**Acknowledgements**

lower concentrations of ATP at 1 and 2 min plasma treatment might be due to 6.8 and 10% increments in calcium concentration, respectively. The increased calcium concentration promoted the hydrolysis of ATP to adenosine diphosphate (ADP) (**Figure 9**). A Ca2+ concentration gradient from 1 to 10 μM, could improve the cell function that regulates cell growth and metabolism to eventually enhance microbial productivity. However, the high concentrations of intracellular Ca2+ can induce cell injury or death [34, 35]. The higher concentrations of ATP in the samples treated by plasma for 3–5 min might be due to an inhibition of ATP hydrolysis caused by the higher cytoplasmic calcium concentration (**Figures 8** and **9**). In addition, any disturbance in environmental conditions would influence the activities of catabolic enzymes, thereby accelerating the accumulation of ATP or ADP [35]. Air cold plasma might lead to the accumulation of ADP in the treated samples within 1–2 min of treatment, and of ATP in the treated samples within 3–5 min of treatment, as suggested by the data in **Figure 9**. The accumulation of ATP or ADP might have immediately affected the glycolysis rate [36], producing different ATP concentrations at the 9- or 21-h period of fermentation, depending

Air cold plasma produces different reactive species in the gas phase [37]. These active species further react with water and produce a variety of biologically active reactive species (RS) in the liquid phase, including long-lifetime RS (ozone, hydrogen peroxide and nitrate ions) and short-lived RS (superoxide, hydroxyl radicals and singlet oxygen) [38]. In our research, these reactive species could increase or decrease the cell membrane potential and open Ca2+

supplementations of 0.5 and 1.5 mM have been shown to induce the increment in ATPase activity [29]. The enhanced ATPase activity would then promote the generation of proton motive force through hydrolysis of ATP [29, 39]. A reduction in the intracellular ATP level can result in the up-regulation of the activities of phosphofructokinase (PFK) and pyruvate kinase (PK) [40]. This would accelerate the glycolytic flux and enhance the NADH level in the central metabolic pathway [41]. At the same time, NADH-dependent alcohol dehydrogenase (ADH) activity might be improved, leading to up-control of the oxidation of NADH to NAD+ [40, 42] (**Figure 1**). Therefore, the NADH concentration obtained from 1 min treatment was reduced over the control because of the lower level of ATP (**Figure 10** 1 min versus **Figure 9**

eraldehyde-3-phosphate dehydrogenase (GAPDH), causing decreased glycerol production and ultimately causing more carbon flux from glycolysis being funneled to ethanol [42–44].

Experimental parameters associated with cold plasma discharge at atmospheric air pressure for enhancing ethanol yield of *S. cerevisiae* has been successfully optimized in this research. The maximum theoretical ethanol yield of 0.49 g/g was predicted by the response model under three optimized parameters (1 min of exposure time, 26 V of power voltage and 9 mL of test sample volume), which was closely consistent with the experimental yield of 0.48 g/g. The model may be used as a reference for modulating the experimental parameters related with dielectric barrier discharge at air atmospheric pressure and a novel approach for improving

cyt (**Figures 7** and **8**, at the beginning of culture). Ca2+

would lower the activity of NADH-dependent glyc-

on the plasma treatment time (**Figure 9**).

170 Fuel Ethanol Production from Sugarcane

channels, consequently improving [Ca2+]

1 min). The oxidation of NADH to NAD+

ethanol yield in bio-manufacturing industry.

**3. Conclusion**

This work was financially supported by the National Natural Science Foundation of China (grant numbers 21246012, 21306015, and 21476032). The author thanks undergraduate students of X Wang, TT Liu and YQ Xiong for attending some works of experiments.
