## 2 Results  
### Long-Lasting Growth of the E. magnusii Yeast  
Figure 1 shows the growth curve of _E. magnusii_ yeast upon assimilating 1% glycerol. The dynamics of cell growth displayed classical logarithmic dependence with a well-pronounced lag phase (from 0 to 6 h of cultivation), a phase of logarithmic (exponential) growth (from 6 to 20 h of cultivation), a diauxic shift phase defining the transition of the population into the stationary growth phase (from 22 to 30 h of cultivation), early stationary growth phase (from 30 to 48 h of cultivation), and late (48 h) and stationary phase (from 48 to 200 h of cultivation) (Figure 1).  
Microscopic analysis of the morphology of _E. magnusii_ cells in various growth stages showed that it varies greatly. In the logarithmic growth stage, the cells are round and oval, sometimes fusiform or pear-shaped, either with well-visible buds (in the process of division) or budded daughter cells (Figure 2A), whereas in the stationary stage (after 30 h of cultivation) the number of daughter cells decreased to 5% of the total number (Figure 2B). The population of the exponential phase had about 4% actively dividing cells with buds at the poles, about 10% cells with detached immature buds, and about 20% young daughter cells, which are smaller and regularly spherical (Figure 2A). In the stationary growth phase, the number of actively budding and young cells nearly halved, there was some heterogeneity in the cell population, some cells changed their shape, and some single pseudomycial and mycelial forms appeared (Figure 2B). It should be noted that during long-lasting cultivation, the nuclei of _E. magnusii_ cells changed their shape (increased by 1.5 times). In the stationary phase, the nuclei increased in their volume and became more round (Figure 2C,H). The morphometric features of this growth stage indicated the intensive growth and fission of _E. magnusii_ cells (Table S1 in the Supplementary Section). At the same time, the population was highly heterogeneous without any signs of pathological changes or inclusions (Figure 2D). Moreover, the cells at this stage got a distinct thickened cell wall, large vacuoles, and small dense cytoplasmic inclusions, which are likely to be autophagic vacuoles or bodies (Figure 2H). It should be emphasised that the energy status of the cells in the logarithmic and stationary growth phases, confirmed by potentiometric staining, was maintained at a high level (Figure 2E,F).  
Summarising all the obtained results including the changes in morphology, along with the functional heterogeneity of the _E. magnusii_ culture, shown before [21], we studied the lipid amount and composition in the mitochondria at different growth stages upon ageing.  
Figure 1: The growth curve of _E. magnusii_ yeast in glycerol-containing medium grown for 260 h. Absorbance was assayed every two hours in cell suspension at the wavelength of 590 nm. The curve is representative of four biological experiments. (The figure was partially used in the article [Microorganisms 2020, 8, 91; doi:10.3390/microorganisms8010091]).  
Figure 2: Micro images of the _E. magrusii_ cells in the logarithmic (**A**,**C**,**E**,**G**) and late stationary (**B**,**D**,**F**,**H**) growth stages. (**A**,**B**)—The cells were stained with neutral red; (**C**,**D**). Fluorescent micro-images of the cells, labelled with 0.3 \(\upmu\)M DAPI for DNA. (**E**,**F**) the potential-dependent stain of the mitochondria in _E. magrusii_ cells by 5 mM JC-1. The regions of high mitochondrial polarization are bright yellow due to the concentrated dye. The cells were examined after 30 min. The incubation medium contained 0.01 M phosphate buffer saline (PBS) and 1% glycerol, pH 7.4. To examine the Rh123-stained preparations, filters 02, 15 (Zeiss) were used (magnification 100\(\times\)). Photos were taken using an AxioCam MRc camera. (**G**,**H**)-ultrastructure of _E. magrusii_ cells. Nu—nucleus; V—vacuole; Mt–mitochondria; CW–cell wall.