Improvement of lipase production at different stirring speeds and oxygen levels. Alonso I ; E. Oliveira I ; G. Dellamora-Ortiz I ; F.
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Improvement of lipase production at different stirring speeds and oxygen levels. Alonso I ; E. Oliveira I ; G. Dellamora-Ortiz I ; F. E-mail address : meirelle rdc. Lipase production by a Brazilian wild strain of Yarrowia lipolytica at different stirring speeds and air flow rates was studied. The relationship among lipid consumption, cell growth and lipase production by this microorganism is presented.
The most pronounced effect of oxygen on lipase production was determined by stirring speed. An increase in the availability of oxygen at higher air flow rates led to faster lipid uptake and anticipation of enzyme release into the culture medium.
Lipases E. For this reason, these are the most widely used enzymes in organic synthesis Elibol and Ozer, Promising fields for application of lipases include the biodegradation of plastics, such as polyhydroxyalkanoates PHA and polycaprolactone PCL Gombert et al. Owing to their biotechnological interest, many of these enzymes have been identified, cloned and characterized Eggert et al. Nevertheless, the availability of lipases possessing characteristics appropriate for a specific application is still a limiting factor.
Thus, the identification of conditions to improve lipase production and the search for new enzymes with different characteristics continue to be important research topics. Recently attention has been turned to nonconventional yeasts NCY. Interest in these microorganisms may be due to a variety of reasons, ranging from their use in specific technological applications to the treatment of infections caused by some of them Flores et al.
Yarrowia lipolytica , one of these nonconventional yeasts, is used as a model to study dimorphism Herrero et al. It is able to produce several substances of biotechnological importance, including citric acid Finogenova et al. It is also able to grow on specific substrates Flores et al. In spite of the great interest in both lipases and Yarrowia , much in respect to them is as yet unknown.
Lipase production by Yarrowia lipolytica has been studied in shaking flasks with wild cells Pereira-Meirelles et al. For lipase production by Rhizopus arrhizus it has been reported that dissolved oxygen DO concentration is not the intrinsic factor affecting rates and yields Elibol and Ozer, In addition, for lipase production with Aspergillus terreus , Gulati et al.
In order to improve lipase production and acquire additional knowledge of Yarrowia lipolytica metabolism, this work addresses the influence of OTR on lipase production by a Brazilian strain that produces highly stable lipases Pereira-Meirelles et al. Was determined at 30 o C, pH 7. Was estimated according to Charney and Tommarelli One unit U of protease activity was defined as the amount of enzyme that causes a difference of one absorbance nm unit between the sample and the blank per minute under assay conditions.
Total lipid content was determined according to Frings and Dunn The volumetric coefficient of oxygen transfer was determined by the dynamic gassing out method, as described in Stanbury et al. First, oxygen concentration was decreased to zero inside the fermenter. Lipase activity was measured by the titrimetric and spectrophotometric methods Figs. Comparison of activity profiles obtained with both methods indicates whether lipases titrimetric method are really being produced among the esterases spectrophotometric method.
Otherwise, activity values at rpm were higher than at rpm when the spectrophotometric method was used Fig. Protease was detected from the beginning of cultivation for all stirring speeds Fig. The lowest protease levels were observed at rpm.
Cell growth profiles are presented in Fig 2a. The lowest cell growth 4. Biomass concentration 8. The decrease in biomass concentration after h at rpm was possibly due to cell removal from the medium caused by the formation of foam. Increases in stirring speed to and rpm resulted in lower biomass concentrations 5. Parallel profiles for viability and cell dry weight were found at and rpm Figure 3. Viability remained unaffected at rpm, while at rpm a drastic drop was seen after 24 h.
Total lipid content was measured throughout the experiments Fig. At stirring speeds from to rpm, most of the lipid was consumed during the first 24 h and none was left at around h. At rpm, pH remained almost unaltered during h of cultivation Fig. Similarly to lipid profiles, higher stirring speeds resulted in higher decreases of initial pH. Increases in pH values were observed after h of cultivation. Table 1 summarizes the major process parameters when different stirring speeds were employed.
Although k L a increased with stirring speed, the same behavior was not seen for fermentation parameters. However, volumetric productivity P v values were similar at and rpm. Significantly lower values were obtained for all these parameters at and rpm. For the titrimetric method Fig. For the spectrophotometric method Fig. Protease activity profiles Fig. The effect of air flow rate on cell growth was studied Fig.
Biomass concentration was 8. At about 24 h, cells were no longer in the exponential cell growth phase, and afterwards, shorter periods of diauxie were observed for higher air flow rates. Higher air flow rates resulted in higher lipid consumption rates Fig. An initial decrease in pH was observed for each condition Fig. After variable periods of stabilization, the pH increased at similar time intervals.
Figure 5 also shows that, at rpm, cells stop to grow, independently of the air flow rate when the culture media achieved pH values lower than 4.
Process parameters obtained for different air flow rates are presented in Table 2. Consequently, maximum lipase production P m decreased with an increase in air flow rate. The essential role of oxygen in lipid metabolism and cell growth is well known.
Moreover, lipase production by several microorganisms depends on the availability of oxygen. Although in most cases oxygen seems to favor lipase production, low levels of aeration have also been reported to increase production of the enzyme Corzo and Revah, It is often difficult to distinguish the effects of changes in stirring speed from changes in air flow rate.
The OTR, however, is not constant during fermentation, varying with time due to the change in DO in the fermentation medium. It was clearly observed that an increase in stirring speed, rather than air flow rate, resulted in the most pronounced increase in k L a , as expected for a mechanically stirred vessel.
Maximum extracellular lipase activity was achieved when k L a was These results indicate that there is an optimum k L a value for maximum lipase production by Yarrowia lipolytica. No linear correlation was found between volumetric productivity P v and k L a for lipase production by this microorganism, contrary to the previous findings of Elibol and Ozer for Rhizopus arrhizus.
It is possible to state that these different responses are a consequence of the morphophysiological differences between the microorganisms, reflecting different oxygen requirements for maintenance of their physiological functions. When lipase production was conducted at different stirring speeds, maximum activity values were attained at rpm. Stirring speeds of and rpm produced not only reduced lipase activity levels but also slower cell growth.
These effects were more pronounced at rpm, where culture viability drastically decreased after 24 h, probably due to shear stress promoted by the impellers. Although at rpm cell viability was maintained, the occurrence of a metabolic change is suggested by the lower maximum biomass concentration, which is lower than at rpm. An increase in stirring speed up to rpm resulted in a decrease in maximum lipase activity, assayed by the titrimetric method.
These findings agree well with the results reported for Aspergillus terreus Gulati et al. A stirring speed of rpm seemed to limit oxygen levels, impairing culture medium homogenization and reducing lipid availability or uptake by the cells. As a consequence, lower biomass and lipase levels were obtained. On the other hand, activity detected by the spectrophotometric method was higher at rpm than at rpm. The appearance of this peak at rpm spectrophotometric method , which was not observed with the titrimetric method, indicates the release of esterases, other than lipases into the culture medium.
Thus, it can be suggested that, at rpm, shear stress causes cell disruption, resulting in the release of other enzymes, including esterases and proteases. An increase in protease release into the culture medium was observed for Penicillium restrictum Freire et al. These results corroborate the hypothesis that oxygen availability enhances esterase release.
As previously described for shaking flasks Pereira-Meirelles et al. When oxygen availability was enhanced by either higher stirring speeds or higher air flow rates, faster lipid uptake and early lipase and protease release into the culture medium were observed, suggesting once more that some alteration of cell metabolism or the secretion process is occurring.
These results were similar to those reported for Penicillium restrictum Freire et al. Apparently, factors that accelerate carbon source consumption can possibly cause the release of lipase into the culture medium.
Thus, it can be proposed that lipase release into culture medium is triggered by decrease of external lipid under a critical level. Early enzyme release with an increase in stirring speed has also been observed with Penicillium restrictum Freire et al. Dependence between protease production and pH has been previously demonstrated Mc Ewen and Young, An increase in air flow rate produced an increase of pH at the beginning of cultivation. Thus, it is possible to suggest that procedures that lead to increase of external pH would favor protease production by Yarrowia lipolytica.
A reduction in initial pH of the culture medium was observed when either stirring speed or air flow rate was increased. In spite of this, no deleterious effects lysis, for instance on cell growth, were observed. Despite of pH decrease, cell growth is observed at the beginning of cultivation, even at rpm.
Laboratório de Enzimologia Industrial
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