Scaling biological pretreatment from the bench scale to the production scale may be more economical if unsterilized feedstock are used, however these allow for microbial competition from contaminates. An accurate and rapid method for identifying the desired biological pretreatment organism is necessary to confirm the presence of the desired organism when contaminates are morphologically similar to the target organism. Traditional methods, such as visual identification, sequencing, and selective plating can be time consuming and are sometimes still inconclusive. Based on methods described in the literature, plasmid DNA containing the marker genes gus (�-glucuronidase), LacZ, and gfp (green fluorescence protein) incorporated into the lignin-degrading basidiomycete Phanerochaete chrysosporium would result in a rapid genetic test for the desired organism. The presence of these genes can be confirmed either through an X-Gluc (cyclohexylammonia salt), X-Gal histochemical assay or observing the gfp’s fluorescence by a specially equipped confocal microscope. Each reporter systems will allow for rapid, reliable identification of the target species. This study will report on the success of the transformation methods in creating a transformed fungus to be used in the context of a large-scale fermentation operation. Additionally, a novel in-harvest lignocellulose feedstock biological pretreatment inoculation trial was performed comparing lignolytic performance between fungal inoculum application techniques. Optimization of carbohydrate availability for enhanced saccharification was determined by analyzing glucose release by treated and non-treated unsterilized switchgrass. This study also focused on identifying parameters to enhance saccharification efficacy at the farm-scale.

The following full-factorial study compared fungal activity on lignocellulosic biomass that was inoculated with three different amounts of fungus, and grown using three different airflow rates. These treatments were compared to a control which consisted of biomass that was not inoculated but was exposed to the same growth conditions in the environmental chamber. The objectives of the following experiment were to determine the inoculum density and airflow rate required to optimize Phanerochaete chrysosporium lignin degradation. Additionally, this study quantifies the saccharification yield from the pretreated switchgrass. The impact of substrate bulk density and substrate particle size on fungal growth were compared to determine if the particle size or the substrate bulk density has the predominant influence on the growth of the fungus, and subsequent pretreatment effectiveness quantified as an increase in glucose yields and lignin degradation. The particle size tests were controlled for bulk density; all three particle sizes were tested at a bulk density of 80 kg/m3. To test the density, three different bale densities were prepared controlling for particle size. The density tests were performed on small-scale bales made of 4 inch cut pieces of switchgrass compressed to the correct density. Therefore; density tests had the same particle size throughout all treatments, and particle size tests had the same density through all treatments. Carbohydrate accessibility post-pretreatment was examined through enzymatic saccharification and determination of glucose yields in the treatments and controls.

AN ABSTRACT OF THE THESIS OF Thara G. Siddaramu, for the Master of Science degree in Civil and Environmental Engineering, presented on February 5, 2011, at Southern Illinois University Carbondale. TITLE: EVALUATION OF DIFFERENT PRETREATMENT APPROACHES FOR DISRUPTING LIGNOCELLULOSIC STRUCTURES MAJOR PROFESSOR: Dr. Yanna Liang There are two major steps in biofuel production- pretreatment of lignocellulosic materials and enzymatic hydrolysis. The present study investigated the ability of two pretreatment methods, namely traditional oven and microwave oven treatments for disrupting lignocellulosic structures. The substrates tested were Jatropha seed cake and sweet sorghum bagasse. In recent years, Jatropha curcas also known as physic nut or purging nut has attracted extensive attention due to its several unique characteristics. Similarly, sweet sorghum has the potential to provide great value to energy sectors and food industries being that the entire plant is rich in various sugars and nutrients. Both crops can adapt to various climates, and can withstand extended drought conditions compared to other crops. Additionally, both Jatropha seed cakes and sweet sorghum bagasse are good sources of lignin and carbohydrates, which could be used for production of biofuels only if the sugars can be unlocked. Several treatment methods such as mechanical, physical, chemical and biological treatments have been reported to breakdown the cellulosic structure of biomass. However, other low cost and quicker methods, such as ovenpretreatment and microwave irradiation have not been evaluated for Jatropha seed cake and Sweet Sorghum Bagasse (SSB), respectively. Composition change of Jatropha seed cake samples was evaluated upon lime pretreatment at 100 oC with different parameters. With a lime dose of 0.2 g and a water content of 10 ml per gram of cake and a treatment period of 1 h, 38.2 ± 0.6% of lignin was removed. However, 65 ± 16% of hemicellulose was also lost under this condition. For all the treatments tested, cellulose content was not affected by lime supplementation. Through further examining total reducing sugar (TRS) release by enzymatic hydrolysis after lime pretreatment, results indicated that 0.1 g of lime and 9 ml of water per gram of cake and 3 h pretreatment produced the maximal 68.9% conversion of cellulose. Without lime pretreatment, the highest cellulose conversion was 33.3%. Finally, this study shows that Jatropha seed cake samples could be hydrolyzed by enzymes. Even though the cellulose content was not high for this Jatropha cake sample, the fractionation by lime presented in this study opened the door for other applications, such as removal of lignin and toxicity for use as animal feed and fertilizer. The microwave radiation pretreatment of SSB was evaluated with or without lime (0.1 g/g bagasse) at 10 ml water/g bagasse for 4 min. TRS release over 72-h enzymatic hydrolysis was different for samples treated differently and at different solid loadings. The TRS concentration was increased by 2 and 5-fold from 0 to 24 hours in non lime-pretreated and lime-pretreated samples, respectively. Further incubation of samples for 48 and 72 h did not result in increased TRS. Comparing different solid loadings of samples treated with or without lime, 1% solid content resulted in 1.4 times higher TRS increase than that of 5% solid concentration. Therefore, lime was effective in disintegrating lignocellulosic structures and making cellulose more accessible for saccharification. Higher solid loadings which can lead to higher sugar concentrations are desired for downstream biofuel production. But, as shown in this study, higher concentration of bagasse samples decreased rate of cellulose hydrolysis due to poorer mixing efficiency and hindrance to interactions between enzymes and solid materials. Thus, an optimal solid content needs to be determined for maximal cellulose hydrolysis and for preparing the hydrolysates for downstream processes, either bioethanol or lipid production.

Made available in DSpace on 2014-06-11T19:31:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-04-28Bitstream added on 2014-06-13T19:20:47Z : No. of bitstreams: 1 saito_im_dr_botfca.pdf: 984135 bytes, checksum: d68af5e5076154cc8f38f5517fb4da20 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Recentemente há uma crescente busca de maior utilização de resíduos agroindustriais visando recuperar substâncias e/ou materiais e deste modo aumentar a eficiência econômica dos processos de produção. Diversos processos são desenvolvidos para utilização desses materiais convergindo em compostos químicos e produtos finos. A aplicação de resíduos agroindustriais em bioprocessos é uma alternativa observada na forma de substratos, e uma ajuda para solucionar o problema da poluição nos processos de agroindustrialização. O farelo de mandioca é um resíduo sólido produzido nas agroindústrias que extraem o seu amido (fecularias) que contém alto teor de amido (60 a 70% em peso seco) que é descartado e causa problemas ao meio ambiente. A recuperação do amido residual por tratamento físico ou biológico do material que tem sido objeto de pesquisas visando a melhoria dos processos, com o objetivo de agregar valor à matéria prima consumida. A produção de fécula de mandioca gera cerca de 930 Kg de farelo com aproximadamente 85% de umidade para cada 1000 Kg de raiz processada apresentando uma concentração de amido residual de 60 a 75% em massa seca. A fim de se obter um melhor aproveitamento do amido residuário existente no farelo de mandioca, o presente trabalho desenvolveu uma metodologia adequada para produção de hidrolisado, rico em glicose originário de tratamento hidrotérmico com fluido subcrítico. O tratamento do farelo residuário originário de uma agroindústria com umidade de 85% foi realizado em reator agitado em bateladas de 2,0 Kg, utilizando como catalisador o ácido sulfúrico a temperatura de 140 a 170°C. Os melhores resultados foram obtidos para as seguintes condições de tratamento: tempo de processo 35 minutos; temperatura de 140°C; concentração do catalisador de 2,5 % (m/s); umidade de 90,5% e rotação de 50 rpm. As respostas foram:... / Recently there has been an increase search for a large utilization of agro-industrial residues based on recovering substances and/or materials to increase the economical efficiency of the production. Several processes were developed for production fine chemicals compounds and other products. The application of agro-industrial residues on bioprocesses is an alternative observed in the substract form, and also a help to solve the pollution problem on the processes of agro-industrialization. The cassava waste is a solid residue produced in agro-industry that extracts its starch which has a high starch content which is wasted and causes environmental problems. The recovery of these residual starch by physical or biological treatment of the material require research aiming the optimization of the chosen processes, as well as for aggregating value to the consumed raw material. The cassava starch production generates about 930 Kg of solid waste with approximately 85% of moisture to each 1000 Kg of processed root, presenting a concentration of starch residue from 60 to 70% on a dry weight. In order to obtain a better use of the residual starch present on the cassava waste, in the present work an optimized methodology for hydrolysate production, was developed with production of a glucose rich solution from a hydrotermic treatment with subcritical fluid. The residual waste treatment originated from an agro-industry with 85% of moisture was performed in a stirred reactor with agitator in amounts of 2 Kg using sulphuric acid as a catalyst at temperatures between 140 and 170°C. The optimum results were obtained to the following treatment conditions: ...(Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:24:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-27Bitstream added on 2014-06-13T19:31:25Z : No. of bitstreams: 1 morais_aps_me_botfca.pdf: 687506 bytes, checksum: baad0742c0a39bca3a4a2b5908c9cedf (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O bioetanol é um combustível produzido por meio da fermentação do açúcar do caldo da cana, que representa apenas um terço do carbono (energia), presente na planta. Atualmente, estão sendo feitos esforços para o aproveitamento do restante da biomassa, divididos entre as folhas e bagaço do colmo. Esta biomassa lignocelulósica poderia ser aproveitada para produção de etanol, desde que submetida a processos hidrolíticos químicos (ácidos e bases) e enzimáticos gerando açúcares fermentescíveis. Pela fermentação alcoólica é possível a produção de etanol a partir da mistura de açúcares liberados. Neste trabalho procurou-se a padronização de procedimentos para avaliar o pré-tratamento físico e químico do bagaço da cana-de-açúcar, aliado a diferentes tratamentos térmicos a partir de duas granulometrias de bagaço (1,1 e 2,5 mm). Para o delineamento experimental, utilizou-se tratamentos ajustados em fatorial 4 X 5, sendo que as variáveis foram a influência do tempo de pré-tratamento (0, 15, 30, 45 e 60 minutos) e concentração de ácido sulfúrico (H2SO4) a 7 e 9%. Estes fatores exerceram influência sobre o desempenho da pré-hidrólise, medido pela liberação açúcares redutores (AR) na solução e a % de espécies químicas no bagaço prétratado / Bioethanol fuel is produced through the fermentation of sugar cane juice, which representes only a third of the carbon (energy) present in the plant. Currently, efforts are being made to the use of the remaining biomass, divided among the leaves and seed stalk. This lignocellulosic biomass could be used for ethanol production, provided that undergo hydrolytic process chemicals (acids and bases) and enzymatic generating fermentable sugars. For fermentation is possible to produce ethanol from mixed sugars released. This research is the standardization of procedures to assess the pre-treatment physical and chemical properties of bagasse from sugar cane, coupled with different thermal treatments from two particle sizes of mulch (1,1 and 2,5 mm). For this experiment, we used adjusted treatments in a factorial 4 x 5, and the variables were the influence of time of pretreatment (0, 15, 30, 45 and 60 minutes) And concentration of sulfuric acid (H2SO4) 7 and 9%. These factors have exerted influence on the performance of pre-hydrolysis, measured by the release sugars (RS) in the solution, and% of chemical species in the pretreated bagasse

Normalmente, a causa da toxicidade do efluente de uma estação de tratamento de esgotos não é conhecida. Devido à complexidade dos efluentes não domésticos por ela recebidos, um programa de redução de toxicidade deve ser implementado, contemplando desde a descarga no corpo d?água até a fonte de poluição. A identificação do(s) poluente(s) que causa(m) impacto na estação e o mecanismo de toxicidade, tipicamente, não são simples de serem determinados a partir de dados de caracterização tradicional de águas residuárias. Além disso, a toxicidade pode estar ligada à presença de compostos refratários ou sub-produtos gerados na própria estação de tratamento de esgotos. Devido ao efeito sinérgico dos poluentes, a metodologia dos limites locais nem sempre pode ser aplicada. Desta forma, o presente trabalho foi desenvolvido com o objetivo de determinar as classes de poluentes presentes na água residuária de uma indústria química, que causa toxicidade e inibição na estação de tratamento de esgotos de Suzano (ETE Suzano), sistema convencional de lodos ativados, localizada na Região Metropolitana de São Paulo, e propor, a partir dos resultados obtidos, tecnologias de pré-tratamento para o referido efluente. Ensaios de fracionamento da água residuária industrial seguidos de testes de avaliação da toxicidade refratária modificados por Ferraresi (2001) foram realizados, utilizando-se um reator que simulava as condições atuais da estação e outros dois que avaliavam a condição futura, nas condições críticas de máxima vazão do efluente industrial pré-tratado e mínima da ETE-Suzano. Os resultados dos ensaios de fracionamento indicaram que compostos voláteis, adsorvíveis e material particulado eram os principais responsáveis pela inibição à nitrificação e pela toxicidade conferida ao efluente final da ETE-Suzano. Desta forma, arraste com ar, adsorção em carvão e coagulação, floculação e sedimentação ou flotação com ar dissolvido são tecnologias que podem ser utilizadas como pré-tratamento do efluente industrial estudado. / Typically, the cause of effluent aquatic toxicity is unknown. Due to this and the complex makeup and varying toxicants in individual waste streams, a toxicity reduction program must usually begin at the discharge and proceed upstream to find the source. The key toxicant(s) and the actual toxic mechanism are normally not apparent from traditional characterization data and may consist of refractory organics or by-products generated in biological treatment process. Due to the complex interaction between toxicants, threshold concentration for specific compounds cannot be universally applied. The research work was carried out to identify classes of pollutants from chemical industry effluent that causes toxicity and inhibition at Suzano Wastewater Treatment Plant (Suzano WTP), a conventional activated sludge process, located at São Paulo Metropolitan Region and to suggest based on results, pretreatment technologies to this wastewater. Methods applied in effluent treatment scenarios (e.g., air stripping, powdered carbon, coagulation, flocculation and sedimentation) and Refractory Toxicity Assessment (RTA) according Ferraresi (2001) were used. A reactor simulated the real condition of Suzano WTP and two others, future condition with maximum flow rate of treated industrial wastewater and minimum flow rate of sewage. Volatile compounds, adsorbed and particulate material present in industrial effluent caused inhibition in activated sludge process and effluent WTP toxicity. Thus, air stripping, carbon adsorption and coagulation, flocculation and sedimentation or air dissolved flotation were efficient technologies to pretreat this industrial effluent.

Neste trabalho, fibras de eucalipto pré-tratadas diferentemente com ácido e/ou enzima foram investigadas visando se verificar alterações estruturais decorrentes dos pré-tratamentos. Na caracterização foram empregadas técnicas de microscopia eletrônica, de fluorescência (campo de claro), de fluorescência confocal de varredura a laser (CLSM) e de tempo de vida de imagem de fluorescência (FLIM). Além disso, foi também empregada espectrocopia de fluorescência, absorção no ultravioleta e vísivel (UV-Vis), no infravermelho (FT-IR) e ressonância magnética nuclear (RMN). Para a caracterização das fibras via microscopia e espectroscopia de fluorescência foi necessário a produção de filmes de fibras de eucalipto. Os resultados demonstraram um aumento na intensidade da fluorescência das fibras em função dos pré-tratamentos. As imagens de microscopia permitiram conhecer a estrutura morfológica das fibras. Para aumentar a fluorescência das fibras e facilitar a visualização por microscopia de fluorescência, nós depositamos o polieletrólito poli(cloreto de tetraidrotiofeno de xililideno) (PTHT), precursor do poli(p-fenileno vinileno) (PPV). Pelas imagens CLSM observou-se a afinidade do PTHT-PPV pelas as fibras que, foi investigada pelo método arraste. A distribuição de tempos de vida associado à imagem FLIM da fibra de eucalipto indicou a presença do PTHT-PPV nas superfícies da fibra e a lignina no interior da parede da mesma. O PTHT-PPV apresentou um tempo de decaimento de fluorescência mais curto do que a lignina. As análises composicionais das fibras de eucalipto e do bagaço de cana-de-açúcar in natura evidenciaram sinais relacionados à lignina, hemicelulose e celulose. / In this dissertation, eucalyptus fibers pretreated with different acid and / or enzyme were investigated in order to study the resulting structural changes of the fibers. The characterization was made using several microscopy techniques such as, electronic microscopy, confocal laser scanning microscopy (CLSM) and fluorescence lifetime imaging microscopy (FLIM). In addition, we used fluorescence spectroscopy, absorption in the ultraviolet and visible (UV-Vis), infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR). It was necessary to produce films of eucalyptus fibers for the characterization of fibers via microscopy and fluorescence spectroscopy. The results showed changes in the fluorescence intensity of the fibers as function of the pre-treatments. The microscopy images were used to study the morphological structure of fibers. To increase the fluorescence of the fibers and facilitate its visualization via fluorescence microscopy, e deposited a polyelectrolyte precursor of PPV poly(p-phenylenevinylene), the poly-(xylylidenetetrahydro-thiophenium chloride). CLSM images revealed an affinity between the PTHT-PPV and the fibers. This affinity was investigated by a method consisting of spinning a solution containing the fibers and subsequent optical analysis. The results confirmed the affinity of PTHT-PPV with eucalyptus fibers. The lifetime distribution associated with the FLIM image of eucalyptus fiber indicated the presence of PTHT-PPV on the fiber surface and lignin in the inner wall of the fibers. Also, the PTHT-PPV showed fluorescence decay time shorter than the lignin. The analysis of the composition of eucalyptus fibers, we used sugar cane bagasse in natura to facilitate the identification of functional groups. The results showed signs related to lignin, cellulose and hemicellulose.

Doctor of Philosophy / Department of Biological & Agricultural Engineering / Donghai Wang, Scott Bean / The performance of soybean hulls and forage sorghum as feed stocks for ethanol production was studied. The main goal of this research was to increase fermentable sugars' yield through high-efficiency pretreatment technology. Soybean hulls are a potential feedstock for production of bio-ethanol due to their high carbohydrate content ([approximately equals]50%) of nearly 37% cellulose. Soybean hulls could be the ideal feedstock for fuel ethanol production, because they are abundant and require no special harvesting and additional transportation costs as they are already in the plant. Dilute acid and modified steam-explosion were used as pretreatment technologies to increase fermentable sugars yields. Effects of reaction time, temperature, acid concentration and type of acid on hydrolysis of hemicellulose in soybean hulls and total sugar yields were studied. Optimum pretreatment parameters and enzymatic hydrolysis conditions for converting soybean hulls into fermentable sugars were identified. The combination of acid (H[subscript]2SO[subscript]4, 2% w/v) and steam (140 °C, 30 min) efficiently solubilized the hemicellulose, giving a pentose yield of 96%. Sorghum is a tropical grass grown primarily in semiarid and dry parts of the world, especially in areas too dry for corn. The production of sorghum results in about 30 million tons of byproducts mainly composed of cellulose, hemicellulose, and lignin. Forage sorghum such as brown midrib (BMR) sorghum for ethanol production has generated much interest since this trait is characterized genetically by lower lignin concentrations in the plant compared with conventional types. Three varieties of forage sorghum and one variety of regular sorghum were characterized and evaluated as feedstock for fermentable sugar production. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM) and X-Ray diffraction were used to determine changes in structure and chemical composition of forage sorghum before and after pretreatment and enzymatic hydrolysis process. Up to 72% of hexose yield and 94% of pentose yield were obtained using "modified" steam explosion with 2% sulfuric acid at 140°C for 30 min and enzymatic hydrolysis with cellulase (15 FPU/g cellulose) and [Beta]-glucosidase (50 CBU/g cellulose).

Energy utilization is a determining factor for the standards of living around the world, and the current primary source of energy is fossil fuels. A potential source of liquid fuels that could ease the strain caused by diminishing petroleum resources is bioethanol. Effective exploitation of biomass materials requires a pretreatment to disrupt the lignin and cellulose matrix. The pretreatment utilized for this research was oxygen delignification, which is a standard process stage in the production of bleached chemical pulp. The model substrate utilized as a feedstock for bioethanol was recovered fibre. An analysis of the substrates digestibility resulted in a hexose yield of approximately 23%, which justified the need for an effective pretreatment. An experimental design was performed to optimize the delignification conditions by performing experiments over a range of temperature, caustic loadings, and reaction times. Equations were developed that outline the dependence of various response parameters on the experimental variables. An empirical model that can predict sugar concentrations from enzymatic hydrolysis based on the Kappa number, enzyme loading, and initial fibre concentration was also developed. A study of hydrolysis feeding regimes for untreated recovered fibre (87 Kappa), pretreated recovered fibre (17 Kappa), and bleached pulp (6 Kappa) showed that the batch feeding regime offers reduced complexity and high sugar yields for lower Kappa substrates. In order to evaluate the possibility of lignin recovery, the pH of delignification liquor was reduced by the addition of CO₂ and H₂SO₄, resulting in up to 25% lignin yield. An experiment that looked at effect of post-delignification fibre washing on downstream hydrolysis found that a washing efficiency of approximately 90% is required in order to achieve a hexose sugar yield of 85%. / Applied Science, Faculty of / Chemical and Biological Engineering, Department of / Graduate

The rapid accumulation of municipal solid waste is a significant environmental concern in our rapidly growing world. Due to its low cost, high energy recovery and limited environmental impact anaerobic digestion (AD) is a promising solution for stabilizing the organic fraction of municipal solid waste (OFMSW). Hydrolysis is often the rate-limiting step during AD of wastes with high solid content; this step can be accelerated by pretreatment of waste prior to AD. This thesis presents the results of alkaline pretreatment of OFMSW using NaOH and KOH. Four different pH levels 10, 11, 12 and 13 at two temperatures 23±1°C and 80±1°C were examined to study the effects of the pretreatment on (i) enhancing the solubility of the organic fraction of the waste, and (ii) enhancing the AD process and the biogas production. The effects on solubility were investigated by measuring changes in the soluble COD (SCOD) concentrations of pretreated wastes and the enhanced AD was investigated by measuring volatile solids (VS) destruction, total COD (TCOD) and SCOD removal in addition to biogas and methane production using biochemical methane potential (BMP) assay and semi-continuous laboratory reactor experiments. Pretreatment at pH 13 at 80±1°C demonstrated the maximum solubility for both NaOH and KOH pretreated samples; however the BMP analysis demonstrated that pretreatment at pH 12 at 23±1°C showed the greatest biogas yield relative to the removed VS for both chemicals. Thus pretreatment at pH 12 at 23±1°C using NaOH and KOH were examined using semi-continuous reactors at three different HRTs: 10, 15 and 20 days. Pretreatment demonstrated a significant improvement in the AD performance at SRTs of 10 and 15 days.