Susana Juliano Kalil

Response surface analysis and simulation as a tool for bioprocess design and optimization

Abstract In the present work, factorial design and response surface techniques were used in combination with modeling and simulation to design and optimize an industrial bioprocess. Alcoholic fermentation process with multiple stages was considered. The fermentation system is composed of four ideal continuous-stirred tank reactors (ICSTR), linked in series, with cell recycling. Operational conditions for maximal yield and productivity were determined with ten parameters under consideration: temperature (four stages), residence time for each stage, cell recycling concentration, and the fraction of fresh medium fed into the second fermentation stage. Initially, screening design methodology was used to evaluate the process variables which were relevant in relation to yield and productivity. Five statistically significant parameters for each response were selected and utilized in factorial design in order to optimize the process. With the models obtained from the factorial design, response surfaces were generated, and the productivity improved to 12 g/l·h (an increase of 52% in relation to the control version of the bioprocess), while maintaining the high yield of 86.28% (99.1% conversion).

The production of rhamnolipid by a Pseudomonas aeruginosa strain isolated from a southern coastal zone in Brazil.

The production and properties of a rhamnolipid-type biosurfactant, synthesized by the Pseudomonas aeruginosa LBM10 strain, isolated from a southern coastal zone in Brazil, were investigated. The assays were conducted in a rotary shaker at 30 degrees C and 180 rpm for a period of 96 h. Soybean oil and sodium nitrate were the best sources of carbon and nitrogen, respectively. A nitrogen-limiting condition (C/N ratio of 100) was favorable to biosurfactant production. The formation of stable emulsions was better in saline concentrations below 0.5%, pH values in the range from 6 to 9 and temperatures in the range from 35 to 40 degrees C, maintaining about 80% of its original activity for salinity up to 3% and 120 min of exposure at 100 degrees C. The biosurfactant may be produced with this microorganism using renewable substrates that are readily available, reaching values of 1.42 g l(-1) measured as rhamnose. This biosurfactant has interesting and useful properties for many industrial applications.

Optimization of inulinase production by Kluyveromyces marxianus using factorial design.

Factorial design and response surface techniques were used to optimize the culture medium for the production of inulinase by Kluyveromyces marxianus. Sucrose was used as the carbon source instead of inulin. Initially, a fractional factorial design (25–1) was used in order to determine the most relevant variables for enzyme production. Five parameters were studied (sucrose, peptone, yeast extract, pH, and K2HPO4), and all were shown to be significant. Sucrose concentration and pH had negative effects on inulinase production, whereas peptone, yeast extract, and K2HPO4 had positive ones. The pH was shown to be the most significant variable and should be preferentially maintained at 3.5. According to the results from the first factorial design, sucrose, peptone, and yeast extract concentrations were selected to be utilized in a full factorial design. The optimum conditions for a higher enzymatic activity were then determined: 14 g/L of sucrose, 10 g/L of yeast extract, 20 g/L of peptone, 1 g/L of K2HPO4. The enzymatic activity in the culture conditions was 127 U/mL, about six times higher than before the optimization.

C-phycocyanin extraction from Spirulina platensis wet biomass

C-Phycocyanin is a natural blue dye used in food and pharmaceutical industry. In the present study, a simple and efficient method to extract C-phycocyanin from Spirulina platensis wet biomass is reported. The extractions were carried out using six different methods, including chemical (organic and inorganic acid treatment), physical (freezing and thawing, sonication, homogenization) and enzymatic (lysozyme treatment) methods. The extraction using ultrasonic bath in the presence of glass pearls in the biomass proved to be the most efficient method, 56% higher than using freezing and thawing (the method most frequently used), and presented a extraction yield of 43.75 mg.g-1 and a C-phycocyanin concentration of 0.21 mg.mL-1.

Strategy for a protein purification design using C-phycocyanin extract.

A variety of techniques have been developed for the separation and recovery of proteins. The cost of purifying the product is frequently determined by the desired quality of the final product, which is evaluated by measuring the purity. In this work the design of a protein purification process for C-phycocyanin, a phycobiliprotein that can be used in the food and medical industries, was established. The study evaluated the use of ammonium sulfate precipitation, ion exchange chromatography and gel filtration to purify C-phycocyanin in a variety of sequences. The final design included the C-phycocyanin extraction step, precipitation with ammonium sulfate and ion exchange chromatography. When the elution step was studied, the kind of elution and pH were considered in order to obtain a product with a final purity of 4.0 with a purification factor of 6.35, so that, at the end of the strategy, C-phycocyanin of analytical grade would be obtained.

Mathematical modeling and simulation of inulinase adsorption in expanded bed column

A mathematical model for an expanded bed column was developed to predict breakthrough curves for inulinase adsorption on Streamline SP ion-exchange adsorbent, using a crude fermentative broth with cells as the feedstock. The kinetics and mass transfer parameters were estimated using the PSO (particle swarm optimization) heuristic algorithm. The parameters were estimated for each expansion degree (ED) using three breakthrough curves at initial inulinase concentrations of 65.6UmL(-1). In sequence, the model parameters for an ED of 2.5 were validated using the breakthrough curve at an initial concentration of 114.4UmL(-1). The applicability of the validated model in process optimization was investigated, using the model as a process simulator and experimental design methodology to optimize the column and process efficiencies. The results demonstrated the usefulness of this methodology for expanded bed adsorption processes.

Parameters optimization for enzymatic assays using experimental design

The conditions for maximization enzymatic activity were determined using experimental design and inulinase from Kluyveromyces marxianus ATCC 16045. The effects of substrate concentration (sucrose and inulin), pH and temperature on inulinase activity were verified using four factorial design and surface response analysis. Using sucrose as substrate. It has bean shown that the effects sucrose on enzymatic activity is not statistically significant and the best condition for the highest activity (110 U/mL) was achieved with temperature between 60°C and 68°C and pH between 4.5 and 5.0. Using inulin as substrate it was verified that temperature is the only variable statistically significant and the maximum activity was 7.3 U/mL at temperature between 50°C and 51°C.

Purification of carbonic anhydrase from bovine erythrocytes and its application in the enzymic capture of carbon dioxide

This work presents a study of industrially applicable techniques to obtain a biologically supported carbon dioxide capture system, based on the extraction of carbonic anhydrase from bovine blood. Carbonic anhydrase is a metalloenzyme which catalyzes the reversible hydration of carbon dioxide. The objective of this study was to establish conditions to obtain carbonic anhydrase from bovine erythrocytes and apply it in the capture of carbon dioxide. To achieve this, two different purification techniques were evaluated: one by extraction with the organic solvents chloroform and ethanol, where different solvent proportions were studied; and the other by ammonium sulfate precipitation, testing percent saturations between 10% and 80%. Carbon dioxide was enzymatically captured by its precipitation as calcium carbonate with the enzyme obtained by both techniques. The enzyme extracted by ethanol and chloroform showed an activity of 2623 U mL(-1), recovery of 98% and purification factor of 104-fold. That precipitated by ammonium sulfate showed an activity of 2162 U mL(-1), recovery of 66% and purification factor of 1.4-fold using 60% ammonium sulfate saturation. The results obtained in the carbon dioxide capture experiments showed that the carbonic anhydrase extracted in this study not only enhanced the hydration of CO(2), but also promoted the formation of CaCO(3).

Ultrasonic waves and glass pearls: a new method of extraction of beta-galactosodade for use in laboratory

The optimization of a traditional technique of cellular disruption by abrasion was carried out and a process using ultrasonic waves associated with glass pearls to extract b-galactosidase from Kluyveromyces marxianus proposed. In the first case, the effects of the diameter and weight of the pearls in relation to the volume of cellular suspension and amount of time for cellular disruption were evaluated. The efficiency of the new process of cellular disruption was evaluated by varying the length of time of sonification and comparing with the method of abrasion under the same conditions. The proposed method can be efficiently applied to obtain b-galactosidase at laboratory scale.

Optimization of glucoamylase production by Aspergillus niger in solid-state fermentation.

Glucoamylase production by Aspergillus niger in solid-state fermentation was optimized using factorial design and response surface techniques. The variables evaluated were pH and bed thickness in tray, having as response enzyme production and productivity. The bed thickness in tray was the most significant variable for both responses. The highest values for glucoamylase production occurred using pH 4.5 and bed thickness in the inferior limits at 2.0–4.2 cm. For productivity, the optimal conditions were at pH 4.5 as well and bed thickness from 4.4 to 7.5 cm. The optimal conditions for glucoamylase production while obtaining high activity without loss of productivity were pH 4.5 and bed thickness in tray from 4.0 to 4.5 cm, which resulted in an enzyme production of 695 U/g and productivity of 5791 U/h.