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Cells of Aspergillus oryzae IPT-301 rich in fructosyltransferase (FTase) were successfully immobilized by crosslinking with glutaraldehyde and used for the transfructosylation reaction of sucrose. The glutaraldehyde concentration and pH used in the immobilization process were optimized for maximizing the transfructosylation activity (AT) and minimizing the hydrolytic activity (AH). Also, the operational stability and the influence of temperature, pH and sucrose concentration on the enzymatic activities of the free and crosslinked cells were evaluated. Both the maximum AT and minimum AH were obtained for cells immobilized with glutaraldehyde concentration of 2.1% (v/v) and pH 7.9. Crosslinked cells showed considerably higher AT/AH ratio than free cells at several temperatures, pH and sucrose concentrations in the reaction media. Kinetics data suggested that crosslinked cells present higher substrate-enzyme affinity and transfructosylation rate than free cells. Furthermore, after 12 batch reaction cycles the FTase present in the immobilized cell kept 88.9% of its initial AT, demonstrating a considerably higher operational stability than the FTase present in the free cell, which showed 50.3% of its initial AT. These results suggest the potential use of crosslinked cells of Aspergillus oryzae IPT-301 for the large-scale production of fructooligosaccharides (FOS).

Extracellular fructosyltransferase (FTase, E.C.2.4.1.9) from Aspergillus oryzae IPT-301 was immobilized on silica gel by adsorption and biochemically characterized aiming at its application in the transfructosylation reaction of sucrose for the production of fructooligossaccarides (FOS). The transfructosylation activity (AT) was maximized by the experimental design in function of the reaction pHs and temperatures. The AT of the immobilized enzyme showed the kinetics behavior described by the Hill model. The immobilized FTase showed reuse capacity for six consecutive reaction cycles and higher pH and thermal stability than the soluble enzyme. These results suggest a high potential of application of silica gel as support for FTase immobilization aiming at FOS production.

Made available in DSpace on 2021-06-25T10:11:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Objective: Fructooligosaccharides (FOS) are prebiotic substances that have been extensively incorporated in different products of food industry mostly for their bifidogenic properties and economic value. The main commercial FOS production comes from the biotransformation of sucrose and intracellular and extracellular microbial enzymes–fructosyltransferases (FTase). Aspergillus oryzae IPT-301 produces FTase. In order to increase its production, this study focuses on evaluating the effects of different agitation speed and aeration rates which affect yields in a stirred tank bioreactor. Results: Agitation had more influence on cell growth than aeration. The maximum intracellular FTase activity and the volumetric productivity of total intracellular FTase were obtained at 800 rpm and 0.75 vvm, and reached values of 2100 U g−1 and 667 U dm−3 h−1, respectively. The agitation speed had a strong influence on the activity of extracellular FTase produced which reached the maximum amount of 53 U cm−3. The higher value of total activity obtained was 22,831 U dm−3 at 0.75 vvm and 800 rpm. Conclusion: Aeration rates and agitation speed showed strong influence upon the growth and production of fructosyltransferase from Aspergillus oryzae IPT-301 in media containing sucrose as carbon source. The control of aeration rate and agitation speed can be a valuable fermentation strategy to improve enzyme production. Bionanomanufacturing Center Institute for Technological Research (IPT), Av. Prof. Almeida Prado 532 Instituto Cubano de Investigaciones de los Derivados de la Caña de Azúcar (ICIDCA), Vía Blanca 804, Carretera Central, San Miguel del Padrón Institute of Science and Technology Federal University of Alfenas (ICT/UNIFAL-MG), José Aurélio Vilela Road, 11999 BR 267 Km 533 Biosciences Institute São Paulo State University (UNESP), Praça Infante Dom Henrique s/no Instituto de Estudos Avançados do Mar (IEAMar) São Paulo State University, Av. João Frascisco Bensdorp 1178 Biosciences Institute São Paulo State University (UNESP), Praça Infante Dom Henrique s/no Instituto de Estudos Avançados do Mar (IEAMar) São Paulo State University, Av. João Frascisco Bensdorp 1178

Made available in DSpace on 2020-12-12T01:35:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-10-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Abstract: Arabinanases from glycoside hydrolase family GH93 are enzymes with exo-activity that hydrolyze the α-1,5 bonds between arabinose residues present on arabinan. Currently, several initiatives aiming to use byproducts rich in arabinan such as pectin and sugar beet pulp as raw material to produce various compounds of interest are being developed. However, it is necessary to use robust enzymes that have an optimal performance under pH and temperature conditions used in the industrial processes. In this work, the first GH93 from the thermophilic fungus Thermothielavioides terrestris (Abn93T) was heterologously expressed in Aspergillus nidulans, purified and biochemically characterized. The enzyme is a thermophilic glycoprotein (optimum activity at 70 °C) with prolonged stability in acid pHs (4.0 to 6.5). The presence of glycosylation affected slightly the hydrolytic capacity of the enzyme, which was further increased by 34% in the presence of 1 mM CoCl2. Small-angle X-ray scattering results show that Abn93T is a globular-like-shaped protein with a slight bulge at one end. The hydrolytic mechanism of the enzyme was elucidated using capillary zone electrophoresis and molecular docking calculations. Abn93T has an ability to produce (in synergism with arabinofuranosidases) arabinose and arabinobiose from sugar beet arabinan, which can be explored as fermentable sugars and prebiotics. Key points: • Thermophilic exo-arabinanase from family GH93 • Molecular basis of arabinan depolymerization. Department of Biotechnology Lorena School of Engineering University of São Paulo Departamento de Física e Ciências Aplicadas Instituto de Física de São Carlos Universidade de São Paulo Departamento de Bioquímica Instituto de Biologia Universidade Estadual de Campinas Programa de Processos Tecnológicos e Ambientais Universidade de Sorocaba Instituto de Pesquisas Tecnológicas do Estado de São Paulo Diretoria de Operações e Negócios Núcleo de Bionanomanufatura Department of Physics – Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP) Department of Physics – Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP) FAPESP: 2014/06923-6 FAPESP: 2014/18714-2 FAPESP: 2017/00525-0 FAPESP: 2019/01165-0 CNPq: 2019/06663-8 FAPESP: 2019/06663-8 FAPESP: 2019/22284-7 CNPq: 302627/2018-9 CNPq: 443916/2014-4

In this work, it was determined the influence of temperature, pH and storage time on the enzymatic activity and stability of an extracellular fructosyltransferase (FTase E.C.2.4.1.9) from Aspergillus oryzae IPT-301 produced by submerged fermentation. The thermodynamic parameters showed a tendency for increasing enzyme denaturation with the rise in temperature. The maximum transfructosylation activity was obtained at the incubation pH 5.5. During storage at 4 °C, the transfructosylation activity decreased, whereas the hydrolytic activity increased, especially in the first nine hours, a time in which the enzyme presented 45.6% of its initial transfructosylation activity. These results contributed to the improvement of the conditions of storage, immobilization and use of the soluble fructosyltransferases (FTase) in fructooligosaccharide (FOS) production. Resumo Neste trabalho foi determinada a influência da temperatura, do pH e do tempo de armazenamento na estabilidade enzimática de frutosiltransferase (E.C.2.4.1.9) extracelular de Aspergillus oryzae IPT-301, produzida por fermentação submersa. Os parâmetros termodinâmicos mostraram tendência de aumento da desnaturação da enzima com o aumento da temperatura. A atividade de transfrutosilação máxima foi obtida para a enzima incubada em pH 5,5. Durante o armazenamento a 4 °C, a atividade de transfrutosilação diminuiu, enquanto a atividade hidrolítica aumentou, principalmente nas primeiras nove horas, período em que a enzima apresentou 45,6% de sua atividade inicial de transfructosilação. Esses resultados contribuem para a melhoria das condições de armazenamento, imobilização e utilização das frutosiltransferases solúveis na produção de frutooligossacarídeos.

BACKGROUND: Fructooligosaccharides (FOS) are sweeteners produced through the use of enzymes called fructosyltransferases (FTases) (EC 2.4.1.9). However, FTase's reuse is meager and the cost of the enzyme production is high, which turns the FOS production economically unfeasible. In order to lessen these issues, the study aims the immobilization of cells from Aspergillus oryzae IPT301, containing FTase enzyme previously glutaraldehydecrosslinked, by means of entrapment in calcium alginate beads. The conditions to obtain the particles, such as alginate type, mycelium mechanical dispersion, degree of crosslinking, size of the beads, and their reflections on transfructosylation activity (AT) were previously evaluated. RESULTS: The highest AT used low viscosity alginate solution (4.0% w/w) crosslinked with calcium chloride (0.2 molL1) and beads mean diameter of 2.15mm. The entrapment conditions were complemented by conducting the reaction under different temperatures, pH values, substrate concentrations and by reusing the beads. The highest transfructosylation activities were obtained under reaction conditions of 50 °C, pH 5.5, and substrate concentration of 400 gL1. CONCLUSION: The AT levels found with the glutaraldehydecrosslinked and entrapped cells in calcium alginate were about 70% higher than the ones obtained with free cells, not glutaraldehydecrosslinked. Moreover, the immobilized cells (crosslinked+entrapped) retained their AT up to the third cycle of total sucrose depletion in the reaction medium, attesting the potential use of the beads in continuous FOS production., The authors gratefully acknowledge The Foundation for Researchof the State of Minas Gerais–FAPEMIG (Process APQ-02131-14)and National Council for Scientific and Technological Develop-ment–CNPq (Process 421540/2018-4) for providing financial support., info:eu-repo/semantics/publishedVersion

The anthraquinone dye Remazol Brilliant Blue R is largely used in the textile industry. However, its removal from wastewaters is costly and complex. Many methods have been tested to solve this ecological problem, but there is still a need for efficient methods. We propose here an alternative use of a two-chambered microbial fuel cell (MFC), fuelled with domestic wastewater in the anodic chamber, to degrade a simulated textile dye effluent made of Remazol Brilliant Blue R inoculated with an immobilised fungal strain, Pleurotus ostreatus URM 4809, as a laccase producer, in the cathodic chamber. The MFC showed continuous synthesis of laccase in the cathodic chamber, which, in turn, promoted the rapid decolourisation, of more than 86\% of the textile dye effluent. The yield was further increased by the addition of glycerol. Electrochemical monitoring also indicated an increase in power density and current density. After 20Â days of MFC operation, 62.1\\% of organic matter was removed in the anodic compartment, thus leaving the effluent with a much lower toxicity., Authors would like to acknowledge the technician and fnancial support of Programa Novos Talentos provided by the Instituto de Pesquisa Tecnológica do Estado de São Paulo (IPT) and Instituto de Estudos Avançados do Mar (IEAMar)., info:eu-repo/semantics/publishedVersion

β-Fructofuranosidase production by Aspergillus oryzae IPT301 was maximized in shake flasks. Response Surface Methodology (RSM) involving Small Central Composite Design was adopted to evaluate the fructosyltransferase (FTase) activity by changing three medium component concentrations: sucrose, urea and yeast extract. The optimal set of conditions for maximum fructosyltransferase production was as follows: sucrose 320.5 g/L, urea 7.13 g/L and yeast extract 2.11 g/L. In this optimal condition, the following improvements were achieved: an increase of 48.8% in cell growth, 112% and 62% in micelial and free FTase activities, respectively, 62.8% in the ratio of fructosyltransferase/hydrolytic activities for enzyme linked to mycelium and 67.5% for free enzyme.

Fructooligosaccharides (FOS) are prebiotic substances found in several vegetable or natural foods. The main commercial production of FOS comes from enzymatic transformation of sucrose by the microbial enzyme fructosyltransferase. The development of more efficient enzymes, with high activity and stability, is required and this has attracted the interest of biotechnologists and microbiologists with production by several microorganisms being studied. This article reviews and discusses FOS chemical structure, enzyme characteristics, the nomenclature, producer microorganisms and enzyme production both in solid state fermentation and submerged cultivation.

This work aimed at the study of purification of biopolymers produced by Burkholderia cepacia IPT64, through a chemical attack like an adjuvant procedure through chemical and enzymatic route. Enzymatic lysis using protease was run on an enzyme/cell ratio of 0.02. Chemical attacks were performed as pre or post-treatment for enzymatic attacks, using sodium dodecyl sulphate or hydrogen peroxide. The same chemicals and procedures were used alone as a control. Fenton’s reagent was also tested as a chemical treatment. Using only one of the chemicals H2O2, Fenton’s reagent or SDS, the purity increase achieved values of 14%, 16% or 23%, respectively. When H2O2 or SDS were used as pre-treatment for the enzymatic attack, the results of purity increase achieved values of 58% for H2O2/cell ratio between 0.60 and 1.20, and 57% when SDS/cell ratio at 0.56 was used. In the case when H2O2 or SDS were used as post-treatment for the enzymatic attack, results of purity increase achieved 60% when the H2O2 was used at H2O2/cell ratio ranging between 0.30 and 0.60 and 71% when SDS was used at a ratio SDS/cell of 0.56.

Seventeen different strains of filamentous fungi were grown in batch cultures to compare their abilities for the production of beta-fructofuranosidase. Three of them, Aspergillus oryzae IPT-301, Aspergillus niger ATCC 20611 and strain IPT-615, showed high production with total fructosyltransferase activity higher than 12,500 units l(-1). In addition, the beta-fructofuranosidases of those strains have a high fructosyltransferase activity-to-hydrolytic activity ratio. The temperature and pH effects on the sucrose-beta-fructofuranosidase reaction rate were studied using a 2(2) factorial experimental design. The comparative analysis of the tested variable coefficients shows that the variable pH contributes mostly to the changes in the fructosyltransferase and hydrolytic rates and in the V (t)/V (h) ratio. At 40 and 50 degrees C, there were no significant differences between the fructosyltransferase and hydrolytic velocities of these enzymes.

Polyhydroxybutyrate (PHB) is the most studied among a wide variety of polyhydroxyalkanoates, bacterial biodegradable polymers known as potential substitutes for conventional plastics. This work aimed at evaluating the use of enzymes to recover and purify the PHB produced by Ralstonia eutropha DSM545. Screening experiments allowed the selection of trypsin, bromelain and lysozyme among six enzymes, based on their efficiency in lysing cells of a non-PHB producing R. eutropha strain. Then, process conditions for high efficiency in PHB purification from the DSM545 cells were searched for the enzymes previously selected. The best result was achieved with 2.0% of bromelain (enzyme mass per biomass), equivalent to 14.1 U ml(-1), at 50 degrees C and pH 9.0, resulting in 88.8% PHB purity. Aiming at improving the process efficiency and reducing the enzyme cost, experiments were carried out with pancreatin, leading to 90.0% polymer purity and an enzyme cost three times lower than the one obtained with bromelain. The molecular mass analysis of PHB showed no polymer degradation. Therefore, this work demonstrates the potential of using enzymes in order to recover and purify PHB and bacterial biopolymers in general.

No presente trabalho foi estudada a influência das condições de aeração no tratamento de efluentes fenólicos com biofilmes. Os ensaios foram realizados com efluente sintético para simular dejetos industriais com elevadas concentrações de fenol. Foram testadas vazões de aeração de 0,7, 1,6, 2,3 vvm na cinética de biodegradação de fenol. Nas melhores condições testadas, quais sejam: reator de leito empacotado, com suporte de esferas de cerâmica e vazão de 2,3 vvm, foi atingida eficiência de remoção de fenol de aproximadamente 96 %.

Aspergillus oryzae IPT-301, previously reported as a β-fructofuranosidase producing microorganism, was successfully mutated using UV irradiation at 253.7 nm followed by the screening of survivors resistant to certain stress conditions. Strains were first subjected to the β-fructofuranosidase activity assay using a portion from the colony grown in Petri dish as the enzyme source. Seven mutants with β-fructofuranosidase activity values relative to the parent culture between 140 - 190% were selected from survivors grown at temperature of 40ºC or 0.018% (w/v) sodium dodecyl sulfate concentration. They were cultivated on a rotary shaker to characterize mycelium and extracellular fructosyltransferase activities. Three mutants named IPT-745, IPT-746 and IPT-748 showed the highest amount of mycelium activity whose values increased 1.5 - 1.8 fold, compared with the parent strain. It was found that more than 55% of total enzyme activity (mycelium- plus extracellular- activity) from these strains was detected in the mycelium fraction. Only one mutant, IPT-747, exceeded the amount of extracellular enzyme exhibited by the parent strain (1.5 times). This mutant also showed the highest value of total fructosyltransferase activity.

The decrease in viscosity is widely used to estimate the activity of pectinolytic enzymes. This method is shown to be influenced by the production strain and this prevents an accurate comparison between the activities of different microorganisms, especially under different conditions.

Made available in DSpace on 2018-12-11T16:45:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-12-01 The present work had the goal of screening a batch of 20 fungal strains, isolated from sugar cane plantation soil, in order to identify those capable of biosynthesis of silver nanoparticles. These nanoparticles are known to have a large and effective application in clinical microbiology. Four strains were found to be capable of biosynthesis of silver nanoparticles. The biosynthesised nanoparticles were characterised by UV–vis spectroscopy, scanning electron microscopy, EDX, and XRD. They were found to have an average size of 30–100 nm, a regular round shape, and potential antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The antimicrobial activity was found to be directly related to the nanoparticles concentration. Mycogenic synthesis of nanoparticles is a green biogenic process preferable to other alternatives. Because fungi are great producers of extracellular enzymes this process makes scaling-up an easier task with high importance for clinical microbiology on the fight against microbial resistance, as well as for other industrial applications. Biosciences Institute São Paulo State University-UNESP, Praça Infante Dom Henrique s/no Biology Department Edge Hill University, St Helens Road, Lancashire Richmond Pharmacology Ltd St George’s University London Laboratório de Biotecnologia Industrial Instituto de Pesquisa Tecnológica do Estado de São Paulo Departamento de Química Universidade Federal do Amazonas, Av. General Rodrigo Octávio, Coroado I, 6200 Biosciences Institute São Paulo State University-UNESP, Praça Infante Dom Henrique s/no

It was the objective of the present study to increase the production of glucoamylase by Aspergillus awamori through solid state fermentation, using wheat bran as the main carbon source and (NH4)2SO4, urea, KH2PO4, glucose, maltose and starch as additional nitrogen, phosphorus, and carbon sources. The production of glucoamylase is strongly influenced by N and C sources. A 100% increase was observed when the (NH4)2SO4 was replaced by urea, with C/N = 4.8, using maltose as the additional carbon source. C/P ratios in a range of 5.1 to 28.7 did not induce glucoamylase production under the studied conditions.

The new elephant grass PCEA (P. purpureum x P. purpureum) was specifically developed to meet Brazilian tropical conditions and is the first seed propagated genotype worldwide to be launched by Embrapa Dairy Cattle (CNPGL). The advancement of lignocellulosic bioethanol in Brazil faces numerous challenges, including efficient pretreatments designed for each raw material. In this context, the effect of alkaline pretreatment on PCEA biomass was evaluated to infer about its potential for lignocellulosic bioethanol. The experimental design approached three factors: NaOH %w/v ranging from 1.0 to 3.0 varying on 0.5 %w/v, Temperature (T) from 80 to 120 varying on 20 °C and Reaction Time (RT) from 15 to 35 min varying on 5 min each. The effect of the factors was evaluated considering the response variables of biomass recovery, cellulose and hemicellulose yield, in addition to lignin removal. The untreated biomass and the 17 samples generated from assays were dried and characterized regarding its composition following NREL protocols. The untreated biomass of PCEA presented 31.79±1.55 %wt. of cellulose, 15.51±0.70 %wt. of hemicellulose and 18.23±0.45 %wt. lignin. The observed ash content of 3.94±0.07 %wt. is also important for bioenergy crops, it is desirable on lower values for energy conversion. In addition, the content of 20.54±0.11 %wt. extractives on PCEA indicates further potential to generate biofuels through thermochemical routes. After the alkaline pretreatment, the highest cellulose yield of 58.59 %wt. was achieved in assay 14 at 100 ºC, 25 min of RT and NaOH 3% (w/v). Contrary to initial expectations, only alkali concentration statistically differed at 95% confidence level analysis for the full quadratic model at a coefficient of determination (R2) of 0.78. In contrast, hemicellulose as response for linear with interactions model was significant at 95% confidence level (p-value 0.022), which suggests that the interaction of the three factors altogether had an effect on hemicellulose yield. The highest hemicellulose yield of 25.52 %wt. was reached in assay 9 at 80 ºC, 25 min of RT and 2 %w/v concentration of NaOH. The alkali alone had a p-value of 0.000 (ANOVA at 95% confidence level), which suggests an effect of this factor on biomass recovery. The same was not true for the square and linear with interactions models that presented p-values of 0.231 and 0.353, respectively. The coefficient of determination (R2) for the full quadratic model was 0.92 and the highest recovery of 67.92 %wt. was obtained at the 1.0 %w/v NaOH, at 100 °C and 25 min (assay 13). In conclusion, the optimal condition of the design was established considering efficiency in means of maximal lignin removal without degradation of cellulose, which was achieved at 100 ºC, 25 RT and 3 %w/v NaOH with 99.03 %wt. recovery of cellulose and 73.76 %wt. of lignin removal. These results suggest an exciting potential of the new PCEA for lignocellulosic bioethanol production via alkaline pretreatment to be further explored by the Brazilian agroenergy sector. However, further studies should be carried out with enzymatic hydrolysis to confirm high yield of fermentable sugar and consequently, bioethanol. O novo capim elefante PCEA (P. purpureum x P. purpureum) foi desenvolvido para atender às condições de clima tropical do Brasil, sendo o primeiro genótipo propagado por semente no mundo a ser lançado pela Embrapa Gado de Leite (CNPGL). No Brasil, o avanço do bioetanol lignocelulósico enfrenta inúmeros desafios, incluindo pré-tratamentos eficientes designados para cada matéria-prima. Neste contexto, o efeito do pré-tratamento alcalino foi avaliado para biomassa de PCEA a fim de inferir sobre o seu potencial para bioetanol lignocelulósico. O delineamento experimental analisou três fatores: NaOH %w/v variando de 1,0 a 3,0 em 0,5 %w/v, Temperatura (T) de 80 a 120 variando em 20 °C e Tempo de Reação (RT) de 15 a 35 min variando em 5 min cada. O efeito dos fatores foi avaliado para resposta de recuperação da biomassa, rendimentos de celulose e hemicelulose, além da remoção da lignina. A biomassa não tratada e as 17 amostras geradas a partir dos ensaios foram secas e caracterizadas quanto à sua composição de acordo com protocolos NREL. A biomassa não tratada de PCEA apresentou 31,79±1,55 %wt. de celulose, 15,51±0,70 %wt. de hemicelulose e 18,23±0,45 %wt. de lignina. O teor de cinzas observado foi de 3,94 %wt. (±0,07) deve ser considerado para as culturas bioenergéticas, uma vez que é desejavel em menor quantidade possível para usos energéticos. Além disso, o teor de 20,54±0,11 %wt. de extrativos no PCEA indica um potencial adicional de geração de biocombustíveis através de rotas termoquímicas. Após o pré-tratamento alcalino, o maior rendimento de celulose de 58,59 %wt. foi alcançado no ensaio 14 a 100 ºC, 25 min de RT e NaOH 3% (p/v). Ao contrário das expectativas iniciais, apenas a concentração alcalina diferiu estatisticamente na ANOVA nível de confiança de 95% para o modelo quadrático completo com um coeficiente de determinação (R2) de 0,78. Em contraste, a hemicelulose como resposta para o modelo linear com interações foi significativa ao nível de confiança de 95% (p-valor 0,022), o que sugere que a interação dos três fatores teve efeito sobre o rendimento da hemicelulose. O maior rendimento de hemicelulose de 25,52%wt. foi alcançado no ensaio 9 a 80 ºC, 25 min de RT e 2%w/v de concentração de NaOH. Somente o álcali teve um p-valor de 0,000 (ANOVA a 95% de confiança), o que sugere um efeito deste fator na recuperação da biomassa. O mesmo não ocorreu com os modelos quadrático e linear com interações que apresentaram p-valores de 0,231 e 0,353, respectivamente. O coeficiente de determinação (R2) para o modelo quadrático completo foi de 0,92 e a maior recuperação de 67,92 %wt. foi obtida a 1,0 %w/v NaOH, T of 100 °C e TR of 25 min (ensaio 13). Com isso, a melhor resposta do design considerando a eficiência quanto à máxima remoção de lignina sem degradar a celulose foi observada a 100 ºC, 25 RT e 3 %w/v NaOH com 99,03 %wt. de recuperação da celulose e 73,76 %wt. de remoção da lignina. Estes resultados sugerem um potencial animador do novo PCEA para a produção de bioetanol lignocelulósico através de pré-tratamento alcalino a ser explorado pelo sector agroenergético brasileiro. No entanto, estudos futuros de hidrólise enzimática se fazem necessários, a fim de confirmar o elevado rendimento de açúcares fermentáveis e, consequentemente, de bioetanol.

A conversão de biomassa vegetal em produtos químicos e energia é essencial a fim de sustentar o nosso modo de vida atual. O bagaço de cana-de-açúcar, matériaprima disponível em abundância no Brasil, poderá tanto ajudar a suprir a crescente demanda pelo etanol combustível como ser empregado para obtenção de produtos de valor agregado, tais como xilitol, além de trazer vantagens econômicas para o setor sucroalcooleiro. O xilitol, um poliol com poder adoçante semelhante ao da sacarose e com propriedades peculiares, como metabolismo independente de insulina, anticariogenicidade e aplicações na área clínica, no tratamento de osteoporose e de doenças respiratórias, é obtido em escala comercial por catálise química de materiais lignocelulósicos. A produção biotecnológica de xilitol como alternativa ao processo químico vem sendo pesquisada e os resultados revelam que a presença de compostos tóxicos nos hidrolisados hemicelulósicos resultantes do processo de hidrólise ácida contribui para sua baixa fermentabilidade. Isto se deve à inibição do metabolismo microbiano causada principalmente por compostos tais como ácidos orgânicos, fenólicos e íons metálicos. No presente trabalho foi avaliado o efeito de diferentes fontes de carbono (xilose, glicose e mistura de xilose e glicose) empregadas no preparo do inóculo de Candida guilliermondii FTI 20037 sobre a bioconversão de xilose em xilitol a partir de fermentações em frascos Erlenmeyer de hidrolisados hemicelulósicos submetidos a procedimentos de destoxificação. A condição de favorecimento deste bioprocesso foi empregada para a avaliação da ampliação de escala em fermentadores de 2,4L para 16L, utilizando como critério de ampliação o KLa (igual a 15h-1). De acordo com os resultados, os máximos valores dos parâmetros fermentativos como fator de conversão de xilose em xilitol e produtividade em xilitol foram alcançados com a utilização de inóculo obtido em xilose durante fermentação do hidrolisado destoxificado por resinas (YP/S = 0,81 g g-1 e QP = 0,60 g L-1 h-1, respectivamente), embora o emprego de carvão ativado tenha gerado valores de rendimento próximos para as diferentes fontes de carbono (YP/S variando de 0,78 a 0,80 g g-1). Considerando o valor de fator de conversão e que o procedimento de destoxificação com carvão ativado é o de menor custo e de mais fácil manipulação em comparação ao processo com resinas, os experimentos de ampliação de escala da produção de xilitol por C. guilliermondii foram realizados nesta condição de destoxificação e empregando-se xilose como fonte de carbono para o inóculo. Nesta etapa ficou evidente a viabilidade de ampliação de escala de produção de xilitol de fermentador de 2,4L para 16L, já que os valores dos parâmetros fermentativos avaliados foram semelhantes entre os fermentadores (valores médios: YP/S ≈ 0,68 g g-1 e QP ≈ 0,28 g L-1 h-1). No entanto, tais valores foram inferiores aos obtidos em frascos Erlenmeyer, possivelmente devido às condições de disponibilidade de oxigênio diferirem nos fermentadores de bancada, uma vez que o oxigênio é o parâmetro mais crítico neste bioprocesso. The conversion of vegetable biomass into chemicals and energy is essential to sustain our current style of life. Sugarcane bagasse, a raw material abundantly available in Brazil, greatly contributes to the supply of the evergrowing demand for ethanol. Furthermore, biomass can be employed for obtaining value-added products, such as xylitol, as well as bring economical advantages for the sugar-ethanol sector. Xylitol, a polyol with sweetener power similar to that of saccharose and peculiar properties such as insulin-independent metabolism, anticariogenic power, and applications in the clinical area, in the treatment of osteoporosis and respiratory diseases, is obtained on a commercial scale by chemical catalysis of lignocellulosic materials. The biotechnological production of xylitol as an alternative to the chemical process has been researched and the results reveal that the presence of toxic compounds in hemicelllosics hydrolysates resulting from acid hydrolysis process contributes to its low fermentability. Such toxicity could be due to the inhibition of microbial metabolism promoted mainly by compounds such as organic acids, phenols and metallic ions. In the present work, the effect of different carbon sources (xylose, glucose and a mixture of xylose and glucose) used in the inoculum preparation of Candida guilliermondii FTI 20037 for the xylose-to-xylitol bioconversion by fermentation of hemicellulosics hydrolysates submitted to detoxification procedures in Erlenmeyer flasks was evaluated. The best condition for this bioprocess was employed to evaluate the scale up from the 2.4L to 16L fermentors, using KLa (equal to 15h-1) as scale-up criteria. According to the results the highest values of fermentative parameters such as xylitol yield and productivity were achieved with the use of inoculum cultivated on xylose during the fermentation of hydrolysate detoxified with resins (YP/S = 0.81 g g-1 and QP = 0.60 g L-1 h-1, respectively), although with the use of charcoal the yield value was similar (YP/S ranging for 0.78 to 0.80 g g-1), regardless of the carbon source employed. Considering the value of xylitol yield and that detoxification with activated charcoal is less expensive and more easily manipulated when compared to detoxification procedure with resins, the experiments for scale up xylitol production by C. guilliermondii were performed in such detoxification condition with xylose as the carbon source for the inoculum. At this stage it was evident the scale up xylitol production from a fermenter of 2.4L to 16L was feasible, since the values of fermentative parameters evaluated were similar to those of the fermentors (medium values YP/S ≈ 0.68 g g-1 e QP ≈ 0.28 g L-1 h-1). However, these values were lower than those obtained in Erlenmeyer flasks, maybe due to conditions of oxygen availability for they differ from those in fermentors, since oxygen is the most critical parameter in this bioprocess.

O principal objetivo deste trabalho foi avaliar o impacto de diferentes condições de processo no preço mínimo de venda do etanol produzido via hidrólise enzimática de bagaço da canade- açúcar. Para alcançar este objetivo o trabalho foi separado em três partes, a primeira para a modelagem das reações envolvidas no processo, a segunda para o dimensionamento dos equipamentos principais e a terceira para a avaliação econômica do projeto. Dessa forma, foi realizada a modelagem cinética das reações de pré-tratamento por explosão a vapor do bagaço de cana, da hidrólise enzimática do bagaço pré-tratado e da fermentação de xilose, os modelos obtidos foram utilizados para o dimensionamento de cada sistema reacional. Os dados experimentais para o pré-tratamento, hidrólise enzimática e fermentação de xilose foram obtidos da literatura. Todos os modelos propostos ajustaram adequadamente os dados experimentais de tal forma que os parâmetros cinéticos calculados foram estatisticamente significativos. O sistema de pré-tratamento foi projetado para operar em regime contínuo em reatores tubulares (PFR) e a hidrólise enzimática para operar em regime contínuo em reatores tipo tanque agitado (CSTR) e tubular em série. O sistema CSTR-PFR para a hidrólise enzimática é promissor para a realização desta reação, pois permite obter elevadas conversões em tempos de reação menores que aqueles necessários para os reatores CSTR. Neste sistema é possível alcançada a conversão de 74,3 % da celulose operando o CSTR e o PFR com tempo espacial de 24 h e 72 h, respectivamente. Foi possível avaliar o impacto das condições de pré-tratamento no dimensionamento do sistema de hidrólise enzimática. Além disso, o efeito da concentração inicial de bagaço e enzimas no rendimento de hidrólise enzimática foi avaliado frente aos resultados econômicos. A avaliação dos custos de capital foi realizada via curvas de custo de equipamentos. As diferentes condições de processo estudadas foram comparadas pela determinação do preço mínimo de venda do etanol (PMVE) obtido através da metodologia do lucro do empreendimento (LE). A avaliação econômica do processo mostrou que as condições que podem minimizar o PMVE são: realização do pré-tratamento a 200 °C por 5 min; realização da hidrólise enzimática com concentração inicial de celulose igual a 9,1 % (m/m) ou aproximadamente 17 % (m/m) de bagaço de cana, tempo espacial nos reatores CSTR e PFR de 24 h e 48 h, respectivamente; dosagem de enzimas igual a 40 FPU/g para celulase e 15 U/g para -glicosidase; capacidade de processamento da planta maior que 500 t/dia de bagaço (base seca) e necessariamente a utilizar da fração hemicelulósica para a produção de etanol. Operando nestas condições para uma planta com capacidade para 1000 t/dia o PMVE foi estimado em R$ 5,79 por litro. Dessa forma, este trabalho atingiu seus objetivos fornecendo subsídios para a escolha das condições mais favoráveis para a redução do PMVE, no entanto para a viabilização deste processo de produção de etanol outras configurações de processo devem ser estudas visando a redução dos custos de investimento e produção. The main objective of this study was to evaluate the impact of different process conditions on the minimum selling price of ethanol via enzymatic hydrolysis of sugarcane bagasse. To achieve this objective the work was separated into three parts, the first to model the reactions involved in the process, the second for the design of major equipment and the third for the economic evaluation of the project. Thus, we performed kinetic modeling of reactions pretreatment by steam explosion of bagasse, the enzymatic hydrolysis of pretreated bagasse and fermentation of xylose, the obtained models were used for the design of each reaction system. The experimental data for the pretreatment, enzymatic hydrolysis and fermentation of xylose were obtained from literature. All the proposed models adequately fitted the experimental data so that the kinetic parameters calculated were statistically significant. The system of pretreatment is designed to operate under a continuous tubular reactor (PFR) and enzymatic hydrolysis to operate in a continuous stirred tank reactor (CSTR) and tubular in series. The CSTR-PFR system for the enzymatic hydrolysis is promising to carry out this reaction, because it offers high conversions in shorter reaction times than those required for CSTR reactors. This system can achieved 74.3% conversion of cellulose operating the CSTR and PFR with space-time 24 h and 72 h, respectively. It was possible to assess the impact of the conditions of pretreatment in the design system of enzymatic hydrolysis. Furthermore, the effect of initial concentration of bagasse and enzymes on yield of enzymatic hydrolysis was evaluated in the face of economic results. The evaluation of capital costs was done through cost curves of equipment. The different process conditions studied were compared by determining the minimum ethanol selling price (MESP) obtained through the methodology of the venture profit (VP). The economic evaluation process showed that the conditions that can minimize the MESP are: completion of pre-treatment at 200 ° C for 5 min; completion of the enzymatic hydrolysis with initial concentration of cellulose of 9.1% (w / w) or about 17% (w / w) of bagasse, time in the reactors CSTR and PFR 24 h and 48 h, respectively, enzyme dosage of 40 FPU / g for cellulase and 15 U / g for -glucosidase; capacity processing plant more than 500 tonnes per day of bagasse (dry basis) and necessarily to use the hemicellulose fraction to ethanol production. Operating in these conditions for a plant with a capacity of 1000 t / day, the MESP was estimated at R$ 5.79 per liter. Thus, this work achieved its objectives by providing subsidies to select the most favorable conditions for the reduction of MESP, but for the viability of this process of ethanol production process of other settings should be studied in order to reduce investment costs and production.

Os polihidroxialcanoatos (PHAs) são poliésteres acumulados por microrganismos como material de reserva. O conhecimento das vias bioquímicas e enzimas envolvidas na biossíntese e degradação dos PHAs é uma importante ferramenta para auxiliar na produção industrial. A linhagem Burkholderia cepacia IPT64 é capaz de acumular uma blenda composta de P(3HB) e P(3H4PE) a partir de sacarose. Este trabalho está focado em duas das principais enzimas envolvidas na biossíntese de PHAs: a b-cetotiolase (phaA) e a PHA sintase (phaC). A primeira está associada à especificidade pelo substrato, e a segunda é considerada a enzima chave na síntese de PHAs. Neste trabalho a linhagem mutante phaC foi avaliada quanto à atividade enzimática de PHB sintase, que se constatou ter sido perdida. A presença de mais de uma tiolase no genoma de B. cepacia foi detectada. A inativação do gene phaABc identificado anteriormente, bloqueou totalmente a síntese de P(3HB), e não promoveu o aumento da quantidade total de polímero. Este resultado indica que a tiolase identificada é responsável direta do acúmulo de P(3HB). Outra indicação é que não há uma competição das vias de síntese dos dois polímeros P(3HB) e P(3H4PE), já que não houve alteração na quantidade de P(3H4PE) acumulado, mesmo quando P(3HB) deixou de ser acumulado. The polyhydroxyalkanoates (PHAs) are polyesters accumulated by microorganisms as storage compounds. Knowing the biochemistry pathway and enzymes involved in the biosynthesis and degradation of PHAs is an important tool to help industrial production. The Burkholderia cepacia IPT64 strain is able to accumulate a blend of P(3HB) and P(3H4PE) from sucrose. The focus of this work is on the two main enzymes involved in PHA biosynthesis: the b-ketothiolase (phaA) and the PHA synthase (phaC). The first one is associated with substrate specificity, and the second one is considered the key enzyme in PHA synthesis. In this work a mutant strain phaC was evaluated on its PHB synthase enzymatic activity, that was discovered to have been lost. The presence of other thiolases in the B. cepacia genome was detected. The inactivation of phaABc gene identified previously, blocked totally the P(3HB) synthesis, and didnt increase the polymer content. This result indicates that the identified thiolase is directly responsible for P(3HB) accumulation. Another indication is that the synthesis pathways of the two polymers, P(3HB) and P(3H4PE), dont compete with each other, because the content of P(3H4PE) was not altered, even when the P(3HB) was not accumulated.

A glicose 6-fosfato desidrogenase (G6PDH) é a primeira enzima do processo oxidativo da via das pentoses fosfato e apresenta diversas aplicações industriais. Foram avaliadas as influências de algumas variáveis sobre a produção de G6PDH. No Processo Descontínuo variaram-se a concentração da fonte de carbono, relação carbono-nitrogênio, concentração de aminoácidos e nucleotídeos , pH e a vazão de ar fornecida. A variação na atividade enzimática indicou que as condições do meio são importantes na obtenção desta enzima, tendo sido obtida atividade específica máxima de 86 U/g cél e produtividade de 10,5 U/L.h. No Processo Descontínuo-Alimentado variaram-se o sistema de adição e os nutrientes alimentados. A atividade específica máxima obtida foi 72 U/g cél e a produtividade foi 47 U/L.h. Os resultados indicaram o controle da concentração de glicose em 5g/L para uma melhor produtividade e a necessidade de maiores estudos a fim de otimizar o processo. The glucose 6-phosphate dehydrogenase (G6PDH) is the first enzyme of the pentose phosphate pathway, converting glucose-6-phosphate into 6-phosphogluconate. Besides its importance in biochemistry and medical studies, this enzyme is used in several analytical methods, industrial and commercial application. In this work the influence of several variables in the production of Glucose-6-Phosphate Dehydrogenase in batch and fed-batch processes were studied. In batch cultures the variables were the glucose concentration (10 and 20 g/L), the C:N ratio (3.5 and 6.7 g/g), the aminoacids and nucleotides concentrations (10 and 20 mg/L), pH (4.6 and 5.7) and the aeration (0, 0.8, 1.7 and 2.2 vvm). It was observed that the G6PDH activity varied according to the conditions of the culture. Specific Activity value of 86 U/g cell and productivity of 10,5 U/L.h were attained when the test was carried out as follows: 30oC, pH 5.7, 400 rpm, 2.2 vvm, C:N ratio of 6.7, glucose concentration of 10 g/L, aminoacids (Tryptophan and Hystidine) and nucleotides (Adenine and Uracil) concentrations of 20 mg/L. Nevertheless, the fed-batch process was more efficient and productive than the batch process. The productivity obtained in the best fed-batch condition (glucose addition by an increasing exponential mode) was 47 U/L.h, more than four folds the productivity of the batch culture. So that, by maintaining the glucose concentration in the medium below 5 g/L, the productivity should be improved. However, more studies are needed for optimizing the G6PDH production in a Fed-Batch process. In the fed-batch cultures the feeding conditions and kind of feeding were studied. It was observed that the best fed-batch G6PDH specific activity (72 U/g of cell when the glucose was added in a decreasing linear mode) was lower than that attained in the batch cultures.