Recuperação e purificação de enzimas usando adsorção em leito expandido

Orientadores: Telma Teixeira Franco, Reginaldo GuirardelloTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia QuimicaResumo: O presente trabalho refere-se ao uso da técnica de adsorção em lejto expandido para recuperar e purificar enzimas. Aspectos fundamentais da adsorção em leito expandido são abordados, utilizando lisozima e soro albumina bovina como proteínas modelo, e as enzimas, gliceraldeído-3-fosfato desidrogenase de Saccharomyces cerevisae, xilanase (extrato comercial) e quitosanase produzida por Bacil/us cereus. A avaliação da influência do uso de dois distribuidores, poroso e do tipo prato perfurado, mostrou que o distribuidor do tipo prato perfurado favoreceu mais à adsorção em leito expandido e que os adsorventes Streamline@ SP e Streamline@ DEAE possuem uma ampla distribuição de tamanho de partículas favorecendo ao fenômeno de segregação (Capítulo 2 - Artigo publicado no IEX 2000 (Cambridge/UK)). O uso de um processo integrado para recuperar e purificar a enzima intracelular Gliceraldeído 3-fosfato desidrogenase mostrou-se bem sucedido. O desempenho hidrodinâmico e cromatográfico foi estudado usando um adsorvente de estrutura pelicular (Pellicular) e dois adsorventes porosos comerciais (Stream1ine e Macrosorb) . Os resultados mostraram que o adsorvente Pellicular apresentou as melhores propriedades hidrodinâmicas e cromatográficas. (Capítulo 3 - Artigo em parceria e que será submetido à periódico internacional). O estudo da influência do uso de uma altura do leito empacotado, de 0,050 m e de 0,075 m na ALE operando-se em 10% da curva de ruptura, mostrou que uma altura de 0,075 m foi mais eficiente. Para o sistema lisozima - Streamline@ DEAE o rendimento aumentou com o aumento da velocidade linear enquanto que para o sistema BSA - Streamline@ SP esse fato não foi observado. Neste caso, para o sistema BSA - Streamline@ SP, a transferência de massa parece ser limitada pela menor densidade de carga acarretando assim em menores valores de eficiência em 10% de ruptura. (Capítulo 4 - Artigo aceito para publicação no periódico Biosepara_ion). O estudo da influência do conteúdo de células na adsorção em leito expandido mostrou que quando foi utilizado o extrato com o conteúdo de 5% de células (peso úmido), em leito expandido, o desempenho da purificação foi comprometido. (Capítulo 5 - Artigo aceito para publicação no periódico Joumal of Chromatography A). A adsorção de quitosanase de um caldo de fermentação de Baci/lus cereus em leito empacotado permitiu à obtenção de um pico com um fator de purificação de 7,6 e uma recuperação de 67,4% que eluiu com 0,51 M de NaCL Valores muito próximos a estes foram obtidos com o uso da adsorção em leito expandido com ambos os caldos, bruto e clarificado. Eletroforese em gel de poliacrilamida (SDS­P AGE) realizada para o tubo que exibiu a maior atividade, quando o leito foi no modo expandido e com células, mostrou a presença de duas bandas. Este fato sugere três possibilidades, a existência de duas quitosanases, a presença de um proteína contaminante ou a existência de uma quitosanase com duas sub-unidades (dímera). Entretanto, grande parte dos contaminantes foram separados da quitosanase em uma única etapa. (Capítulo 6 - Artigo que será submetido à um periódico internacional)Abstract: This work deals with the application of Expanded Bed Adsorption (EBA) to recovery and purify enzymes. Model proteins, lisozyme and bovine serum albumin (BSA), as well as some enzymes, glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from baker's yeast, xylanase (from a commercial extract) and chitosanase from Bacil/us cereus were used to study the EBA background and application. The influence of two distributor (porous and perfurated plate) showed that the perfurated plate distributor was more favorable for the EBA. The Strearnline@ SP and the Strearnline@ DEAE adsorbents have a wide size distribution favourable for the segregation phenomena. (Chapter 2 - artic1e published in IEX 2000 (Carnbridge/UK». An integrated process was successful to recover and to purify an intracellular enzyme, Glyceraldehyde 3-Phosphate Dehydrogenase (G3PDH). Performance of the hydrodynamic and chromatographic properties using a pellicular adsorbent (Pellicular) and two porous commercial adsorbents (Stream1ine and Macrosorb) showed that the former presented the best hydrodynarnic and chromatographic properties. (Chapter 3 - paper that wiil be submmited to an international journal). The influence of two settled bed height, 0.050 m and 0.075m, respectively, in the EBA operating at 10% of the breaktrough curve, showed that the former was more efficient. For the lisozyme - Strearnline@ DEAE system yield increased with the increase of the velocity while for the BSA - Strearnline@ SP system this behaviour was not observed. In this case, mass transfer seems to be limited problably due to its lower charge density. (Chapter 4 - paper accepted for publication in the Bioseparation Journal). The influence of cell contents in the EBA using a 5% (wet weight) cell content showed that the purification perfonnance was hampered. (Chapter 5 ­paper accepted for publication in the Journal ofChromatography A). The chitosanase adsorption of fermentation broth from Bacillus cereus, in packed mode, showed a chitosanase peak that eluted with 0.51 M NaCl, in this case a 7.6-fold purification factor with 67.4% of activity recovery were obtained. Similar values were obtained for both c1arified and unc1arified fermentation broth using the bed in the expanded mode. Experiment in expanded mode showed that two bands were present in the peak showing the highest activity, this suggests three possibilities, the existence of two chitosanases, the existence of a contaminant protein or the existence of a chitosanase with two sub-units. However, it was possible to separate the chitosanase from the-main contaminants in just one step. (Chapter 6 - paper that will be submitted to an international journal)DoutoradoDesenvolvimento de Processos QuímicosDoutor em Engenharia Químic

Estudos dos fenomenos de mistura e segregação em leitos fluidizados de particulas polidispersas

Orientador: Katia TannousDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia QuimicaResumo: Neste trabalho são considerados os fenômenos de mistura e segregação em leitos fluidizados de partículas polidispersas (quaternárias), pertencentes aos grupos A e B da classificação de Geldart. A coluna de fluidização utilizada possuía um diâmetro interno de 0,92m e uma altura de 2 m. Foi analisada a influência dos fatores operacionais, como a velocidade de excesso (U-U IND. E) e o tempo de fluidização, bem como de projeto, representada pela fração de área livre do distribuidor, e das características dos constituintes da mistura (diâmetro médio da mistura/concentração de imergíves) no grau de mistura axial do leito. Foram utilizadas quatro misturas que possibilitaram o estudo dos fenômenos de mistura e segregação, em leitos de partículas de mesma ou de diferentes massas específicas. Foi efetuado um mapeamento radial do leito. Os resultados para os perfis axiais mostram que o uso de uma velocidade suficiente para fluidizar todas as partículas e de um distribuidor de maior área livre favorece a mistura em um curto intervalo de tempo. O aumento da velocidade de excesso melhora a mistura nos leitos, principalmente pela maior quantidade de bolhas presentes. Uma maior fração de área livre do distribuidor favorece a homogeneização do leito, reduzindo as zonas mortas próximo ao distribuidor. O aumento da concentração das partículas imergíveis desfavorece o processo de mistura. O mapeamento radial confirma que para a velocidade de fluidização completa as partículas estão bem misturadasAbstract: In this work the mixing and segregation phenomena of polydisperse particles (quaternaries) belonging to the A and B Geldart' s groups are investigated. The fluidization column have been constructed with a internal diameter of 0.92 m and height of 2 m. The influence of operating factors in the degree axial mixing of the bed, like excess velocity (U-'U IND. E¿) and fluidization time, as well as design, like distributor free area, and of the characteristcs of the constituents of mixture (mean diameter of mixture/jetsam concentration) were analised. Four mixtures that allowed the study of the phenomena of mixing and segregation in beds of same or different densities were used. A radial mapping of the bed was performed. The axial profile results show that the use of a velocity high enough for complete fluidization and the use of a distributor with large free area promote the mixing in a small time interval. The increment of excess velocity improves the mixing in the beds, mainly by larger quantity of bubbles. A larger free area improves the bed homogenezation, reducing the dead zones near the distributor. The increment ofjetsam concentration hampers the mixing process. The radial mapping confirm that for the velocity of complete fluidization the particles are well mixed.MestradoEngenharia de ProcessosMestre em Engenharia Químic

Otimização da hidrólise enzimática para produção de bioetanol por sacarificação e fermentação semi-simultâneas usando fibra de coco maduro

Alternative substrates to produce useful chemicals such as biofuel have been attractive, in particular, for cellulosic ethanol production. In this context, the objective of this work was optimized the synergistic mixture of enzymes and bioethanol production. The enzymes of Trichoderma reesei and crude enzyme extract from Lichtheimia ramosa were used in the hydrolysis of mature coconut fibre pretreated by sequential process of alkaline hydrogen peroxide (Alk-H2O2)-sodium hydroxide (NaOH). Furthermore, these enzymes and pretreated vegetable biomass were applied in the bioethanol production by Saccharomyces cerevisiae in semi-simultaneous saccharification and fermentation strategy (SSSF). Resulting in the yields and conversions of delignified mature coconut fibre into reducing sugars between 12.7-82.14% and 0.09-0.64 g reducing sugars/g dry biomass, respectively, with an initial hydrolysis rate at 12 h between 0.10-0.89 g/(L.h). Yields and conversions of delignified mature coconut fibre into glucose between 10.16-83.78% and 0.06-0.43 g glucose/g dry biomass, in that order, with an initial hydrolysis rate at 12 h between 0.03-0.35 g/(L.h). Bioethanol production by S. cerevisiae using delignified mature coconut fibre, enzymes from T. reesei and crude enzyme extract from L. ramosa resulted in the production of 4.62 g/L, yield of 0.41 g ethanol/g glucose and volumetric productivity of ethanol of 0.13 g/(L.h), respectively. The results showed synergistic effects between enzymes from T. reesei and crude enzyme extract from L. ramosa, without promoting inhibition in the alcoholic fermentation. Therefore, allowing to formulate an optimized enzymatic preparation aiming cellulosic ethanol production.Substratos alternativos para produzir produtos químicos úteis, como biocombustível, têm sido atraentes, principalmente para a produção de etanol celulósico. Neste contexto, o objetivo deste trabalho foi otimizar a mistura sinérgica de enzimas de Trichoderma reesei e extrato de enzima bruta de Lichtheimia ramosa para a hidrólise da fibra de coco maduro pré-tratado com peróxido de hidrogênio alcalino (Alk-H2O2) - hidróxido de sódio (NaOH) e produção de bioetanol por Saccharomyces cerevisiae em estratégia de sacarificação e fermentação semi-simultânea (SSSF). Resultando nas conversões e rendimentos de fibra de coco maduro deslignificada em açúcares redutores entre 12,0-82,3% e 0,09-0,64 g de açúcares redutores/g de biomassa seca, respectivamente, com uma taxa de hidrólise inicial em 12 h entre 0,10-0,89 g/(Lh) . E as conversões e rendimentos de fibra de coco maduro deslignificado em glicose entre 10,4-83,9% e 0,06-0,43 g de glicose/g de biomassa seca, nessa ordem, com uma taxa de hidrólise inicial em 12 h entre 0,03-0,35 g/(L.h). A produção de bioetanol por S. cerevisiae utilizando fibra de coco maduro deslignificado, enzimas de T. reesei e extrato de enzima bruta de L. ramosa resultou na produção, rendimento e produtividade volumétrica de etanol de 4,62 g/L, 0,41 g de etanol/g de glicose e 0,13 g/(Lh), respectivamente. Os resultados mostraram efeitos sinérgicos entre as enzimas de T. reesei e extrato bruto de L. ramosa, além de não promover inibição na fermentação alcoólica. Portanto, permitindo formular uma preparação enzimática otimizada visando a produção de etanol celulósico

EVALUATION OF ALKALINE PRETREATMENT ON THE ENZYMATIC HYDROLYSIS OF CARNAUBA STRAW RESIDUE

Carnauba (Copernicia prunifera) straw residue generated from production of its wax is rich in cellulose, thus showing a potential use in the production of second generation ethanol. However, the high lignin and hemicellulose load associated with cellulose makes it difficult the enzymatic attack, thus having the need of an adequate pretreatment of this material. The objective of this study was to optimize the enzymatic hydrolysis of carnauba straw residue, focusing on the alkaline biomass pretreatment. Therefore, NaOH solutions at concentrations of 1.0% (w/v) (PA1), 2.0% (w/v) (PA2), 3.0% (w/v) (PA3) and 4.0% (w/v) (PA4) were used. The chemical and physical characterization of natural and pre-treated carnauba straw were according to the NREL, and DRX and FTIR performed analyzes. The materials chemical characterization showed that all the used pretreatments were able to remove a significant amount of lignin and hemicellulose, which can improve the enzymes access, favoring the increase of cellulose conversion. In relation to DRX analysis an increase in crystallinity index happens reaching up to 55.15% after the pretreatment PA4, which may be associated to the removal of hemicellulose and amorphous lignin, related to cellulose. After a period of 96 hours of enzymatic hydrolysis, the PA4 pretreated residue showed the best performance with a cellulosic conversion of 78%. Spite of a slightly lower performance of the residue that presented higher cellulose conversion, the pretreated material PA2 is an alternative to reduce costs in the cellulosic ethanol production

PRESSURIZED WATER PRETREATMENT TO INCREASE SUGAR PRODUCTION FROM GREEN COCONUT

Pretreatment of lignocellulosic industrial waste is necessary to promote the cellulose accessibility. Thus, this study evaluated the production of green coconut sugars during pressurized hydrothermal pre-treatment and enzymatic hydrolysis of the pretreated biomass. Pretreatment of the green coconut shell was carried out at 70 °C, 150 bar and water flow rate of 1 mL/min for 4 h. Samples from the system output stream were analyzed by HPLC and Folin Ciocalteu method. The solid fraction was characterized (chemical composition, XRD and FTIR) and subjected to enzymatic hydrolysis. The liquid fractions of pretreatment provided a defined profile of released glucose, xylose, phenolic components and acetic acid over time. After 40 min, most of fermentable sugars were released, forming a liquor with a sugar content above 10 g/L. Post-treated biomass showed a reduction of cellulose (26 % to 17 %) and hemicellulose (23 % to 18 %) and an increase in lignin content (32 % to 44 %). Despite this new chemical composition of the biomass, the pretreated material had lower crystallinity indexes and modifications in its chemical groups. This favored cellulosic conversion from 15 % to 55 %, producing 63.5 % more glucose than the untreated green coconut shell during enzymatic hydrolysis. Thus, the pressurized pre-treatment favored the fermentable sugar production from the green coconut shell, and there is also the possibility of utilizing the sugars present in the pre-treatment liquor

Endoglucanase production by trichoderma reesei cultivated in solid state fermentation using lignocellulosic waste / Utilização de resíduos lignocelulósicos para a produção de endoglucanases por trichoderma reesei cultivado sob fermentação em estado sólido

Trichoderma reesei is a fungus that has been widely explored for its potential as cellulolytic enzyme producer and has diverse industrial applications. However, obtaining the enzymes is still considered a costly and, sometimes, inefficient process. This study aimed to produce endoglucanases by cultivating T. reesei (CCT-2768) in solid state fermentation, using cashew apple bagasse (CAB), sugarcane bagasse (SCB) and green coconut fiber (GCF) residues as substrates. The influence of moisture and pH on enzyme production was evaluated using a factorial design. T. reesei showed viability for producing endoglucanases in all of the three lignocellulosic residues tested, with maximum activity (2.29 ± 0.01 U/g) observed when cultivated in the SCB substrate and using moisture of 60% and pH 5.5. Thus, use of lignocellulosic residues proves to be a viable alternative for producing endoglucanases by cultivation of Trichoderma reesei, which contributes to the recycling of waste and the reduction of environmental impacts.

Bioactive properties and evaluation of the prebiotic potential of cashew apple fiber using Bifidobacterium Lactis / Propriedades bioativas e avaliação do potencial prebiótico da fibra do pedúnculo de caju utilizando Bifidobacterium Lactis

Agro-industrial byproducts possess biotechnological potential due to the presence of bioactive compounds. These have positive metabolic effects, such as the reduction of the risk of chronic diseases, inflammation, among others. The cashew apple is one of these byproducts and stands out for its levels of phenolic compounds, flavonoids and ascorbic acid, which provide high antioxidant capacity, in addition to being an excellent source of pectin, a dietary fiber that stimulates the growth of bifidobacteria. The aim of this study was to evaluate the bioactive characteristics and the in vitro prebiotic effect of processed cashew apple when subjected to Bifidobacterium lactis. Anaerobic fermentation was carried out in a shaker at 100 rpm, 37 °C for 24 hours, using processed cashew apple as the substrate, which was enriched with a nutrient solution containing mineral salts. The growth was estimated in colony-forming units (CFU/mL), during strategic intervals of fermentation times on selective bifidobacteria agar medium, and the medium’s pH was monitored. The prebiotic fructooligosaccharide (FOS) was used as a control. The cashew apple showed bioactive properties, with ascorbic acid values of 4.58 ± 0.00 mg/100 g, total phenolic compounds of 366.85 ± 3.43 mg GAE/100 g, flavonoids of 85.03 ± 4.15 mg CE/100 g and antioxidant capacity of 17.78 ± 0.20 µM TEAC/g. Plate counts showed small white colonies and complete regular edges. The growth of bifidobacteria in FOS was 10 Log CFU/mL-1 after 24 hours of fermentation, while in the cashew apple the growth was 8.8 Log CFU mL-1 after 12 hours of fermentation, with a reduction in pH medium (pH 5.08) that left it slightly more acidic than the standard (pH 5.98). Thus, it can be inferred that the cashew apple presents important functional properties, with possible benefits to human health

Bioethanol production by Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis from delignified coconut fibre mature and lignin extraction according to biorefinery concept

In search to increase the offer of liquid, clean, renewable and sustainable energy in the world energy matrix, the use of lignocellulosic materials (LCMs) for bioethanol production arises as a valuable alternative. The objective of this work was to analyze and compare the performance of Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis in the production of bioethanol from coconut fibre mature (CFM) using different strategies: simultaneous saccharification and fermentation (SSF) and semi-simultaneous saccharification and fermentation (SSSF). The CFM was pretreated by hydrothermal pretreatment catalyzed with sodium hydroxide (HPCSH). The pretreated CFM was characterized by X-ray diffractometry and SEM, and the lignin recovered in the liquid phase by FTIR and TGA. After the HPCSH pretreatment (2.5% (v/v) sodium hydroxide at 180 °C for 30 min), the cellulose content was 56.44%, while the hemicellulose and lignin were reduced 69.04% and 89.13%, respectively. Following pretreatment, the obtained cellulosic fraction was submitted to SSF and SSSF. Pichia stipitis allowed for the highest ethanol yield 90.18% in SSSF, 91.17% and 91.03% were obtained with Saccharomyces cerevisiae and Zymomonas mobilis, respectively. It may be concluded that the selection of the most efficient microorganism for the obtention of high bioethanol production yields from cellulose pretreated by HPCSH depends on the operational strategy used and this pretreatment is an interesting alternative for add value of coconut fibre mature compounds (lignin, phenolics) being in accordance with the biorefinery concept.Brazilian research funding agencies CNPq (Proc:470356/2011-1) and CAPES (Proc:BEX5951/11-9) for financial suppor