Daniel P. Silva

Evaluation of sugar-cane vinasse treated with Pleurotus sajor-caju utilizing aquatic organisms as toxicological indicators

Toxicity tests with aquatic organisms constitute an effective tool in the evaluation, prediction and detection of the potential effect of pollutants from environmental samples in living organisms. Vinasse, a highly colored effluent, is a sub-product rich in nutrients, mainly organic matter, with high pollutant potential when disposed in the environment. Assays for vinasse decolorization were performed using the fungus Pleurotus sajor-caju CCB020 in vinasse biodegradation study, were occurred reductions of 82.8% in COD, 75.3% in BOD, 99.2% in the coloration and 99.7% in turbidity. The vinasse toxicity reduction was determined by the exposition to the following organisms: Pseudokirchneriella subcapitata, Daphnia magna, Daphnia similis and Hydra attenuata. This work concluded that the systematic combination of P. sajor-caju and vinasse can be applied in the bioprocess of color reduction and degradation of complex vinasse compounds, with reduction in the toxicity and improving its physical-chemical properties.

Continuous immobilized yeast reactor system for complete beer fermentation using spent grains and corncobs as carrier materials

Despite extensive research carried out in the last few decades, continuous beer fermentation has not yet managed to outperform the traditional batch technology. An industrial breakthrough in favour of continuous brewing using immobilized yeast could be expected only on achievement of the following process characteristics: simple design, low investment costs, flexible operation, effective process control and good product quality. The application of cheap carrier materials of by-product origin could significantly lower the investment costs of continuous fermentation systems. This work deals with a complete continuous beer fermentation system consisting of a main fermentation reactor (gas-lift) and a maturation reactor (packed-bed) containing yeast immobilized on spent grains and corncobs, respectively. The suitability of cheap carrier materials for long-term continuous brewing was proved. It was found that by fine tuning of process parameters (residence time, aeration) it was possible to adjust the flavour profile of the final product. Consumers considered the continuously fermented beer to be of a regular quality. Analytical and sensorial profiles of both continuously and batch fermented beers were compared.

Partition Behavior and Partial Purification of Hexokinase in Aqueous Two-Phase Polyethylene Glycol/Citrate Systems

This study dealt with the partition behavior and partial purification of hexokinase (HK) from bakers yeast by liquid-liquid extraction using aqueous two-phase polyethylene glycol (PEG)/citrate systems. First, we investigated the effect of agitation type (vortex and 8 rpm rotation) on the stability of the system, and then the effects of sodium citrate concentration, PEG concentration, and molar mass of PEG on the partition coefficient of this enzyme by using a 25 factorial experimental design. The results of this factorial experiment showed the possibility of a partial purification of HK by using two extraction steps, since the enzyme preferentially migrated to the top phase and the total proteins (mainly contaminants) remained in the bottom phase. The purification factor (PurTOP) of the enzyme in the top phase was 1.87, and the partition coefficient of the total proteins (KProt) was 0.47.

High gravity batch and continuous processes for beer production: Evaluation of fermentation performance and beer quality

This study deals with two innovative brewing processes, high gravity batch and complete continuous beer fermentation systems. The results show a significant influence of the variables such as concentration and temperature on the yield factor of the substrate into ethanol and consequently on the productivity of the high gravity batch process. The technological feasibility of continuous production of beer based on yeast immobilization on cheap alternative carriers was also demonstrated. The influence of process parameters on fermentation performance and quality of the obtained beers was studied by sensorial analysis. No significant difference in the degree of acceptance between the obtained products and some traditional market brands was found.

Overexpression of Glucose-6-Phosphate Dehydrogenase in Genetically Modified Saccharomyces cerevisiae

Glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49) is an abun dant enzyme in Saccharomyces cerevisiae. This enzyme is of great interest as an analytical reagent because it is used in a large number of quantitative assays. A strain of S. cerevisiae was genetically modified to improve G6PD production during aerobic culture. The modifications are based on cloning the G6PD sequence under the control of promoters that are upregulated by the carbon source used for yeast growth. The results showed that S. cerevisiae acquired from a commercial source and the same strain produced by aerobic cultivation under controlled conditions provided very similar G6PD. However, G6PD production by genetically modified S. cerevisiae produced very high enzyme activity and showed to be the most effective procedure to obtain glucose-6-phosphate dehydrogenase. As a consequence, the cost of producing G6PD can be significantly reduced by using strains that contain levels of G6PD up to 14-fold higher than the level of G6PD found in commercially available strains.

Processing of byproducts to improve nisin production by Lactococcus lactis

In the last years, disposal from dairy industries have received a special attention due its polluting power in the environment. For this reason, studies have obtained a positive support to develop different alternatives to recycle milk whey components. One of them is its utilization as culture media, aiming to produce biomolecules with noble applications. Nisin is an extracellular peptide, produced by Lactococcus lactis , this peptide has been applied as a natural additive once it presents broad antibacterial activity. Applications of this bacteriocin include dental care products, pharmaceutical products such as stomach ulcers and colon infection treatment and potential birth control. In batch cultures, L. lactis was performed in two different groups of assays. The first group milk whey was prepared in distilled water in four different concentrations: 100 g/l (S100); 50 g/l (S50); 30 g/l (S30); 10 g/l (S10). In the second group of assays, two supplements were added in milk whey with concentration 100 g/l (S100): (1) 5 g/l yeast extract (A1); (2) 5 g/l yeast extract and 10 ml (v/v) tomato extract. Nisin activity was assayed through agar diffusion utilizing Lactobacillus sakei . The results show that the utilization of powder milk whey with concentration of 100 g/l can be used as a culture medium with supplementation. This media is favorable to develop L. lactis cells and nisin production, reaching an activity of about 4 logAU. Biological processing of milk byproduct can be considered as one of the profitable utilization alternatives, generating high-value bioproducts and stimulates researches for its use. Key words: Nisin, byproducts, Lactococcus lactis, batch culture, powder milk whey.

Effect of Agitation and Aeration on Production of Hexokinase by Saccharomyces cerevisiae

A batch culture of Saccharomyces cerevisiae for the production of hexokinase was carried out in a 5-L fermentor containing 3 L of culture medium, which was inoculated with cell suspension (about 0.7 g/L), and left fermenting at 35 degrees C and pH 4.0. The aeration and agitation were adjusted to attain k(L)a values of 15, 60, 135, and 230 h(-1). The highest hexokinase productivity (754.6 U/[L x h]) and substrate-cell conversion yield (0.21 g/g) occurred for a k(L)a of 60 h(-1). Moreover, the formation of hexokinase and cell growth are coupled events, which is in accordance with the constitutive character of this enzyme. Hexokinase formation for kLa > 60 h(-1) was not enhanced probably owing to saturation of the respiratory pathway by oxygen.

Effect of kLa on the production of glucose 6-phosphate dehydrogenase from Saccharomyces cerevisiae grown by fermentation process

In a 5-L fermentor (NBS-MF 105), Saccharomyces cerevisiae (0.7 g/L) was inoculated into a liquid medium (pH 4.0) containing 17 g/L of glucose, 2.55 g/L of yeast extract, 4.25 g/L of peptone, 2.04 g/L of Na2HPO4 x 12H2O, 4.34 g/L of (NH4)2SO4 and 0.064 g/L of MgSO4 x 7H2O and aerobically cultivated at 35 degrees C for 22 h. Agitation and aeration were adjusted to attain initial kLa values of 15, 60, 135, and 230 h(-1). The glucose 6-phosphate dehydrogenase (G6PDH) productivity (PrG6PDH) obtained for kLa values of 15, 60, 135, and 230 h(-1) was 10.6, 31.8, 30.3, and 23.3 U/([Lx h]), respectively, whereas the cell productivity (Pr(x)) for the same kLa values were 0.24, 0.69, 0.69, and 0.49 g/[L x h], respectively. Thus, both events are coupled and depend on the dissolved oxygen in the medium.

Effect of pH on the stability of hexokinase and glucose 6-phosphate dehydrogenase.

Hexokinase (HK) and glucose 6-phosphate dehydrogenase (G6PDH) are important enzymes used in biochemical studies and in analytical methods. The stability of the enzymes can be affected by several variables, pH being one of them. The effect of pH on the stability of HK and G6PDH was evaluated in this work. Bakers yeast cells were suspended in 50 mM Tris-HCl buffer (pH 7.5) containing 5.0 mM MgCl2, and submitted to disruption by agitation with glass beads and in the presence of protease inhibitors. The cell-free extract was obtained by centrifugation (2880g; 10 min), followed by dilution into the buffers: 0.1 M acetate-acetic acid (pH: 4.0, 4.5, 5.0, or 5.5), 0.1 M phosphate buffer (pH: 6.0, 6.5, or 7.0), and 0.1 M Tris-HCl buffer (pH: 7.5, 8.0, 8.5, 9.0 or 9.5). The residual activity of HK and G6PDH, expressed as micromol of NADPH formed per min, were measured through a period of buffer-enzyme contact from 0 to 51 h at 4 degrees C. It was observed that up to 4 h both enzymes were stable in all buffers used. However, after 51 h HK was stable at pH 6.0 and 7.5, whereas G6PDH was stable at pH 7.0, 9.5, and between 4.5 and 5.5.

Fermentation pH in stirred tank and air-lift bioreactors affects phytase secretion by Aspergillus japonicus differently but not the particle size

Abstract Phytases are mainly produced by filamentous fungi and have great potential for biotechnological use in animal feed treatment, because this enzyme hydrolyzes ester bonds of the phytic acid releasing inositol and inorganic phosphate. The aim of this work was to evaluate the effect of pH on the production of phytase by Aspergillus japonicus in two different bioreactors, known to have different mixing patterns—stirred tank and air-lift bioreactors. The maximum phytase production—53 U/mL—was obtained at 120 h in the stirred tank while in the air-lift the maximum value was 41 U/mL, observed at 144 h. In fermentations evaluated at controlled pH values (3.5, 6.0, and 7.5), the stirred tank was more efficient for production of phytase than the air-lift. Under these conditions, the highest value was measured at 24 h and pH 3.5. These results were not closely related to fungi particle size, because hyphae with a similar diameter (0.51–0.63 mm) and sphericity (0.78–0.87 mm) secreted different amounts of phytase under the conditions studied.