Orientadora: Profa. Dra. Graciette Matioli

 Data da Defesa: 26/04/2016



This study was carried out in two research lines with interest to the food industry, converging in regard to the immobilized microorganisms in loofa sponge and other economically feasible substrates. Therefore, the general summary will be presented in two stages.


Stage 1: Biosynthesis of succinoglycan by Agrobacterium radiobacter NBRC 12665 immobilized on loofa sponge and cultivated in sugar cane molasses. Structural and rheological characterization of biopolymer

Introduction: Succinoglycans are acidic extracellular heteropolysaccharides composed of repeated units of octosaccharides with type β connections comprising galactose and glucose, pyruvic acid and succinate. These exopolysaccharides are produced by microorganisms of the Rhizobium, Agrobacterium and Pseudomonas genus. Reveal properties as thickening, stabilizing, emulsifying and texturizing agents. Agro-industrial materials can be used as low-cost carbon sources. For optimization of the production of bioproducts, the technique of cell immobilization has been used in the biochemical processes for various applications.

Aims: Evaluate the production of succinoglycan using free A.radiobacter NBRC 12665 cells and cells immobilized in loofa sponge, with both a conventional carbon source, and agro-industrial waste as a source of substrate. The structural characteristics and rheological properties of the biopolymer obtained were also evaluated.

Material and Methods: Production was performed with different carbon sources, sucrose, glucose, lactose and sugar cane molasses in concentrations of 2.5%, 5.0% and 7.5%. After identifying which carbon source and concentration resulted in the best production, a new assay was performed using immobilized cells. Mixed ion exchange resin was used for depigmentation of succinoglycan obtained from sugar cane molasses. Production operating cycles using free and immobilized cells were performed. Scanning electron microscopy and Fourier Transform Infrared Spectroscopy (FTIR) were used to confirm the immobilization of the microorganism. Succinoglycan characteristics were examined using FTIR techniques, molecular weight determination and rheological properties as a function of concentration (0.5 to 2.0%), temperature (5 to 75 ° C) and pH (2.5 to 10.0).

Results and Discussion: The best results were obtained using immobilized cells and sugar cane molasses (14 g/L) and lactose (9.3 g/L) substrates at 7.5%. Under optimum conditions, it was possible to re-use the matrix to produce in five consecutive operating cycles. Scanning electron microscopy and FTIR confirmed the immobilization of the microorganism in the loofa sponge. FTIR of the biopolymer revealed the presence of carbohydrate residues and carboxylic groups. A depigmentation of 74% in the color of the biopolymer and a reduction of 65% in its turbidity was possible. Succinoglycan obtained from lactose and sugar cane molasses exhibited molecular weights of 2.734 × 106 g/mol and 2,326×106 g/mol, respectively. Rheological analysis of the succinoglycans revealed non-Newtonian and shear thinning behavior of the biopolymer.

Conclusions: The process of producing succinoglycan in operating cycles using immobilized cells and low cost carbon sources, such as sugar cane molasses, may be valuable and feasible for a range of industries.

Keywords: Agrobacterium radiobacter; FTIR; Immobilization; Rheology; Succinoglycan.


Stage 2 - Biosynthesis of industrial enzymes by Alicyclobacillus free and immobilized in different matrices and the use of ultrafiltration in the concentration of the enzymes

Introduction: Enzymes from Alicyclobacillus genus have been studied in many industrial applications due its thermostable characteristics, including proteases and amylases. Amylases hydrolyze α-1,4 glycosidic bonds and represent about a quarter of the market for industrial enzymes. It is desirable that current substrates for the action of amylases can be replaced by economically feasible resources, such as agricultural and agro-industrial by-products. Collagen is an insoluble protein which represents approximately 30% of the total weight of animal proteins. Several cell immobilization techniques have been used to optimize biotechnological processes, such as gel entrapment, adsorption and covalent bonding, which show advantages compared to the use of free cells.

Aims: Production of two industrial enzymes, amylase and collagenase, obtained from Alicyclobacillus acidocaldarius and Alicyclobacillus sendaiensis, respectively, and the immobilization of these microorganisms in order to optimize its enzymatic production capacity. The use of agricultural and agro-industrial by-products as substrates for enzyme production was also in the focus of this research.

Material and Methods: Biosynthesis of industrial enzymes such as amylase and collagenase was evaluated using A. acidocaldarius and A. sendaiensis, respectively. Amylase production was assessed using soluble potato starch, wheat bran and cassava flour substrates. After substrate selection, a characterization study of amylase was performed to analyze the temperature (50 to 85 °C), pH (2.0 to 8.0), activation energy, temperature and pH stability, substrate concentration influence, kinetic parameters by the model Lineweaver-Burk and storage stability under refrigeration. Collagenase production was performed using potato broth as substrate. Different matrices, such as loofa sponge, sponge-alginate and alginate, were evaluated for microorganism immobilization and to optimize the production of enzymes, then it was proceeded their concentration by ultrafiltration membrane.

Results and Discussion: Using wheat bran substrate, amylase enzyme showed an enzymatic activity of 0.45 U/mL. Optimal conditions for temperature and pH were 75 °C and 3.0, respectively. The activation energy was 11.68 kcal/mol. Thermal stability was shown in a range of 55 to 60 °C, and in a wide pH range. Enzyme activity increased with increasing concentration of soluble potato starch in the range of 0.05 to 1% and the kinetic parameters Km and Vmáx were 3.2 mg/mL and 0.5 U/ml, respectively. The production of collagenase by A. sendaiensis was 7.2 U/ml. For amylase, the best results were obtained from the immobilization in loofa sponge and use of ultrafiltration (0.67 U/ml) while, for collagenase extract, free biomass and ultrafiltration were better (13.6 U/ml). The use of ultrafiltration system enabled an average increase of 54% in the activity of both enzymes.

Conclusion: Alicyclobacillus are capable of producing enzymes of industrial interest, with the possibility to use economically feasible substrate. The use of cell immobilization and ultrafiltration were favorable.

Keywords: Alicyclobacillus; Amylase; Collagenase; Cell immobilization; Ultrafiltration; Industrial by-product.



Artigos Publicados Vinculados a Tese:

https://doi.org/10.1016/j.molcatb.2015.08.016 https://doi.org/10.21577/0100-4042.20170109