Tito L.M. Alves

Production and extraction of pectinases obtained by solid state fermentation of agroindustrial residues with Aspergillus niger

In this work soy and wheat bran were employed as raw materials for the production of pectinases by Aspergillus niger through solid-state fermentation. Several fermentation and recovery parameters were studied. The kinetics of enzyme synthesis was investigated in the range from 13 to 96 h with moisture contents varying from 25% to 70% (w/w). A medium moisture content of 40% and a fermentation time of 22 h were selected, as these conditions resulted in high pectolytic activity and enhanced polygalacturonase productivity. In order to optimise the recovery step, the best combination of temperature of extraction, contact time and solvent type was investigated. Acetate buffer (pH 4.4), 35°C and 30 min provided the best recovery. The present results show that optimising the extraction conditions is a simple way of obtaining more concentrated enzyme extracts and could be a useful instrument to extract more selectively a desired biomolecule from fermented solids.

Recovery of pectolytic enzymes produced by solid state culture of Aspergillus niger

Abstract Pectinases are enzymes with a wide range of applications in the food and drink industries. In the present work, the extraction of pectinases produced by Aspergillus niger in a solid state fermentation system was investigated. The purpose was to reduce enzyme losses in the fermented solids and at the same time obtain a crude extract as concentrated as possible. Initially the performances of stirred tank and fixed bed extractors were compared. Polygalacturonase activity and viscosity reducing capacity obtained in the stirred tank system were 105% and 15% superior, respectively. Repeated extractions and multiple stage countercurrent extraction were studied, employing stirred tanks. It was possible to observe that three stages were enough for total recovery of the enzymes contained in the solids. The final enzyme extract obtained by counter-current extraction with three stages showed a polygalacturonase activity 81% higher than the one obtained by one-stage extraction.

Separation of fructose from a mixture of sugars using supported liquid membranes

The main purpose of this study was to investigate the feasibility of using supported liquid membranes to extract fructose from a mixture of sugars contained in a fermentation broth. The membrane consisted of a microporous polypropylene support impregnated with a solution of a phenylboronic acid derivative in 2-nitrophenyl octyl ether. Transport through a flat sheet membrane was studied as a function of carrier and feed concentration. A hollow fiber system was also examined, and the effects of the carrier and feed concentrations, as well as the flow rate through the fiber lumen, on the glucose and fructose fluxes and fructose selectivity were studied. The hollow fiber system is more stable than the flat sheet supported liquid membrane, and produces higher fructose selectivities using a lower carrier concentration. The hollow fiber supported liquid membrane is able to remove fructose from a fermentation broth, although the membrane flux and long term stability need further improvement.

Economic analysis of ethanol and fructose production by selective fermentation coupled to pervaporation: effect of membrane costs on process economics☆

This work presents a preliminary economic analysis of the production of ethanol using different fermentation processes. The installation of a new plant and the adaptation of an existing ethanol plant to the selective fermentation of glucose from sugar cane hydrolysate were considered. In the latter process, glucose is converted to ethanol and fructose accumulates in the fermentation broth. The use of pervaporation for continuous removal of ethanol from the fermentation broth is also considered, in order to minimize inhibition of the microorganism and to facilitate product recovery. The results show that the selective fermentation coupled to membrane processes to removal of ethanol is an attractive process to increase ethanol production economics, although membrane performance and costs should still be improved.

L-DOPA production by immobilized tyrosinase.

The production of L-DOPA using L-tyrosine as substrate, the enzyme tyrosinase (EC 1.14.18.1) as biocatalyst, and L-ascorbate as reducing agent for the o-quinones produced by the enzymatic oxidation of the substrates was studied. Tyrosinase immobilization was investigated on different supports and chemical agents: chitin flakes activated with hexamethylenediamine and glutaraldehyde as crosslinking agent, chitosan gel beads, chitosan gel beads in the presence of glutaraldehyde, chitosan gel beads in the presence of polyvinylpyrrolidone, and chitosan flakes using glutaraldehyde as crosslinking agent. The last support was considered the best using as performance indexes the following set of immobilization parameters: efficiency (90.52%), yield (11.65%), retention (12.87%), and instability factor (0.00). The conditions of immobilization on chitosan flakes were optimized using a two-level full factorial experimental design. The independent variables were enzyme-support contact time (t), glutaraldehyde concentration (G), and the amount of enzyme units initially offered (UC). The response variable was the total units of enzymatic activity shown by the immobilized enzyme (UIMO). The optimal conditions were t = 24 h, G = 2% (v/v), and UC = 163.7 U. Under these conditions the total units of enzymatic activity shown by the immobilized enzyme (UIMO) was 23.3 U and the rate of L-DOPA production rate was 53.97 mg/(L.h).

Evaluation of flat sheet and hollow fiber supported liquid membranes for fructose pertraction from a mixture of sugars

This work investigates the use of supported liquid membranes (SLM) in the pertraction of fructose from a mixture of sugars contained in a fermentation broth. Membranes consisted in a porous polypropylene support impregnated with different kinds of carriers using 2-nitrophenyl octyl ether as solvent. Transport through flat sheet and hollow-fiber membranes was studied as a function of carrier and feed concentration. The results show that a boronic acid derivative yields the highest fructose selectivity and the hollow fiber supported liquid membrane (HFSLM) is more stable than the flat sheet system, also yielding higher fructose selectivities using lower carrier concentration. The HFSLM using a boronic acid as carrier was able to remove fructose selectively from a fermentation broth. Simulations of fructose removal from a fermentation broth were carried out using the experimental results obtained in this work. The results show that fructose removal from the fermentation broth can reduce microorganism inhibition and increase the system performance, although, further improvement in membrane stability and fluxes are still necessary.

Use of Polyhydroxybutyrate and Ethyl Cellulose for Coating of Urea Granules

Fertilizers contain essential nutrients for agricultural growth and development. However, most nitrogen fertilizers are substances with high solubility of ions and are very susceptible to leaching and volatilization. To minimize these losses, an alternative is the creation of a physical barrier around granules. One way is to coat granules with polymers. In the present work urea granules were coated with polyhydroxybutyrate and ethyl cellulose in various conditions in the presence of emulsifiers. The original granules and the final products were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and thermogravimetry, to evaluate the surface morphology, the interaction between the granules and the coating, and the rates of mass change. The rates of urea release in distilled water were measured with a commercial enzyme kit. It is shown that those polymers are effective for coating of granules, leading to reduction of rates of urea dissolution in water.

A HYBRID NEURAL MODEL FOR THE OPTIMIZATION OF FED-BATCH FERMENTATIONS

In this work a hybrid neural modelling methodology, which combines mass balance equations with functional link networks (FLNs), used to represent kinetic rates, is developed for bioprocesses. The simple structure of the FLNs allows the easy and rapid estimation of network weights and, consequently, the use of the hybrid model in an adaptive form. As the proposed model is able to adjust to kinetic and environmental changes, it is suitable for use in the development of optimization strategies for fed-batch bioreactors. The proposed methodology is used to model the processes for penicillin and ethanol production, and the development of an adaptive optimal control scheme is discussed using ethanol fermentation as an example.

An adaptive optimal control scheme based on hybrid neural modelling

A hybrid neural modelling procedure which enables the implementation of an adaptive control scheme for the optimization of fed-batch fermentations is presented. Simulations for the processes of cell mass production and ethanol fermentation by Sacharomyces cerevisae show that, in the presence of modelling errors, the adaptive control leads to nearly optimal results, while open-loop control leads to bad results. Experimental studies show that, for the process of ethanol fermentation by Zymomonas mobilis, a hybrid neural model can be developed with relatively few experimental data and the use of an approximate mathematical model.

Structural Stability of Myoglobin in Organic Media

The structural stability of metmyoglobin in organic solvents and cosolvents was investigated aiming the choice of a suitable medium to perform its dissolution with maintenance of the native folding. The spectroscopic behavior of metmyoglobin solution in UV–Visible and circular dichroism was used to evaluate the solubility and the secondary structure. The results were dependable of the chemical structure of the organic compounds, their polarity and content, in the case of cosolvents. Protic solvents showed better ability than the aprotic ones for the biomolecule dissolution, since they are able to establish hydrogen bonds. Solvents with high polarity usually damage the secondary structure of the protein. Myoglobin was dissolved in pure methanol, ethylene glycol and glycerol. The secondary structure was retained in some extent. The controlled addition of sodium dodecyl sulfate to myoglobin aqueous solution changed the surface moiety of the protein. The complex was extracted to hexane with efficiency of 77%.