Francisco Maugeri

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).

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

Kinetics of Ethanol Fermentation with High Biomass Concentration Considering the Effect of Temperature

A model of ethanol fermentation considering the effect of temperature was developed and validated. Experiments were performed in a temperature range from 28 to 40°C in continuous mode with total cell recycling using a tangential microfiltration system. The developed model considered substrate, product and biomass inhibition, as well as an active cell phase (viable) and an inactive (dead) phase. The kinetic parameters were described as functions of temperature.

Factorial design and simulation for the optimization and determination of control structures for an extractive alcoholic fermentation.

The design, optimization and control of an extractive alcoholic fermentation were studied. The fermentation process was coupled to a vacuum flash vessel that extracted part of the ethanol. Response surface analysis was used in combination with modelling and simulation to determine the operational conditions that maximize yield and productivity. The concepts of factorial design were used in the study of the dynamic behaviour of the process, which was used to determine the best control structures for the process. A good choice of the operational conditions was important to enable efficient control of the process. The performance of a DMC (Dynamic Matrix Control) algorithm was studied to control the extractive process.

Enzymatic biodiesel synthesis from yeast oil using immobilized recombinant Rhizopus oryzae lipase

The recombinant Rhizopus oryzae lipase (1-3 positional selective), immobilized on Relizyme OD403, has been applied to the production of biodiesel using single cell oil from Candida sp. LEB-M3 growing on glycerol from biodiesel process. The composition of microbial oil is quite similar in terms of saponifiable lipids than olive oil, although with a higher amount of saturated fatty acids. The reaction was carried out in a solvent system, and n-hexane showed the best performance in terms of yield and easy recovery. The strategy selected for acyl acceptor addition was a stepwise methanol addition using crude and neutralized single cell oil, olive oil and oleic acid as substrates. A FAMEs yield of 40.6% was obtained with microbial oils lower than olive oil 54.3%. Finally in terms of stability, only a lost about 30% after 6 reutilizations were achieved.

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.

Exploration of Brazilian biodiversity and selection of a new oleaginous yeast strain cultivated in raw glycerol.

The objective of this study was to use glycerol generated from the synthesis of biodiesel to study the oleaginous potential of wild yeasts. An initial selection was performed via a rapid and qualitative technique by staining with Sudan Black B. Initially 129 yeasts were present, from which 5 were selected and cultivated in liquid medium containing pure or raw glycerol. The yeast LEB-M3, isolated from the Pantanal, presented lipid content of 20.46% and 56.58% for cultivation in pure and raw glycerol, respectively. This strain was genotypically identified as Candida sp. The fatty acid profile showed predominance of oleic acid (C18:1), 57.35% for cultivation in pure glycerol, and in raw glycerol linoleic acid (C18:2) was predominant (46.0%). It was possible to select a yeast with high lipid concentrations 9.14 g/L and fatty acid profile similar to vegetable oils commonly used in the synthesis of biodiesel.

Modeling of ion exchange expanded-bed chromatography for the purification of C-phycocyanin.

This work is focused on the experimental evaluation and mathematical modeling of ion exchange expanded-bed chromatography for the purification of C-phycocyanin from crude fermentative broth containing Spirulina platensis cells. Experiments were carried out in different expansion degree to evaluate the process performance. The experimental breakthrough curves were used to estimate the mass transfer and kinetics parameters of the proposed model, using the Particle Swarm Optimization algorithm (PSO). The proposed model satisfactorily fitted the experimental data. The results from the model application pointed out that the increase in the initial bed height does not influence the process efficiency, however enables the operation of expanded-bed column at high volumetric flow rates, improving the productivity. It was also shown that the use of mathematical modeling was a good and promising tool for the optimization of chromatographic processes.