Caracterização de um novo sistema bifasico aquoso e aplicação em extração de proteinas com coluna de discos perfurados rotativos

Orientadores: Elias Basile Tambourgi, Galba Maria de Campos TakakiTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia QuimicaResumo: A partição bifásica aquosa explora a capacidade exibida por soluções de dois polimeros ou de um polímero e um sal de se separarem em duas fases aquosas sempre que certas concentrações mínimas sejam excedidas. As duas fases aquosas assim formadas diferem na sua composição, sendo cada uma delas rica num dos constituintes do sistema, isto é, os dois polimeros ou o polimero e o sal concentram-se em fases distintas. Estas fases apresentam elevados teores em água, podendo em alguns casos atingir valores superiores a 90 %, constituindo ambientes suaves para moléculas e partículas biológicas. O objetivo principal deste trabalho residiu na caracterização do sistema bifásico aquoso formado por um polímero típico do Nordeste brasileiro, o polissacarídeo do cajueiro (Policaju). Sendo o elevado custo dos polímeros envolvidos o principal entrave à aplicação em grande escala da partição bifásica aquosa, este sistema apresentou a possibilidade em reduzir este custo, aumentando a competitividade da partição bifásica aquosa como processo alternativo frente aos processos tradicionais de recuperação de bioprodutos. Mostrou-se ao longo desta tese que o sistema testado apresentou propriedades fisicas (razão e tempo de separação de fases, densidade, viscosidade) e de partição características de sistemas bifásicos aquosos polímero-polímero. Após o isolamento do polissacarídeo, determinou­se o diagrama de fases e estudou-se a partição de uma proteína modelo, a albumina de soro bovino. Verificou-se o efeito exercido pelas variáveis do sistema, peso molecular do PEG (1500, 4000Abstract: Aqueous two-phase partition explores the possibility of solutions of two polymers or one polymer and one salt separating into two immiscible aqueous phases, when certain critical concentrations are exceeded. Each one of these aqueous phases is more concentrated in one of the phase forming chemicals. Both exhibit a high water content, in some cases as much as 90%. Due to this high water content, these byphasic systems provide a gentle environment for molecules and partic1es with biological activity. The main subject ofthis work was the characterisation of a new aqueous two-phase system based on a typical Brazilian north-westem polymer, the polysaccharide of cashew tree (Policaju). Since the cost of the phase forming polymers is the main drawback on the large scale application of aqueous two-phase partition, this system showed the possibility of decreasing this cost by increasing the applicability of biphasic partition as an alternative technique to the traditional recovery processes ofbioproducts. It is shown tOOt physical properties (phase volume ratio, time for phase separation, density, viscosity) and partition properties of the tested system are similar to others aqueous two-phase polymer-polymer systems. Afier the polysaccharide isolation, the phase diagram was determinate and the partition of a model protein, bovine serum albumin, studied. It was analysed the effect of PEG molecular weight (1500, 4000 and 8000), polymer concentration, temperature (25°C e 40°C), pH (6,0, 7,0 and 8,0) and NaCI (O,lM e 1M) addition. It was observed a displacement ofthe binodal towards lower polymer concentrations when PEG molecular weights decreased. Conversely, the variation of pH and temperature did not promote significant alterations on the binodal. It was not possible to stabilise a relation between the protein partition coeflicient and the PEG molecular weight, whi1e the increase in tie-line length and temperature and the decrease in pH promoted the decrease of the partition coeflicient. The results obtained for NaCI addition showed a tendency for the decrease ofthe partition coeflicient with the increase of salt concentration, although in some cases, the partition coefficient decreased on1y for a determina te salt concentration, depending on the system and pH. Continuous operation with aqueous two-phase systems was also tested. The beOOviour of the biphasic system PEG-Policaju was studied using a perforated rotating disc contactor (pRDC). The transfer of bovine serum albumin ftom the dispersed phase to the continuous phase was studied by using the PEG4000-Policaju system selected ftom batch extraction. The column was operated in countercurrent and in a continuous mode during 55 minutes of operatio_ where the dispersed phase was constituted by the PEG-rich phase and the continuous phase by the Policaju­rich phase. Variations in the rotation velocity (60, 140 and 220 rpm), dispersed phase velocity (2, 3 and 5 rnL/min) and discs number (03 and 04) were investigated. The performance of the equipment was characterised by the ana1ysis ofthe ftactional dispersed phase holdup, mass transfer coefticient and KA W ASE recuperation indices. The results showed an increase ofthe dispersed phase holdup, mass transfer coeflicient and recuperation indices with the increase of the rotation velocity. The same effects were observed for the increase of the dispersed phase velocity, except for the recuperation indices, which did not show a regular tendency. The increase of system composition decreased the ftactional dispersed phase holdup and the mass transfer coeflicient, although did not show a significant effect over the recuperation indices. The number discs variation did not demonstrate a significant effect upon the ftactional dispersed phase holdup, although the mass transfer coeflicient and the recuperation indices were higher for 03 discs. The ana1ysis of bovine serum albumin continuous extraction showed the higher protein transfer for the following conditions: 9%PEG4000-18%Policaju system in PRDC with 03 rotary discs with rotation velocity of 220 rpm and dispersed phase velocity of 2 mL/min. The economic ana1ysis showed that PEG-Policaju aqueous two-phase system is very competitive with other polymer-polymer systems described in the literature. This permits the development offuture researches with this new system in recovery processesDoutoradoSistemas de Processos Quimicos e InformaticaDoutor em Engenharia Químic

Production and stability studies of the bioemulsifier obtained from a new strain of Candida glabrata UCP 1002

Evaluation of both tenso-active and emulsifying activities indicated that a biosurfactant was produced by the newly isolated and promising strain Candida glabrata isolated from mangrove sediments. The extracellular water-soluble emulsifying agent was isolated and identified as a heteropolymer. The maximum of bioemulsifier production was observed when the strain was grown on soluble and insoluble substrates cotton seed oil plus glucose, reaching values of 10.0 g/l after 144 hrs at 200 rpm. The cell-free culture broth containing the examined agent lowered the surface tension of the medium to 31 mN/m. Stable and compact emulsions with emulsifying activity of 75% of cotton seed oil were detected. The emulsification capacity remained practically unaltered within a wide pH (2-12), temperature (4-80\ub0C) ranges and under NaCl concentrations up to 10%

Characterization and properties of the biosurfactant produced by Candida lipolytica UCP 0988

Background: Biotechnological processes are costly, especially for the production of biosurfactants. The successful production of a biosurfactant is dependent on the development of processes using low cost raw materials. Considering the importance of the characteristics of a biosurfactant to facilitate its industrial application, the properties of the biosurfactant produced by Candida lipolytica through previously optimized medium have been established. Results: The yeast was grown for 72 h to determine the kinetics of growth and production. The surface tension of the cell-free broth was reduced from 55 to 25 mN/m. The yield of biosurfactant was 8.0 g/l with a CMC of 0.03%. The biosurfactant was characterized as an anionic lipopeptide composed of 50% protein, 20% lipids, and 8% of carbohydrates. Conclusions: The isolated biosurfactant showed no toxicity against different vegetable seeds: Brassica oleracea, Solanum gilo and Lactuca sativa L. and the micro-crustacean Artemia salina. The properties of the biosurfactant produced suggest its potential application in industries that require the use of effective compounds at low cost

Optimization and evaluation of biosurfactant produced by Pantoea sp. using pineapple peel residue, vegetable fat and corn steep liquor

In this study, the authors have investigated the potential of a bacterial strain of Pantoea sp., isolated from wastewater of the textile industry, for the production of biosurfactant. The biosurfactant production was optimized by the combination of CCD (central composite design) and RSM (response surface methodology). To assess the effects and interactions of medium the vegetable fat (1.5, 2.0 and 2.5 v/v), the variables corn steep liquor (2.0, 5.0 and 8.0 v/v) and pineapple peel residue (10.0, 25.0 and 40.0 v/v) on the surface tension were evaluated. The empirical model developed through RSM in terms of the effective operational factors mentioned above was found to be adequate to describe the biosurfactant production. Compositional analysis of the produced biosurfactant has been carried out by FT-IR (Fourier transform infrared spectoscopy) and subjected to the test of removing hydrocarbons. Through the analysis, vegetable fat and pineapple peel residue were found to be the most significant factors, whereas corn steep liquor had less effect within the ranges investigated. A maximum reduction in surface tension of 30.00 mN/m was obtained under the optimal conditions of 2.0% (v/v) vegetable fat concentration, 5.0% (v/v) corn steep liquor and 25.0% (v/v) pineapple peel residue concentration of medium. FT-IR spectrometer analysis of the biosurfactant characterized it as a glycolipid derivative. The biosurfactant exhibited the ability to solubilize the hydrocarbons tested, working between 64% and 92%. According to consists of bars with a length proportional to the absolute value of the estimated effects divided by the standard error. On this chart, ANOVA (analysis of variance) effect estimates are arranged from the largest to smallest absolute value. The chart includes a vertical line at the critical p-value of 0.05. Effects for which the bars are smaller than the critical p-value are considered non-significant and do not have an effect on the response variables. The effects are either positive or negative ANOVA; the determination of regression coefficients and the construction of graphs were performed using the Statistical® program, version 7.0 (Statsoft Inc, RSA). The results, the biosurfactant produced by Pantoea sp. can be a valuable source for application in rapid environmental bioremediation9269279CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À CIÊNCIA E TECNOLOGIA DO ESTADO DE PERNAMBUCO - FACEPEsem informaçã

Assessment of toxicity of a biosurfactant from Candida sphaerica UCP 0995 cultivated with industrial residues in a bioreactor

Background: The aim of the present study was to propose a low-cost method for the production of a biosurfactant by the yeast Candida sphaerica and assess its toxicity and phytotoxicity. The medium was formulated with distilled water supplemented with residue from a soy oil refinery (5%) and corn steep liquor (2.5%) as substrates. These two products were the sources of carbon and nitrogen as well as mineral elements to encourage the growth of the microorganism and production of a biosurfactant. Results: The isolated biosurfactant yield was 6.364 g/l. The biosurfactant exhibited an excellent ability to reduce surface tension (26 mN/m) and demonstrated no toxicity against seeds of Brassica oleracea , Chicoria intybus and Solanum gilo or the micro crustacean Artemia salina employed as a bioindicator. The biosurfactant exhibited no antimicrobial activity against the fungi and bacteria tested. Conclusions: The promising results obtained in this study indicate the feasibility of producing biosurfactants from powerful non-toxic organic residues and their application in the bioremediation of contaminated soil and water

Synthesis of silver nanoparticles using a biosurfactant produced in low-cost medium as stabilizing agent

Background: A biosurfactant produced by Pseudomonas aeruginosa cultivated in a low-cost medium formulated with 2.5% vegetable oil refinery residue and 2.5% corn steep liquor and distilled water was employed to stabilize silver nanoparticles in the liquid phase. The particleswere initially synthesized using NaBH4 as reducing agent in biosurfactant reverse micelles and were extracted from the micellar solution to disperse in heptane. Results: A silver particle size in the range of 1.13 nmwas observed. The UV\u2013vis absorption spectra proposed that silver nanoparticles could be formed in the reverse micelles and relatively stabilized for at least 3 months without passivator addition. The Transmission Electron Microscope (TEM) shows that the silver nanoparticles are of spherical form and relatively uniform. Conclusions: This process provided a simpler route for nanoparticle synthesis compared to existing systems using whole organisms or partially purified biological extracts, showing that the low-cost biosurfactant can be used for nanoparticle synthesis as a non-toxic and biodegradable stabilizing agent

Comparative Evaluation Between a Non-toxic Biodetergent and a Commercial Degreaser for Industrial Use

To clean surfaces contaminated by petrochemicals, industries use products formulated with substances harmful to the environment and the health of workers. Nesse sense, the development of degreasers and non-toxic detergents formulated from natural sources can be a solution. Based on these needs, many companies offer products claiming to be biodegradable, non-toxic and environmentally compatible. Therefore, this work aimed to carry out comparative tests between a biodegradable and non-toxic biodetergent, produced from vegetable inputs and a commercial degreaser with the same purpose. In this sense, the efficiency of the products was evaluated in of tin removing OCB1 fuel oil from smooth and metallic surfaces, the destabilization capacity of heavy oil in was determined in relation to a time of 5 minutes and its toxicity using cabbage seeds (Brassica oleracea) and microcrustacean larvae (Artemia salina) as indicators. The comparative tests between the two formulations demonstrated very satisfactory results in favor of the biodetergent. Regarding the evaluation of the products for OCB1 removal efficiency, the biodetergent showed satisfactory results, with 100% removal of the oil impregnated on smooth and metallic surfaces and 90% for the commercial product. For the destabilization capacity in relation to time, the biodetergent demonstrated total destabilization of the petroderivative in just 2 minutes, whereas the commercial product required twice as long for partial destabilization of the oil. The ecotoxicological tests demonstrated the absence of toxicity of the detergent formulated with biosurfactant and for the commercial degreaser and a mortality rate of 100% was observed for the indicator organisms. Therefore, the biodetergent demonstrates superior efficiency in relation to commercial degreaser and safer and more reliable chemical and toxicological characteristics for human health and the environment

Avaliação do potencial da celulose bacteriana para aplicação em cosméticos / Potential evaluation of bacterial cellulose for cosmetic application

O termo cosmecêutico, associação entre cosméticos e produtos farmacêuticos, surgiu da necessidade da população em aliar os cuidados com o corpo a produtos que utilizam recursos naturais, garantindo um desenvolvimento sustentável das novas tecnologias exploradas pelo mercado de HPPC (Higiene Pessoal, Perfumaria e Cosméticos). Seguindo esta ideia, o presente trabalho consistiu em estudar a utilização de uma bactéria produtora de celulose, cultivada em resíduo do suco de frutas como fonte alternativa vegetal, para a produção de uma película de celulose com características benéficas para a pele. A película de celulose bacteriana, por sua vez, além de servir como suporte para incorporação dos princípios ativos das frutas, possui alta atividade de água quando aplicada na pele, auxiliando na retenção de umidade da mesma. Para a realização dos testes, a bactéria Glucanacetobacter hansenii foi cultivada em onze diferentes meios de cultivo padrão HS (Hestrim-Schramm) modificados, tendo sido selecionado o meio com melhor custo benefício, formulado com suco do resíduo de frutas, fosfato dissódico e ácido cítrico. Os experimentos foram realizados após 10 dias do cultivo da bactéria no meio citado previamente. Os resultados demonstraram a produção de uma película contendo 99,13±0,09% de atividade de água, 2,0±0,1% oBx, 13±1g de ácido cítrico/100g de celulose e 24,4±1,10mg de ácido ascórbico/100g de celulose. Em seguida, foi confeccionada uma máscara de CB a partir da película produzida em resíduo de frutas. Pode-se concluir que a adição de resíduo de frutas no meio de crescimento da bactéria concede à película de celulose características ideais para a hidratação da pele, possibilitando o desenvolvimento de um novo produto biotecnológico para as indústrias farmacêuticas e cosméticas do país.