Maria Isabel Rodrigues

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).

Optimization of extracellular lipase production by Geotrichum sp. using factorial design

Response surface methodology was employed to study the effects of carbon source (soy oil, olive oil and glucose) and nitrogen source concentrations (corn steep liquor and NH(4)NO(3)) on the lipase production by Geotrichum sp. The experiment included a 2(4) central composite rotatable design (CCRD) and four others 2(3) CCRD. According to the responses from the experimental designs, the effects of each variable were calculated and the interactions between them were determined. The response surface methodology was applied for the optimization of the nutrient concentrations in the culture medium for the enzyme production, at 30 degrees C. The optimum medium composition for lipase production by Geotrichum sp. was ammonium nitrate 2.1-2.5%, corn steep liquor 13-15% and soy oil 0.6% as carbon source, which lead to a lipase activity of about 20 U/ml. Using olive oil as carbon source, the optimum composition was ammonium nitrate 0.8-1%, corn steep liquor 13-15% and olive oil 0.6%, leading to an activity of 17 U/ml.

Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation.

The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35-60 degrees C and pH 4.0-6.0, with a maximum activity at 50 degrees C and pH 4.0-5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 degrees C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0-C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.

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.

Dynamic modelling, simulation and optimization of an extractive continuous alcoholic fermentation process

The conventional alcoholic fermentation is a typical inhibitory process, leading to low productivity and yield. The ethanol produced inhibits yeast cells, causing a reduction in the alcohol production rate and cell growth rate. In this work, modelling and simulation have shown that continuous extractive fermentation, coupling a fermenter with an extractive vacuum flash chamber, is technically possible. In this case, the ethanol is partially removed, increasing drastically the productivity. Additionally, temperature control can be performed without using heat exchangers. The optimization was carried out using the method of factorial design and response surface analysis, leading to the determination of the most relevant variables, which were: 1.2 h residence time, 0.4 flash recycle rate, 180 g dm−3 sugar concentration and 0.35 cell recycle rate. The results, using optimized variables, were 98% conversion and 23 g dm−3 h−1 productivity, which represent a three times higher productivity than in a conventional continuous process. © 1999 Society of Chemical Industry

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.

Studies on lipase‐affinity adsorption using response‐surface analysis

Lipases are widely distributed enzymes that can be obtained from animals, plants and micro‐organisms. Coupling lipases with a wide range of substrates allows the opportunity for synthesis of optically pure pharmaceutical preparations, flavour compounds and other food additives. Affinity chromatography owes its power as a purification method to specific biological interactions. Response‐surface analysis was chosen to study column efficiency. This method allows the understanding of interactions between variables with advantages over conventional methods, which involve changing one variable while fixing others at certain levels. The aim of this work was to study the influence of the ratio bed height/column diameter (L/D) and superficial velocity (V) on the column efficiency. The experimental design involved the two variables, L/D (2–10) and v (1–2 cm/min), at five levels. Lipase was obtained from Geotrichum sp. culture in a complex medium composed of 5% corn‐steep liquor, 0.5% NH4NO3 and 1% olive oil at 30 °C, with 1VVM (air volume/medium volume per min) aeration and 400 rev./min agitation. Maximum lipase activity was 19 units/ml after almost 9 h of fermentation. This lipase could potentially be used in esterification reactions to increase the content of γ‐linolenic acid and to produce bioaromas for food industries. The adsorption assays were carried out in a fixed‐bed column with an affinity adsorbent, which was obtained by reaction of a gel with oleic acid as ligand. Breakthrough curves were obtained for all experiments. It has been shown that the lower the values of both L/D and v, the higher the column efficiency (maximum 65.43%). Also, it was observed from the response surface that the efficiency reached a minimum at an L/D of around 8.

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.

In vitro synthesis of oligosaccharides by acceptor reaction of dextransucrase from Leuconostoc mesenteroides

Glucose was used as acceptor to obtain small chain oligossaccharides from sucrose using dextransucrase from Leuconostoc mesenteroides NRRL B-512F. Better conditions for the synthesis of the oligosaccharides were obtained using experimental design and response surface methodology. Yield of oligosaccharides was increased from 5% to 45% following an increase in both sucrose and glucose/sucrose concentrations, from 58 g/l to 142 g/l and from 0.02 to 0.18, respectively. Molecular weight increased from 2800 to 4500 daltons with a temperature shifting from 10°C to 30°C.

Galacto-oligosaccharides production using permeabilized cells of Kluyveromyces marxianus.

Galacto-oligosaccharides are non-digestible carbohydrates and are recognized as important prebiotics for than stimulation of the proliferation of lactic acid bacteria and bifidobacteria in the human intestine. GOS can be produced by a transgalactosylation reaction catalysed by ?-galactosidase enzyme, and microorganisms can be used as a source of ?-galactosidase. In this work, a process for producing GOS using permeabilized cells of Kluyveromyces marxianus CCT 7082 was proposed. The effects of the concentrations of lactose and enzyme, temperature and pH were studied using a fractional design followed by a central composite rotatable design. The optimum conditions for galacto-oligosaccharides production were found to be: lactose concentration 500 g/L, enzyme concentration 10 U/mL, 45°C and pH 7.0. Under optimized conditions, the GOS concentration, yield and productivity were 83 g/L, 16.5% and 27.6 g/L.h, respectively.