## 3 Discussion
### Robust Pullulan Synthesis by Strain ZH27 after Optimization
After optimization, strain ZH27 exhibited a remarkable pullulan production capability, achieving a pullulan titer of 115.4 \(\pm\) 1.82 g/L at 132 h and an improvement of 105% under the OC compared with the IC (Figure 4). Simultaneously, strain ZH27 exhibited a high pullulan yield (0.77 g/g), productivity (0.87 g/L/h), and DCW (21.4 g/L) through batch fermentation (Figure 4). Notably, more than 90% of sugars were utilized for pullulan synthesis and cell growth, reflecting a high efficiency compared with the reported pullulan titers (48.9-66.8 g/L) produced by _A. pullulans_ strains RBF-4A3 and MR using 15% glucose or sucrose within 144 h [29; 37]. This implies that the efficient pullulan synthesis of strain ZH27 may be attributed to its good adaptability to high osmotic tolerance. The pullulan yield (0.77 g/g) of strain ZH27 using sucrose was close to the reported one (~0.80 g/g) from high pullulan-producing wild-type _A. melanogaster_ strains [13; 18]. As for the productivity (0.87 g/L/h), it achieved a high level via batch fermentation among the wild-type strains, even though it was lower than those from fed-batch fermentation or some mutants [10; 11; 13; 14]. Notably, the lower supernatant viscosity under the OC might result from high expressions of some enzymes (e.g., \(\alpha\)-amylase and pullulanase) that hydrolyze pullulan (Table 2), just as reported by a previous study [38]. More importantly, pullulan produced by strain ZH27 was free of melanin during fermentation (Figure S2), making it a promising candidate for the industrial production of melanin-free pullulan. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Strains**} & \multicolumn{2}{c}{**Fermentation**} & \multirow{2}{*}{**Production g/L**} & \multirow{2}{*}{**Yield (g/g)**} & \multirow{2}{*}{**References**} \\ \cline{2-2} \cline{4-6}  & **Carbon** & & & **Mode and Conditions** & **(g/L/h)** & \\ \hline _A. pullulans_ MG271838 & Sucrose & Batch, 28 \({}^{\circ}\)C, 200 rpm, 168 h & 37.6 (0.22) & 0.63 & [2] \\ _A. pullulans_ H31 & Sucrose & Batch, 28 \({}^{\circ}\)C, 200 rpm, 96 h & 51.4 (0.54) & 0.93 & [3] \\ _A. melanogaster_ 13-2 & Sucrose & Batch, 28 \({}^{\circ}\)C, 250 rpm, 6.5 L/min, 120 h & 78.1 (0.65) & 0.56 & [8] \\ _A. melanogaster_ A4 & Maltose & Fed-batch, 30 \({}^{\circ}\)C, 180 rpm, -3 L/min, & 122.3 (1.02) & 0.40 & [10] \\ _A. melanogaster_ TN1-2 & Sucrose & Batch, 28 \({}^{\circ}\)C, 250 rpm, 6.5 L/min, 132 h & 114 (0.86) & 0.81 & [13] \\ _A. melanogaster_ TN3-1 & Glucose & Batch, 28 \({}^{\circ}\)C, 250 rpm, 6.5 L/min, 132 h & 110.3 (0.84) & 0.79 & [18] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Pullulan derived from fermentation of _Aureobasidum_ strains recently. Swollen Cells, By-Products, and High Expressions of Related Genes May Be Required for Efficient Pullululan Production  
Several cell types may coexist in a certain cultivation period, while the rest may not. The differentiation of cells in strain ZH27 under the OC revealed a shift toward mainly swollen cells after 48 h, contrasting with the control under the IC. The occurrence of chlamospores was minimal within 132 h under the OC (Figure 5), and most swollen cells during this period contained 1-2 large vacuoles, which differed from the main single large vacuole with several small ones in the control (Figure 5). These changes in cell differentiation and vacuole characteristics were likely influenced by variations in nutrients and pH values, impacting pullulan synthesis. Swollen cells emerged as a prominent cell type during pullulan synthesis in _A. pullulans_ NG [40], exhibiting a robust stress defense and antioxidative capabilities compared with yeast-like cells [22]. As shown in Figure 5, swollen cells under the OC seemed to have a stronger and more stable ability to withstand environmental stress, which may require for high levels of pullulan production. Simultaneously, the number and volume of vacuoles in the swollen cells of strain ZH27 are speculated to play a crucial role in pullulan synthesis.