Biological Effect Of Dielectric Barrier Discharge (DBD) Air Plasma On Yeast Cells

Atmospheric pressure Dielectric Barrier Discharge (DBD) air plasma composes of electron, ions, excited atoms and molecules, free radicals, and other active particles, as well as electric field and ultraviolet radiation, and thus is an efficient way for non-invasive surface sterilization. In addition, the plasma with low temperature is easy to be operated. Thus, it has attracted more and more attention. However, the mechanism of the plasma on micro-organisms sterilization is still not clear. On the other hand, the plasma, as a source of various active particles, has rarely been used as a mutagen for strain development. In this study, the biological effect of the plasma on the yeast Saccharomyces cerevisiae were investigated, and its mutagesis on ethanologenic strains, S. cerevisiae ATCC 4126 and Candida shehatae CICC1766 were further examined.1. The biological effect induced by the plasma on S. cerevisiae. The plasma was obtained at a root-mean-square (RMS) voltage of 12.0 kV and a frequency of 20 kHz. S. cerevisiae cells were suspended in water with a pool of 2 mm in depth. A discharge gap of 4 mm between the surface of the sample and the tip of the upper electrode was applied. After plasma discharge, the cells showed extensive death. The longer the treatment time, the more intense stained color the cells exhibited. And in the meantime, the plasma-treated cells exhibited significant increases in extracellular protein and nucleic acid concentrations, suggesting that these macromolecules were released from the cells, possibly via the leakages in the cell membranes. In addition, retardance in cell growth also occurred, and the plasma-treated cells showed cell cycle arrested at their G1 phase, and this arrest effect increased with the increase of the treatment time, indicating the presence of intracellular DNA damage.2. Oxidative stress induced by the plasma on S. cerevisiae. For the plasma-treated S. cerevisiae cells, the concentration of reactive oxygen species (ROS) within the cells increased significantly, leading to the activation of total intracellular and extracellular antioxidant capability (T-AOC), and intracellular glutathione reductase (GR). Intracellular malondialdehyde (MDA) content also increased with the increase of the plasma treatment time. After 3 h of re-incubation following plasma treatment, the specific activities of intracellular superoxide dismutases (SODs) and catalase increased. These results indicated that the plasma might have inflicted oxidative damage on the yeast cells and exerted oxidative stress, witch could be the cause of cell damage, or even cell death.3. The stimulation effect of the plasma on ethanol production of S. cerevisiae. The plasma was used to improve the ethanol production of S. cerevisiae isolates when they were cultured under high temperature and substrate concentration conditions. Using the medium containing 30 g/L glucose and incubated at 40℃,8 clones were isolated as positive strains, and their ethanol production was enhanced from 13.7 to 40.4%, compared to their wild type. When they were cultured with the medium containing 100 g/L glucose under 37℃,5 isolates could maintain their improved ethanol production, which was enhanced from 2.5 to 6.6%, compared to their wild type. When these isolates were subsequently cultured under 100 g/L and 300 g/L glucose at 40℃, their ethanol production couldn't maintain. More plasma-treated clones with improved ethanol production were selected with 300 g/L glucose medium and under 40℃; however, they couldn't maintain their improved ethanol production with subcultures, either, indicating that the plasma might have some stimulate effect on S. cerevisiae cells for a while, and further work needs to be done to obtain stable positive mutants.4. The mutation effect of the plasma on the xylose-fermenting yeast C. shehatae. The plasma was used to enhance ethanol production of the xylose-fermenting yeast, C. shehatae CICC1766. Three stable mutants, C80828, C81015 and C81020 were isolated by 15 subcultures that switched between TTC medium incubation and flask culture. Among them, C80828 and C81015, which showed more intense red color on the TTC plate, and exhibited higher ethanol production, compared to the wild type were designated as positive mutants. With the medium containing 50 g/L xylose, C80828 enhanced ethanol production by 8.2%, and C81015 by 36.2%, compared to their wild type (P<0.05). However, the biomass production of C81015 was significantly lower than that of its wild type, indicating its specific ethanol productivity of C81015 increased significantly. At the same time, the specific activities of NADH- and NADPH-linked xylose reductases (XR) and NAD+-linked xylitol dehydrogenase (XDH) of C81015 (as measured in the cell extract) increased by 34.1%(P<0.01),61.5%(P<0.05) and 66.3%(P<0.01), respectively, compared to its wild type. However, no difference in ethanol production from glucose between C81015 and its wild type was detected. In contrast, C81020 was a negative mutant and showed decrease in ethanol production, with significantly lower XR and XDH specific activities. These results indicated that the DBD air plasma might affect with XR and XDH to influent the ethanol production of mutant C81015. In addition, more biomass was harvested in xylose-containing medium of C81020. SDS-PAGE of the total intracellular proteins of C81020 showed bands that differed from its wild type, suggesting that the plasma might have genotoxic effect on the C. shehatae.5. The mutagenesis of the plasma on key enzymes for xylose consumption. The genes XYL1 and XYL2, which encodes for XR and XDH, respectively, in the mutant C81015 and the wild type were sequenced. The XYL1 sequence of C81015 had three nucleotide changes compared to its wild type, resulting in the codon changes ATT->GGT and AAT→AAG, which corresponds to two amino acid substitutions, Ile309→Gly309 and Asn314→Lys314. In the XYL2 sequence of C81015, six nucleotides were found to be different from the wild type, covering the beginning, middle and end parts of the sequence. However, only the nucleotide changes in the middle of the sequence, AGT→GGT and TTC→CTC, resulted in amino acid substitution, Ser185→Gly185 and Phe189→Leu189, respectively.In conclusion, ROS produced in the DBD air plasma was one of the most important factors causing cell damage, even cell death. The DBD air plasma can induce oxidative stress in yeast cells, cause DNA damage, protein leakage and gene changes, which can be used to generate mutants from yeast with improved ethanol production, either regular S. cerevisiae or xylose-fermenting C. shehatae in addition to sterilization.