Avaliação da produção de pectinases por Aspergillus oryzae IPT-301 em processo submerso

Abstract

The cultivation of Aspergillus oryzae IPT-301 in liquid medium was studied to assess the influence of pH and oxygen supply on the fungal growth and the formation of pectinases. A growthlimiting medium was defined with the following concentrations: wheat bran in aqueous extract, 40g/L; glucose, 5g/L; ammonium sulphate, 5g/L; citric pectin (inducer), 20g/L. The inducer was evaluated with respect to the time of addition to the medium. With the addition in 24h of cultivation, maximum biomass concentration was achieved in 70h, almost 48h after reaching the cell growth peak of the control experiment. The maximum enzyme activity (27U/mL) was similar to that of the reference run (24U/mL), but the absence of pectin at the beginning of the process favoured mixing and the control of pH and dissolved oxygen concentration. In this case, the maximum impeller speed in the bioreactor (650 to 600rpm) and the time period that this level of agitation was maintained to provide the best possible medium oxygenation were reduced (30 to 12h), a condition that could favour the preservation of mycelium. Constant and varied medium pH values were evaluated in experiments with the addition of pectin in 24h and without dissolved oxygen concentration control. It was found that a constant pH of 4.0 favoured biomass growth, but the enzyme activity was insignificant (1.4U/mL). When the pH naturally decreased up to a minimum of 2.7, biomass concentration of 4.0g/L and pectinase activity of 24U/mL were attained; in the sequence, however, the activity fell to 2U/mL following the increase of pH observed after 96h. Nevertheless, in the cultivation in which the pH 2.7 was maintained though the final hours of process, the enzyme activity was preserved. These results together with those from enzymatic tests have shown that A. oryzae pectinases loose stability as pH rises. The experiment with constant pH at 2.7 was the only that results in superior pectinase activity with pectin present in the medium from the beginning of the cultivation. Possibly, cell growth limitation (maximum of 3.5g/L) favoured mass transfer and mixing mechanisms and the biomass viability was extended. Cultivations with the pH controlled at 4.0 when the cell growth was more intense and, afterwards, at 2.7 for pectinase production (Experiments T5 and T10) result in high enzyme activities, especially when pectin was added in 24h (43U/mL). In the case, the more effective conditions of oxygen transfer and mixing, allied to the adequate pH for cell growth, allowed that biomass concentration achieved 6.3g/L and, after reducing the pH to 2.7, pectinolytic activity as increased. Experiments with dissolved oxygen concentration at a minimum of 30% of saturation were carried out at two conditions: Experiment B5 - initial pH 4.0 and controlled at 2.7 after reaching this value; Experiment B6 – constant pH up to 24h, followed by reduction to 2.7 and control at this value. The cell concentrations achieved in B5 (6.0g/L) and in B6 (6.8g/L) were similar to that of T10, but their maximum pectinase activities – 106 and 120U/mL, respectively – were the largest in this study. Such results were probably due to the raising fungal metabolism which is associated to the unlimited oxygen availability. Although the time to reach the peaks of biomass and enzyme activity has been increased, the productivity of Experiment B6 was 3.7 times larger than that of the control run. The results of this work have confirmed that, in cultivations of A. oryzae, the pH and the oxygen supply decisively affect both cell growth and pectinase production. The defined operational conditions provided the reduction of cell growth – allowing, however, the achievement of high enzyme activities – and, consequently, an efficient control of the studied parameters.