## Results and Discussion
In accordance with the mentioned results, for the acetate negative _Y. lipolytica_ mutants Wratislavia 1.31 and Wratislavia AWG7 cultivated in batch glycerol-based bioreactor experiments, remarkable agitation into the medium (800 rpm) and cascade aeration ensuring DOT > 40% \(\psi\)/\(\psi\) throughout the culture clearly favored \(CA\) in these strains; for instance, under this aeration regime the strain Wratislavia 1.31 achieved a \(CA_{\text{max}}\) = 92.8 g/L, with yield \(Y_{CA/Gly}\) \(\approx\) 0.63 g/g [66]. Concluding, it may be indicated that a variety of external factors like medium pH, salt addition and aeration may critically influence the physiological and kinetic behavior of _Y. lipolytica_ yeast [10; 11; 12; 14; 15]. However, such high discrepancies in the final spectrum of metabolites between shake-flask and aerated/agitated batch-bioreactor experiment that have been observed in the current investigation, is indeed, of notable importance. Given that _Y. lipolytica_ LMBF Y-46 was revealed as capable at rapidly assimilating glycerol and efficiently converting it into citric acid in bioreactor experiments, a pulsed fed-batch bioreactor trial (like the one performed by [PERSON] et al. [57] and [PERSON] et al. [67]), in which culture conditions (_viz._ aeration, agitation, pH) were the same as in the previous batch, was performed (see Figure 4a-c). Significant glycerol uptake occurred, whereas noticeable DCW quantities were synthesized relatively early (\(X_{\text{max}}\)\(\approx\) 21 g/L) with these values being drastically depleted afterwards due to significant cell autolysis (final DCW quantity when the culture stopped \(\approx\)11 g/L; Figure 4a). Despite significant cell autolysis, yeast biomass maintained its viability, since simultaneously with biomass decrease noticeable glycerol uptake occurred and remarkable quantities of \(CA\) were synthesized. When the culture stopped, a noticeable \(CA\) quantity (= 101.1 g/L) with a simultaneous quite high \(CA\) volumetric productivity (\(\approx\)0.61 g/L/h) were recorded (Figure 4b), with a global conversion of citric acid produced per unit of glycerol consumed (\(Y_{CA/Gly}\)) being = 0.46 g/g (Figure 4c). In the above-mentioned fermentation point, iso-citric acid corresponded to 7.8% _w/w_ of \(CA\) formed (= 7.9 g/L). Although at the early steps of the bioreactor culture (\(t\) = 0-30 h after inoculation), the assimilation of glycerol did not contribute importantly to \(CA\) formation (see Figure 4b), finally the representation of \(CA\) produced \(f\) (glycerol consumed) and the linear regression performed for the whole fed-batch culture carried presents interest, as for most of the experimental points glycerol assimilation mostly contributed to the synthesis of \(CA\), whereas the correlation coefficient of the regression (\(r^{2}\) = 0.97) presented a very satisfactory value (Figure 4c). Moreover, \(CA\) production was connected with a high DOT in the medium (Figure 4a), whereas interestingly and in accordance with a number of reports indicated in the literature [40; 68], \(CA\) production linearly increased with the time (\(r^{2}\) = 0.97), resulting in a global \(r_{\text{CA}}\) = 0.58 g/L/h (Figure 4b). Metrics of citric acid production by wild-type, mutant and genetically modified _Y. lipolytica_ strains cultivated in bioreactor experiments with several types of carbon sourcesemployed as substrates, in various fermentation configurations and their comparisons with the current investigation, are depicted in Table 4.  
The maximum \(CA\) quantity obtained in the current investigation (=101.2 g/L) compares favorably with most cultures of wild-type _Y. lipolytica_ strains, while this value is also higher compared with that obtained for various mutants of genetically engineered strains (see Table 4). Concerning the conversion yield \(Y_{CA,Gly}\) values achieved in the batch and fed-batch bioreactor operations of the present study (=0.41-0.46 g/g), they are slightly lower compared with the relevant studies in which other _Y.