Microalgae surpass the lipid productivity of terrestrial plants by several folds. However, due to the high moisture content and rigidity of algal cell walls, extraction of lipids from algae is still a significant technological challenge. In this research, an attempt was made to develop an algal lipid separation process which is energy efficient and effective. Algal related research requires a unique set of knowledge in areas of algae culturing, measuring cell concentration, harvesting, cell rupturing and lipid quantification. The first section of this thesis focuses on the state of the art as well as knowledge gained during preliminary studies. The second section of this thesis focuses on selecting a suitable measurement technique for quantification of algal cell disruption induced by homogenization. The selected method, hemocytometry was used to measure the degree of algal cell disruption induced by homogenization. In the third section, various homogenization treatments were evaluated for determining the fraction of cells disrupted during the homogenization. Finally, lipid extraction efficiency of homogenized algae was evaluated using different extraction solvents under different homogenization conditions. Preliminary research concluded that using cell counting is the most suitable technique to measure the effect of high pressure homogenization on concentrated microalgae. It was observed that higher pressure and increased number of passes increase the degree of cell disruption. Concentrated, non stressed samples show best response to homogenization. Out of the three solvents used for solvent extraction, chloroform gave a higher extraction yield at low intensity homogenizations. However at harsher homogenization levels the advantage of chloroform was not significant. Lipid extraction efficiency increases with increased levels of homogenization. However, a significant increase in lipid yields was not detected beyond 20 000 psi and 2 passes of homogenization treatment.

Master of Science / Department of Biological & Agricultural Engineering / Lisa R. Wilken / Microalgae has potential as a biofuel feedstock and as a source of valuable bioproducts for a variety of food, feed, nutraceutical, and pharmaceutical industries. However, several challenges are associated with bioproduct extraction from microalgae. The complexity of microalgae cell necessitates use of energy intensive disruption methods but current chemical or mechanical techniques can degrade economically valuable bioproducts. Aqueous enzymatic extraction (AEE), is a non-solvent and environmentally friendly bio-product recovery method that provides an opportunity to design an integrated process for protein and oil fractionation while reducing industrial costs. Based on the mechanistic understanding of biomolecule distribution and compartmentation, an aqueous enzymatic treatment for the release of internally stored proteins and lipid bodies in wild type Chlamydomonas reinhardtii was developed. In this study, we optimized harvesting times that maximized lipid and protein yields in nitrogen depleted cultures of the microalgae Chlamydomonas reinhardtii. Furthermore, an aqueous enzymatic extraction (AEE) treatment was developed. First, four lytic enzymes were tested for their ability to permeate C. reinhardtii cell walls. After cells were permeable, another set of enzymes were tested for their ability to release internally stored bioproducts. Protein recovery and lipid characterization after enzymatic treatment indicated a 54% release of total soluble protein and a localization of lipids to the chloroplast. Additionally, the development of secondary enzyme treatment for chloroplast disruption achieved about 70% total lipids released into the supernatant. Taken together, results indicate the application of an enzymatic treatment scheme for protein and oil recovery as a promising alternative to traditional extraction processes.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Embora todas as etapas do processo de produÃÃo de biodiesel a partir de microalgas sejam essenciais, a separaÃÃo e a ruptura celular da biomassa sÃo particularmente importantes, uma vez que as tecnologias disponÃveis para este fim apresentam elevados custos, comprometendo a viabilidade do aproveitamento energÃtico. Este trabalho teve como objetivo apontar um sistema que conseguisse aliar a separaÃÃo da biomassa algal de efluente de sistemas de lagoas de estabilizaÃÃo à ruptura celular, como uma alternativa de prÃ-tratamento para maximizar a extraÃÃo de lipÃdios pelo mÃtodo modificado de Bligh e Dyer; assim como estudar preliminarmente a comunidade fitoplanctÃnica presente para comparaÃÃo dos rendimentos lipÃdicos. Um reator de eletroflotaÃÃo nÃo-convencional foi confeccionado para operar em batelada e em fluxo contÃnuo, utilizando-se eletrodos nÃo consumÃveis e baixa potÃncia elÃtrica. A metodologia proposta foi comparada com tÃcnicas tradicionalmente utilizadas para ruptura celular: autoclave, micro-ondas e ultrassom. Foram selecionadas seis estaÃÃes de tratamento de esgotos (ETE) com diferentes configuraÃÃes de lagoas de estabilizaÃÃo para anÃlise dos gÃneros fitoplanctÃnicos dominantes e do teor lipÃdico. Observou-se a separaÃÃo da biomassa, sendo obtida eficiÃncia de remoÃÃo de turbidez superior a 80% em 20 minutos de operaÃÃo do reator em batelada. Entre os mÃtodos de prÃ-tratamento tradicionais testados, o micro-ondas foi o que apresentou maior rendimento lipÃdico (33,7  5,3%), diferente estatisticamente da autoclave (15,4  2,26%) e ultrassom (13,3  2,96%). Para a eletroflotaÃÃo nÃo-convencional o rendimento lipÃdico foi de 24,8  7,05%, que nÃo apresentou diferenÃa estatÃstica quando comparado ao micro-ondas. Entretanto, quando os custos energÃticos foram considerados, a eletroflotaÃÃo nÃoconvencional apresentou um custo de 5,6 Wh.g-1, sendo menor que o obtido para o micro-ondas. Os gÃneros de microalgas que predominaram nos efluentes analisados pertenciam Ãs classes Chlorophyceae, Cyanophyceae e Euglenophyceae. Em uma anÃlise quantitativa geral, os efluentes de todas as estaÃÃes analisadas apresentaram rendimento lipÃdico entre 8,5 e 34,6%. Os efluentes das ETE Aquiraz e TupÃ-Mirim apresentaram os maiores teores lipÃdicos (28,4  6,3% e 23,1  3,6%, respectivamente), sem diferenÃa estatÃstica (p=0,12). Os efluentes das ETE Araturi e Tabapuà apresentaram os menores potenciais lipÃdicos, sem diferenÃa estatÃstica entre eles (14,3  5,9% e 15,6  4,9%, respectivamente, p=0,68). A eletroflotaÃÃo nÃo-convencional mostrou-se como uma metodologia promissora para separaÃÃo e ruptura das cÃlulas de microalgas de efluentes de lagoas de estabilizaÃÃo, cujos potenciais lipÃdicos assemelharam-se aos obtidos a partir de biomassa microalgal cultivada em processos convencionais. AlÃm disso, a metodologia desenvolvida neste trabalho soma um motivo para a separaÃÃo e utilizaÃÃo da biomassa algal de lagoas de estabilizaÃÃo evitando, com isso, seu lanÃamento nos corpos dÂÃgua e garantindo um ganho econÃmico e ambiental, uma vez que visa o aproveitamento da biomassa para a produÃÃo de biocombustÃvel, ao passo que minimiza impactos ambientais decorrentes da presenÃa elevada desse material no ambiente. / Although all stages on the production of biodiesel from microalgae cells are essential, harvest and cell disruption biomass are particularly important, since the available technologies for this purpose present high cost, compromising energy recovery viability. This study aimed to develop a system that could combine separation of algal biomass from wastewater stabilization pond systems to cell disruption as an alternative of pretreatment to maximize total lipids extraction by the modified method of Bligh and Dyer, as well as perform a preliminary study of the phytoplankton for comparison of lipid content. A non-conventional electroflotation reactor was designed to operate in batch and continuous flow, using non-consumable electrodes and low electrical power. The proposed methodology was compared to traditional techniques used for cell disruption: autoclave, microwave and ultrasound. Six Wastewater Treatment Plant (WTP) with different pond configuration were selected for analysis of the dominant phytoplankton genera and lipid content. Biomass separation was achieved, with turbidity removal efficiency exceeding 70% in 20 minutes of operation with the batch reactor. Among the traditional pretreatment methods tested, the microwave showed the highest lipid yield (33.7  5.3%), followed by autoclave (15.4  2.26%) and ultrasound (13.3  2.96%). For non-conventional electroflotation methodology, lipid yield was 24.8  7.05%, which showed no statistical difference when compared to the microwave method. However, when energy cost was considered, non-conventional electroflotation presented a cost of 5.6 Wh.g-1, which was smaller than that obtained by the microwave method. The predominant microalgae genera in the analyzed effluents belonged to the classes Chlorophyceae, Cyanophyceae and Euglenophyceae. In a general quantitative analysis, the effluents from the analyzed ponds presented lipid yield between 8.5 and 34.6%. Effluents of the WTP Aquiraz and TupÃ-Mirim achieved the highest lipid content (28.4  6.3% and 23.1  3.6%, respectively), with no statistical difference (p = 0.12). Effluents of the WTP Araturi and Tabapuà achieved the lowest lipidic potential, with no statistical difference (14.3  5.9% and 15.6  4.9%, respectively, p = 0.68). Non-conventional electroflotation proved to be a promising methodology for harvesting and cell disruption of microalgae provided by effluent stabilization ponds, whose potential lipid resembled those obtained from microalgal biomass cultivated in conventional processes. Furthermore, the methodology developed in this work adds a reason for the harvesting and utilization of algal biomass from waste stabilization ponds avoiding, thus, its release into water bodies and ensuring an environmental and economic gain, since it aims the use of biomass for biofuel production, while minimizing environmental impacts due to elevated presence of this material on the environment.

Achieving microbial compliance levels in biosolids storage is complicated by the unpredictable increase of Escherichia coli (E. coli), which serves as an important indicator for pathogen presence risk. Meeting required microbial specifications validates sludge treatment processes and ensures that a safe product is applied to agricultural land. Controlled indicator monitoring provides confidence for farmers, retailers and the food industry, safeguarding the sludge-to-land application route. Following mechanical dewatering biosolids products are stored before microbial compliance testing permits agricultural application. During storage, concentrations of E. coli bacteria can become elevated and prevent the product from meeting the conventional or enhanced levels of treatment outlined in The Safe Sludge Matrix guidelines. Literature research identified innate characteristics of sludge and ambient environmental parameters of storage which are factors likely to influence E. coli behaviour in stored biosolids. The research hypothesis tested whether E. coli growth and death in dewatered sewage sludge can be controlled by the modification of physical-chemical factors in the cake storage environment. Parameters including nutrient availability, temperature, moisture content and atmospheric influences were investigated through a series of laboratory-scale experiments. Controlled dewatering and the assessment of modified storage environments using traditional microbial plating and novel flow cytometry analysis have been performed. At an operational scale, pilot trials and up-scaled monitoring of the sludge storage environment have been conducted enabling verification of laboratory results. Understanding the dynamics of cell health within the sludge matrix in relation to nutrient availability has provided a valuable understanding of the mechanisms that may be affecting bacterial growth post-dewatering. The importance of elevated storage temperatures on E. coli death rates and results showing the benefits of a controlled atmosphere storage environment provide important considerations for utilities.

The obligate stem parasite Cuscuta pentagona is able to take up host plant mRNA through a specialized organ known as the haustorium. Direct cell-to-cell symplastic connections between two different organisms are rare, and the translocation mechanisms and fate of these RNAs in the parasite is not understood. To characterize this phenomenon, mobile Arabidopsis and tomato mRNAs were identified from microarray and transcriptome sequencing projects and quantified in the host-parasite system. Mobile RNAs were quantified using real time (qRT)-PCR and were found to vary substantially in their rate of uptake and distribution in the parasite. Transcripts of tomato Gibberellic Acid Insensitive (SlGAI) and Cathepsin D Protease Inhibitor (SlPI) can be traced over 30-cm of parasite stem. SlPI was abundant in the C. pentagona stem, but the number of copies decreased substantially within the first eight hours post detachment. Additional studies of mobile RNAs from Arabidopsis, Translationally Controlled Tumor Protein (AtTCTP), Auxin Response Factor (AtARF) and a Salt-inducible Zinc Finger Protein (AtSZFP) supported the idea that mRNA molecules differ in their mechanisms of uptake and mobility between host and parasite. Known phloem-mobile RNAs (SlGAI and AtTCTP) have uptake patterns that differ from each other as well as from other RNAs that are not reported to be phloem mobile (SlPI and AtSZF1). The function of RNAs in plants extend beyond protein translation to include post transcriptional gene silencing or long distance signaling, and mobile RNA in C. pentagona systems offers novel insights into this aspect of plant biology. Studies of cell-to-cell trafficking of RNAs and other macromolecules would be facilitated by the ability to manipulate individual cells. To this end, work was initiated to explore alternative approaches to understanding single cell biology using laser-mediated approaches. Optoperforation, or the use of multiphoton processes to form quasi-free electron plasmas to initiate transient pore formation in plasma membranes, has been demonstrated, but not in cells of an intact plant. This work details a protocol for optoperforation of Arabidopsis epidermal cells to allow for uptake of external dye-labeled dextrans and retention for up to 72 hours, and has the potential for transformation and molecular tagging applications. / Ph. D.

Le concept d’une algoraffinerie primaire traitant les principaux composants de microalgues (lipides, protéines, glucides et pigments) a été étudié. Une séquence d'opérations unitaires a été mis en œuvre afin d'obtenir des fractions enrichies de ces biomolécules tout en conservant leur integrité dans le procédé en aval. L'étude a été principalement centrée sur Chlorella vulgaris, une espèce connue pour sa paroi cellulaire rigide. La majorité de la fraction lipophile (lipides et pigments) a été récupérée en utilisant du dioxyde de carbone supercritique avec de l'éthanol en tant que co-solvant, sans opération unitaire de cassage cellulaire préalable. La fraction hydrophile (protéines et polysaccharides) a été récupérée dans la phase aqueuse après broyage à billes comme méthode de cassage cellulaire. Par la suite, la phase aqueuse a été séparée en trois fractions par un procédé d'ultrafiltration en deux étapes. Ainsi, les amidons, les pigments, les protéines et les sucres ont été séparés les uns des autres avec succès. Une analyse du cycle de vie sera nécessaire pour estimer le coût et la durabilité du procédé de fractionnement. / A primary algorefinery, concept that deals with the main components of microalgae (lipids, proteins, carbohydrates and pigments), has been studied. A sequence of unit operations has been implemented in order to obtain separated enriched fractions of these biomolecules by conserving their integrity in the downstream process. The study was mainly centred on Chlorella vulgaris, a species known for its rigid cell wall. Most of the lipophilic fraction (lipids and pigments) was recovered using supercritical carbon dioxide with ethanol as a co-solvent, without a preliminary unit operation of cell disruption. The hydrophilic fraction (proteins and polysaccharides) was recovered in the aqueous phase after bed milling as cell disruption method. Subsequently, the aqueous phase was fractionated into three fractions by means of a process of two-stage ultrafiltration. Thus, starches, pigments, proteins and sugars were successfully separated from each other. A life cycle assessment will be necessary to estimate the cost and the sustainability of the fractionation process

Microbial cell disruption using pressurized gas is a promising approach to improve the lipid extraction yield directly from the wet biomass by eliminating the energy-intensive drying process, which is an integral part of traditional methods. As the process starts with the solubilization of the gas in lipid-rich microbial cells, it is important to understand the solubility of different potential gases in both lipid (triglyceride) and lipid-rich microbial cell culture to design efficient cell disruption processes. In this study, we determined the solubility of different gases (e.g., CO2, CH4, N2, and Ar) in canola oil (triglyceride) using a pressure drop gas apparatus developed in our laboratory. The solubility of different gases in triglyceride followed the trend CO2 > CH4 > Ar > N2. Since the solubility of CO2 was found to be higher compared to other gases, the solubility of CO2 in lipid rich cell culture, cell culture media, and spent media was also determined. It was found that CO2 is more soluble in triglycerides, but less soluble in lipid-rich cell culture compared to CO2 in water. From both thermodynamic models and Monte Carlo simulations, the correlated solubility was found to be in good agreement with the experimental results. CO2 was found to be the most suitable gas for microbial cell disruption because almost 100% cell death occurred when using CO2 whereas more than 85% cells were found to be active after treatment with CH4, N2, and Ar. The optimization of microbial cell disruption was conducted using the combination of Box-Behnken design of experiment (DOE) technique and response surface methodology. The optimized cell disruption conditions were found to be 3900 kPa, 296.5 K, 360 min, and 325 rpm where almost 100% cell death was predicted from the statistical modeling. Finally, it was found that 86% of the total lipid content can be recovered from the wet biomass after treatment with pressurized CO2 under optimized conditions compared to control where up to 74% of the total lipid content can be recovered resulting in 12% increase in the lipid extraction yield using pressurized CO2.

Submitted by Morgana Andrade (morgana.andrade@ufes.br) on 2016-04-11T20:49:17Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação_Jean.pdf: 1797995 bytes, checksum: b603b8c2f1c6e995111923a3d87928d5 (MD5) / Approved for entry into archive by Patricia Barros (patricia.barros@ufes.br) on 2016-05-16T14:48:42Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação_Jean.pdf: 1797995 bytes, checksum: b603b8c2f1c6e995111923a3d87928d5 (MD5) / Made available in DSpace on 2016-05-16T14:48:42Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação_Jean.pdf: 1797995 bytes, checksum: b603b8c2f1c6e995111923a3d87928d5 (MD5) / Financiadora de Estudos e Projetos - FINEP, CNPq, CAPES, FAPES / A meleira, causada pelo Papaya meleira virus (PMeV), é uma doença importante no Brasil e no México devido as grandes perdas econômicas que ela causa. Estudos para avaliar a interação patógeno-hospedeiro em nível histológico são fundamentais para compreender os mecanismos responsáveis pela resistência natural das plantas. Neste estudo foram coletadas folhas de mamoeiros saudáveis e sintomáticos para meleira e observou-se a topografia e a dureza da parede celular dessas plantas por microscopia de força atômica (AFM). As imagens em duas dimensões obtidas a partir de diferentes áreas da parede celular mostraram que as paredes celulares das plantas saudáveis são mais uniformes do que as paredes de plantas doentes. As plantas saudáveis também apresentaram características constitutivas da parede celular mais elevadas do que as de plantas doentes e a média da força de adesão máxima observada foi maior em plantas saudáveis do que em plantas doentes. Estes resultados indicam que o PMeV promove alterações nas paredes das células, tornando-as mais frágeis e suscetíveis à ruptura. Estas alterações, associadas ao aumento da captação de água e aumento da pressão interna dos laticíferos, provocam o rompimento celular que leva à exsudação espontânea do látex e facilita a disseminação de PMeV para outros laticíferos. Os resultados deste trabalho fornecem novas percepções sobre a interação mamoeiro-PMeV que podem revelar-se úteis no que se refere a entender e controlar a meleira do mamoeiro. / Papaya sticky disease, caused by Papaya meleira virus (PMeV), is an important papaya disease in Brasil and Mexico due the severe economic losses it causes. Studies to assess the pathogen-host interaction at a histological are fundamental in order to understand the mechanisms that underlie natural resistance. In this study we collected leaves of healthy and symptomatic papaya sticky diseased plants and observed the topography and mechanical properties of plant cell walls by atomic force microscopy (AFM). Two-dimensional images obtained from different areas of the cell wall showed that the cell walls of healthy plants are smoother than the walls of sticky diseased plants. Also healthy plants displayed higher constitutive characteristics of the cell wall than diseased plants and the average maximum adhesion force was higher on healthy plants than on diseased plants. PMeV promotes changes on cell walls, making them more fragile and susceptible to breakage. These changes, associated with increased water uptake and internal pressure of laticifers causes cell disruption that leads to spontaneous exudation of latex and facilitates the spread of PMeV to other laticifers. The results of this work provide new insights on the interaction papaya-PMeV which could prove helpful when trying to understand and control the papaya sticky disease.

The techniques of optoperforation, spectral characterization of alloy disorder, and metal-enhanced fluorescence were applied to previously unconsidered or disregarded systems in order to demonstrate that such applications are both feasible and consequential. These applications were the subject of three disparate works and, as such, are independently discussed. Despite being ostensibly restricted to mammalian cells, optoperforation was demonstrated in intact plant cells by means of successful femtosecond-laser-mediated infiltration of a membrane impermeable dextran-conjugated dye into cells of vital Arabidopsis seedling stems. By monitoring the rate of dye uptake, and the reaction of both CFP-expressing vacuoles and nanocellulose substrates, the intensity and exposure time of the perforating laser were adjusted to values that both preserved cell vitality and permitted the laser-assisted uptake of the fluorophore. By using these calibrated laser parameters, dye was injected and later observed in targeted cells after 72 hours, all without deleteriously affecting the vital functions of those cells. In the context of alloy disorder, photoluminescence of excitonic transitions in two InAsxP1-x alloys were studied through temperature and magnetic field strength dependencies, as well as compositionally-dependent time-resolved behavior. The spectral shape, behavior of the linewidths at high magnetic fields, and the divergence of the peak positions from band gap behavior at low temperatures indicated that alloy disorder exists in the x=0.40 composition while showing no considerable presence in the x=0.13 composition. The time-resolved photoluminescence spectrum for both compositions feature a fast and slow decay, with the slow decay lifetime in x=0.40 being longer than that of x=0.13, which may be due to carrier migration between localized exciton states in x=0.40. In order to achieve broadband metal-enhanced fluorescence in upconverting NaYF4:Yb,Er nanocrystals, two nanocomposite architectures were proposed that retrofit metallic nanoshells to these lanthanide-doped nanocrystals. The typical monometallic construction was rejected in favor of architectures featuring Au-Ag bimetallic concentric surfaces, a decision supported by the considerable overlap of the calculated plasmon modes of the metallic structures with the emission and absorption spectrum of the nanocrystals. Furthermore, precursors of these nanocomposites were synthesized and photoluminescence measurements were carried out, ultimately verifying that these precursors produce the requisite upconversion emissions. / Ph. D.

A utilização de resíduos agrícolas visando à produção de insumos por via biotecnológica tem se mostrado importante uma vez que estes resíduos são fontes renováveis de carbono. A fração hemicelulósica destes resíduos apresenta como componente principal a xilose, que pode ser utilizada como substrato em processos de bioconversão para a obtenção de produtos com valor agregado. Um destes produtos é a enzima glicose-6-fosfato desidrogenase (G6PD), primeira enzima da via das pentoses fosfato que pode ser utilizada como reagente analítico em análises quantitativas, sobretudo em estudos bioquímicos e médicos. O presente trabalho visou estudar o processo de purificação dessa enzima empregando a extração em sistemas de duas fases aquosas convencional (sem integração) e integrado ao rompimento celular, em duas escalas, reduzida e ampliada. A enzima foi produzida por Candida guilliermondii FTI 20037 cultivada em meio constituído de hidrolisado hemicelulósico de palha de arroz, sob condições pré-determinadas. Inicialmente, foram realizados ensaios para avaliar o efeito das variáveis volume de suspensão celular, velocidade de agitação do moinho de esferas de vidro e tempo sobre o rompimento das células. Os valores destas variáveis foram, então, estabelecidos em: 100 mL, 400 rpm e 25 minutos, respectivamente. Posteriormente, a influência da massa molar de PEG e comprimento de linha de amarração sobre a extração da G6PD foram investigados no sistema convencional (homogeneizado obtido a partir do rompimento celular, em presença ou ausência de fragmentos) e integrado (rompimento na presença dos componentes da extração), empregando-se a metodologia do planejamento experimental. Nos ensaios realizados em escala reduzida, sob condições otimizadas, alcançou-se um fator de purificação na fase rica em sal (FPf), ou fase fundo, de 2,8 e em maior escala, ou seja, em moinho de rompimento, de 1,3. Com isso, realizou-se o estudo cinético e termodinâmico empregando a enzima presente no homogeneizado antes da purificação e após purificada no processo integrado em escala reduzida, nas seguintes condições: TLL 40% e PEG 1500 mol/L. Os valores determinados para os parâmetros cinéticos foram Km, 0,07 e 0,05 mM, Vm, 34,8 e 19,1 U/L e dos parâmetros termodinâmicos ΔG, -13,71 e -13,64 KJ/mol; ΔH, -2,49 e -2,50 KJ/mol; ΔS, 37,02 e 36,77 J/mol.K; Ea, 24,18 e 15,02 KJ/mol, da enzima presente no homogeneizado celular antes e após purificação, respectivamente. / The employment of agricultural residues aiming the attainment of biotechnological products has been shown its importance since these residues are renewable and low cost sources of carbon. The hemicellulosic fraction of these residues presents xylose as main component, which can be utilized as substrate for different bioconversion processes for the acquisition of high value products. As an example, glucose-6-phosphate dehydrogenase, the first enzyme of pentose phosphate pathway which can be used as analytical reagent in several quantitative analysis, mainly in biochemical and medical studies. The present work contemplated the study of glucose-6-phosphate (G6PD) purification process by a conventional aqueous two phase systems extraction and integrated with cell disruption, in two scales, reduced and increased. The enzyme was obtained from cells of Candida guilliermondii FTI 20037 grown in hemicellulosic rice straw hydrolysate, using conditions established in previous work. Initially, assays in bead mill were performed to determine the effect of cell suspension volume, agitation speed and time on cell disruption. The determined conditions were: 100 mL, 400 rpm and 25 minutes, respectively. After this, the influence of molar mass of PEG and tie line lenght (TLL) on the G6PD recovery were investigated in the conventional system (with previous disrupted cells, with or without cell fragments) and integrated (disruption in the presence of extraction components), using the experimental design methodology. In the reduced scale assays, in optimized conditions, a purification factor in salt rich phase (FPf), or bottom phase, of 2,8 was reached while in the increased scale, this means in bead mill, a FPf of 1,3 was attained. In addition, kinetic and thermodynamic studies were performed, employing the enzyme present in the homogenate before and after purification in reduced scale, in the following conditions: TLL of 40% and PEG 1500 mol/L. The established values for the kinetics parameters were Km, 0,07 and 0,05 mM, Vm, 34,8 and 19,1 U/L and of thermodynamics ΔG, -13,71 and -13,64 KJ/mol; ΔH, -2,49 and -2,50 KJ/mol; ΔS, 37,02 and 36,77 J/mol.K; Ea, 24,18 and 15,02 KJ/mol, of the enzyme present in the homogenate before and after purification respectively.