cultivars are released from each other and from the woody core. A strong correlation was found between the ability to perform retting and the degradation of sparsely esterified pectin, a substrate of polygalacturonase. This led to the conclusion that polygalacturonase plays a key role in the enzymatic retting of flax. We purified and characterized an extracellular polygalacturonase produced by Rhizopus oryzae, a very potent retting organism. The purified enzyme which appeared to be the single active component in retting, has non-methylated polygalacturonan as its preferred substrate. Peptide sequences indicate that the enzyme, like another polygalacturonase (EC. 3.2.1.15), belongs to glycosyl hydrolase family 28. It contains, however, an N-terminal sequence absent from other fungal pectinases, but present in an enzyme from the phytopathogenic bacterium, Ralstonia solanacearum. Our finding that removal of calcium ions from the plant material by pre-incubation in dilute acid in enzymatic retting could reduce enzyme consumption by several orders of magnitude, improves the economical feasibility of the enzymatic retting process. Comparisons with different acids showed that the action was mainly pH dependent. Pectinases were employed as analytical tools in a study of stored wood discoloration and, together with cellulases, in a mechanical process for making pulp from flax and hemp in paper production.

Fiber-yielding plants is an area of increased interest due to the potential use in a variety of green-based materials. These biocomposites can be incorporated into multiple uses; for example, to replace building materials and interior vehicular paneling. The research here aims to focus in on the crop Hibiscus cannabinus for utilization into these functions. H. cannabinus is economically attractive due to the entire process being able to be accomplished here in the United States. The plant can be grown in a relatively short growth period (120-180 days), and then processed and incorporated in a biocomposite. The plant fiber must first be broken down into a useable medium. This is accomplished by the retting process, which occurs when microbial constituents breakdown the heteropolysaccharides releasing the fiber. The research aims to bridge the gap between the primitive process of retting and current techniques in molecular and microbiology. Utilizing a classical microbiological approach, which entailed enrichment and isolation of pectinase-producing bacteria for downstream use in augmented microbial retting experiments. The tracking of the bacteria was accomplished by using the 16S rRNA which acts as “barcodes” for bacteria. Next-generation sequencing can then provide data from each environment telling the composition and microbial diversity of each tested variable. The main environments tested are: a natural environment, organisms contributed by the plant material solely, and an augmented version in which pectinase-producing bacteria are added. In addition, a time-course experiment was performed on the augmented environment providing data of the shift to an anaerobic environment. Lastly, a drop-in set was performed using each isolate separately to determine which contributes to the shift in microbial organization. This research provided a much needed modernization of the retting technique. Previous studies have been subject to simple clone libraries and growth plate assays and next-generation sequencing will bring the understanding of microbial retting into the 21st century.

Lignocellulose is the most abundant biopolymer on earth and offers excellent potential for sustainable manufacturing. Because lignocellulose is structurally complex and resistant to decomposition, innovative degradation strategies are necessary to unlock its value. In this dissertation, a green manufacturing process through enzyme-triggered self-cultured bacteria retting for lignocellulosic fiber was developed and investigated. The mechanism of the lignocellulosic fiber retting at a controlled degradation strategy was studied. This enzymatic degradation strategy utilizes a small amount of enzyme to trigger a large aggregation of specific bacteria to obtain clean fibers. Industrial hemp (Cannabis sativa L.) fiber was successfully retted with this strategy. The degradation of pectin was proved through an environmental scanning electron microscope and reducing sugar analysis. The bacterial successions were identified by 16S rRNA gene metagenomic sequencing. The results showed that Bacillaceae dominated the hemp retting conditions containing 1% pectinase, suggesting that pectinase can manipulate bacterial community succession by changing the nutrients available to bacteria through the degradation of pectin. This degradation strategy has 20-25% less environmental impact than the thermochemical degradation strategy, resulting in better fiber consistency and much shorter processing time (3-5 days) than the traditional water degradation strategy. The study on the degradation of lignin-rich lignocellulose also contributes to the understanding of the natural formaldehyde release mechanisms from wood.

Cellulosic fibers sized from the macro-scale to the nano-scale were prepared hierarchically from kenaf bast fibers using chemicals. The process began with a hermetical alkaline retting followed by a bleaching treatment. The bleached fibers were hydrolyzed using inorganic acid, from which microfibers and cellulose nanowhiskers (CNWs) were fabricated. Inorganic nanoparticle impregnation (INI) was used to treat the retted fibers for the improvement of the interfacial compatibility between the fiber and polypropylene (PP) matrix. The retted fibers and INI-treated fibers were used as reinforcement for the PP polymer composites. Film casting process was used to make CNW/PVA composites. The hermetical retting process used in this study produced fibers with high cellulose contents (81-92%) by removing the lignin and hemicelluloses. Higher retting temperature resulted in higher fiber surface hardness and elastic moduli. The tensile strengths and tensile moduli of the fibers decreased as the temperature increased. The SEM images showed the micropores in the cell wall structure for the fibers retted at over 130°C, providing the possibility to anchor nanoparticles into the cell wall. Surface morphology of the INI-treated fibers was examined with SEM, and showed that the CaCO3 nanoparticle crystals grew onto the fiber surface. Energy-dispersive X-ray spectroscopy (EDS) was used to verify the CaCO3 particle deposits on the fiber surface. As the size scale of the fibers decreased, the fiber crystallinity increased from 49.9% (retted fibers) to 83.9% (CNWs). About 23% á-cellulose in the raw kenaf bast fibers had been converted into CNWs. The retted fibers without INI treatment had poor compatibility with the polypropylene matrix. The INI treatment improved the compatibility between the fibers and the PP matrix, resulting in an improvement in kenaf fiber/PP composite tensile moduli and tensile strengths. The CNWs prepared from kenaf bast fiber gave excellent reinforcement for PVA composites. A nine percent increase of CNWs in the CNW/PVA composites yielded significant improvements in tensile strength and modulus of about 46% and 152%, respectively, compared with pure PVA.

Presently there are two forms of biological fibre extraction, water retting or dew retting, which use bacteria or fungi, respectively. Microbial action results in release of the cellulose fibres due to modification of the pectin, hemicellulose and lignin content from parenchyma cells and the middle lamellae. Water retting results in pollution, high costs associated with labour and drying, as well as significant waste water production, while disadvantages to dew retting include the need for appropriate climates, variable and inferior fibre quality, risks of over-retting as well as health effects due to dust and fungal contaminants. The overall objective of this research was to explore silage retting as a new pre-processing technique allowing use of available farm infrastructure and contained retting conditions to produce plant-derived fibres with improved physical and chemical characteristics suitable for application in biocomposites. The corn processing ability of the hemp retting agents Clostridium felsineum and Bacillus subtilis was also investigated. Pleiotropic and/or crop management practices were assessed by comparing the physico-mechanical properties and the microbial populations during silage fermentation of genetically equivalent conventional, Roundup Ready® (RR) and Bt-Roundup Ready® (Bt-RR) corn isolines. Potential recovery of volatile organic acids in silage retting effluent as value-added chemicals was also explored. The results indicated that C. felsineum is an effective corn retting agent given the effective release of the fibre bundles from the corn pith, with B. subtilis contributing to the retting process by reducing the oxygen content and providing the required anaerobic conditions for clostridial growth. The native microflora present in the plant phyllosphere also showed some retting ability. Composition, thermostability and mechanical properties of the biocomposites produced using the fibres from the retted corn were all found to vary depending on the variety of corn. Specifically, retted Bt-RR cornstalk showed a 15°C increase in onset of degradation. Divergences between corn silage microbial communities analyzed by community-level physiological and enzyme activity profiling indicated that metabolic shifts were time-, region-, and contaminant-sensitive. Acetic and butyric acid production in silage retting effluent was found to be highest under anaerobic conditions and was also influenced by corn hybrid variety, although a specific variety was not identified as most or least favourable for organic acid production due to high variability. Bt-RR cornstalk material was found to have higher cellulose content and better thermostability with an onset of degradation of up to 45°C higher than its genetic RR and conventional counterparts. However, fibres from the RR corn isoline produced biocomposites with the highest flexural strength and modulus. RR cornstalk-reinforced polypropylene showed a 37 and 94% increase in flexural strength and modulus, respectively when compared to the mechanical properties of the pure polypropylene. The Bt-RR and conventional varieties produced biocomposites with an average increase of 26.5% in flexural strength and 83.5% in flexural modulus. The thermostability of ensiled corn biomass was found to be influenced by region, use of inoculants and silage treatment, while the silage treatment accounted for most of the variability in corn biomass composition. Polypropylene matrix biocomposites produced with (30 wt%) pre- and post-silage corn did not show significant differences in mechanical properties. However, ensiled corn resulted in an increase in fibres and potential microbial biomass of smaller particle sizes with more optimal thermostability and purity, producing biocomposites with higher flexural strength and modulus especially at higher extrusion temperatures. Cornstalk is an effective reinforcement material, producing biocomposites with higher flexural strength, flexural modulus and impact strength. Whole corn biomass presents a potential alternative to other plant fibres, especially as filler material. Silage retting resulted in fibres with a higher thermostability and smaller particle size distribution that, given their already smaller aspect ratio, could result in better mechanical properties in thermoplastics with a higher melting temperature or biocomposites requiring higher shear for mixing.

Presently there are two forms of biological fibre extraction, water retting or dew retting, which use bacteria or fungi, respectively. Microbial action results in release of the cellulose fibres due to modification of the pectin, hemicellulose and lignin content from parenchyma cells and the middle lamellae. Water retting results in pollution, high costs associated with labour and drying, as well as significant waste water production, while disadvantages to dew retting include the need for appropriate climates, variable and inferior fibre quality, risks of over-retting as well as health effects due to dust and fungal contaminants. The overall objective of this research was to explore silage retting as a new pre-processing technique allowing use of available farm infrastructure and contained retting conditions to produce plant-derived fibres with improved physical and chemical characteristics suitable for application in biocomposites. The corn processing ability of the hemp retting agents Clostridium felsineum and Bacillus subtilis was also investigated. Pleiotropic and/or crop management practices were assessed by comparing the physico-mechanical properties and the microbial populations during silage fermentation of genetically equivalent conventional, Roundup Ready® (RR) and Bt-Roundup Ready® (Bt-RR) corn isolines. Potential recovery of volatile organic acids in silage retting effluent as value-added chemicals was also explored. The results indicated that C. felsineum is an effective corn retting agent given the effective release of the fibre bundles from the corn pith, with B. subtilis contributing to the retting process by reducing the oxygen content and providing the required anaerobic conditions for clostridial growth. The native microflora present in the plant phyllosphere also showed some retting ability. Composition, thermostability and mechanical properties of the biocomposites produced using the fibres from the retted corn were all found to vary depending on the variety of corn. Specifically, retted Bt-RR cornstalk showed a 15°C increase in onset of degradation. Divergences between corn silage microbial communities analyzed by community-level physiological and enzyme activity profiling indicated that metabolic shifts were time-, region-, and contaminant-sensitive. Acetic and butyric acid production in silage retting effluent was found to be highest under anaerobic conditions and was also influenced by corn hybrid variety, although a specific variety was not identified as most or least favourable for organic acid production due to high variability. Bt-RR cornstalk material was found to have higher cellulose content and better thermostability with an onset of degradation of up to 45°C higher than its genetic RR and conventional counterparts. However, fibres from the RR corn isoline produced biocomposites with the highest flexural strength and modulus. RR cornstalk-reinforced polypropylene showed a 37 and 94% increase in flexural strength and modulus, respectively when compared to the mechanical properties of the pure polypropylene. The Bt-RR and conventional varieties produced biocomposites with an average increase of 26.5% in flexural strength and 83.5% in flexural modulus. The thermostability of ensiled corn biomass was found to be influenced by region, use of inoculants and silage treatment, while the silage treatment accounted for most of the variability in corn biomass composition. Polypropylene matrix biocomposites produced with (30 wt%) pre- and post-silage corn did not show significant differences in mechanical properties. However, ensiled corn resulted in an increase in fibres and potential microbial biomass of smaller particle sizes with more optimal thermostability and purity, producing biocomposites with higher flexural strength and modulus especially at higher extrusion temperatures. Cornstalk is an effective reinforcement material, producing biocomposites with higher flexural strength, flexural modulus and impact strength. Whole corn biomass presents a potential alternative to other plant fibres, especially as filler material. Silage retting resulted in fibres with a higher thermostability and smaller particle size distribution that, given their already smaller aspect ratio, could result in better mechanical properties in thermoplastics with a higher melting temperature or biocomposites requiring higher shear for mixing.

Conselho Nacional do Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / Extratos, óleos essenciais e compostos químicos isolados de origem vegetal têm sido cada vez mais utilizados, não só como matéria-prima para indústrias de diversos ramos, mas também como alternativa no tratamento de problemas da saúde pela população em geral. Considerando a expressiva importância de produção destas substâncias com qualidade elevada e preço acessível, o presente trabalho teve como objetivo a análise técnica e econômica na obtenção de compostos bioativos a partir das espécies vegetais Artemisia annua e Varronia curassavica. Visando selecionar e testar três tecnologias de extração ambientalmente sustentáveis, dióxido de carbono supercrítico, maceração a frio com o solvente etanol e destilação com arraste a vapor, um protocolo de alto rendimento e baixo custo para obtenção de compostos bioativos de plantas foi proposto. Para a extração com dióxido de carbono supercrítico foi realizada a investigação da influência da temperatura, pressão e volume de cossolvente (etanol) no rendimento de extração e pureza dos extratos. Três experimentos com delineamento inteiramente casualizado em arranjo fatorial foram realizados. Para o experimento com A. annua foram testadas 3 temperaturas (40/50/60 ºC), 3 pressões (200/250/300 bar) e 3 níveis de cossolvente (0/15/25%); e para V. curassavica 2 temperaturas (40/60 ºC), 5 pressões (100/150/200/250/300 bar) e extração fracionada. As extrações com destilação por arraste a vapor foram conduzidas à pressão ambiente por 3 - 4 h. E a maceração a frio com etanol foi realizada em série, a pressão ambiente por 6 h, condições estas as ótimas referenciadas. Ao fim dos experimentos os rendimentos e pureza dos extratos e óleo essencial foram comparados e utilizados para a implementação de indústrias de produção de biocompostos utilizando as tecnologias testadas. Por meio de pesquisa de custos envolvidos na implementação do projeto, uso de planilhas no Microsoft Office Excel, do Software SuperPro Designer® e do Aplicativo Web $AVEPI, foi possível estimar os Custos de Produção e Manutenção, Capital Fixo Direto, e, na sequencia simular computacionalmente os cenários econômicos, gerando os indicadores de viabilidade e riscos de implantação das indústrias de extração de artemisinina à partir de A. annua, e óleo essencial à partir de V. curassavica. Observou-se que a extração de artemisinina com dióxido de carbono supercrítico obteve menores rendimentos de extrato que a extração com etanol, porém com maior pureza do composto alvo e custos menores de produção. Na produção de óleo essencial de V. curassavica a extração com dióxido de carbono supercrítico alcançou os mesmos rendimentos e pureza obtidos por destilação com arraste a vapor. No cenário estudado os custos de operação e manutenção da produção de extratos com etanol, dióxido de carbono supercrítico, para A. annua, e dióxido de carbono supercrítico para V. curassavica, revelaram ser maiores que o preço de revenda dos extratos. Para a planta A. annua tais resultados foram atribuídos aos altos custos da matéria-prima, e para V. curassavica aos baixos rendimentos de extrato. O projeto de investimento utilizando destilação com arraste a vapor para obtenção de óleo essencial de V. curassavica mostrou ser promissor devido à alta rentabilidade e baixo risco associado nos senários previstos. / Extracts, essential oils and isolated chemical compounds of vegetable origin have been increasingly used, not only as raw material for industries of different branches, but also as an alternative in the treatment of health problems by the population in general. Considering the expressive importance of producing these substances with high quality and affordable price, the present work had the objective of the technical and economic analysis in obtaining bioactive compounds from the plant species Artemisia annua and Varronia curassavica. Aiming to select and test three environmentally sustainable extraction technologies, supercritical carbon dioxide, cold maceration with the ethanol and steam distillation, a protocol of high yield and low cost to obtain bioactive compounds of plants was proposed. For the supercritical carbon dioxide extraction, the influence of temperature, pressure and volume of cosolvent (ethanol) on the extraction yield and purity of the extracts was investigated. Three experiments with a completely randomized design in factorial arrangement were performed. For the A. annua, three temperatures (40/50/60 ºC), 3 pressures (200/250/300 bar) and 3 levels of cosolvents (0/15/25%) were tested; and for V. curassavica 2 temperatures (40/60 ºC), 5 pressures (100/150/200/250/300 bar) and fractional extraction. The extractions with steam distillation were conducted at ambient pressure for 3 - 4 h, and cold maceration with ethanol was carried out in series, at ambient pressure for 6 h, conditions being the best referenced. At the end of the experiments the yields and purity of the extracts and essential oil were compared and used for the implementation of industries of biocomposites production using the technologies tested. Through the research of costs involved in the project implementation and the use of spreadsheets in Microsoft Office Excel, it was calculated the Costs of Production and Maintenance and Direct Fixed Capital; on the sequence, using the Software SuperPro Designer® and the Web Application $AVEPI, the computational simulations were conducted generating the economic indicators on the feasibility and risks of implantation of the industries of extraction of artemisinin from A. annua, and essential oil from V. curassavica. It was observed that the extraction of artemisinin with supercritical carbon dioxide obtained lower yields of extract than the extraction with ethanol, but with higher purity of the target compound and lower costs of production. In the production of essential oil of V. curassavica the extraction with supercritical carbon dioxide reached the same yields and purity obtained by steam distillation. In the scenario studied the costs of operation and maintenance of the production of extracts with ethanol, supercritical carbon dioxide for A. annua, and supercritical carbon dioxide for V. curassavica, were shown to be greater than the revenue price of the extracts. For the A. annua plant, these results were attributed to the high costs of the raw material, and to V. curassavica the low yields of the extract. The investment project using steam distillation for the obtaining of V. curassavica essential oil showed to be promising due to the high profitability and low risk associated in the expected scenarios.