Sterilization Mechanism And Mutation Of Klebsiella Pneumoniae By Cold Plasma At Atmospheric Pressure

Recently, cold plasmas at atmospheric pressure have been applied widely in microbial extermination. Most research on the interaction of plasma discharges with bacteria has concentrated on germicidal efficiency and discharge conditions. However, little attention is paid on sterilization mechanism and positive effect of plasma on microbial cells when microbial cells are treated by plasma. Klebsiella pneumoniae is widely used to produce 1,3-propanediol (1,3-PD), an important chemical. It can be used as a monomer of polyesters, polyethers or polyurethanes due to its superior characterics of stretching and stretch recovery. Therefore, it has a large commerical potential. Microbial production of 1,3-PD is receiving more and more extensive attention due to its advantages, such as mild fermentation condition, simple operation, less byproducts, protected environment and so on. However, there still remain some disadvantages with using the wild-type strain to produce 1,3-PD, such as its low concentration and productivity of 1,3-PD owing to the multiple inhibitions by glycerol and other metabolic products, e.g. acetate, lactate and ethanol. In this paper, research on physical-chemical factor in discharge of cold plasma at atmospheric pressure and effect of plasma on biomacromolecule were performed, which not only explained the mechanism of microbial sterilization by plasma, but also provided more opportunities to apply plasma in microbial mutagenesis. Additionally, dielectric barrier discharge (DBD) plasma in air at atmospheric pressure was used to induce and mutagenize cells of K. pneumoniae for increasing the concentration and productivity of 1,3-PD.Firstly, physical and chemical factors produced by plasma discharge were investigated at 13.0 kV of peak voltage,7.0 kHz of discharge frequency and 3 mm of discharge gap. The results indicated that ultraviolet (UV) radiation and reactive oxygen species (ROS) may be produced during plasma discharge. Plasma discharge also may induce the change of ROS concentration in treated cells.Secondly, inactivation mechanism of DBD plasma at atmospheric pressure was studied. The results showed that polysaccharides and proteins on cell membrane could be degraded by plasma in helium at atmospheric pressure, forming amino acids, polypeptides, maltose, glucose, acetic acid and so on. Effects of DBD plasma in air at atmospheric pressure on plasmid DNA and base guanine were also investigated. It is evident that superhelical form of plasmid DNA could be converted to relaxed circular form and linear form by plasma exposure, leading to loss of order property of DNA. In addition, the melting temperature (Tm) of plasmid DNA was decreased owing to increase of damaged hydrogen bond with plasma treatment time. Corresponding, mutation frequency increased firstly, and then decreased with a decrease of transformation rate of the plasmid treated by plasma discharge. Effects of plasma on guanine were investigated by Fourier Transform Infrared (FTIR) spectroscopy. The results indicated that the peak heights of N-H and C=O bond in guanine were decreased with plasma treatment time, while the peak height of C-H was increased. Additionally, a new peak of O-H bond was observed.Thirdly, inoculums of K. pneumoniae CGMCC 2028 were pre-treated by DBD plasma at air atmospheric pressure. The results showed that the highest production of 1,3-PD was observed when cells treated by DBD plasma for 4 min were cultured in seed medium containing 6% glycerol. The concentration of 1,3-PD in flask fermentation was 16.95 g/L. Corresponding, the specific activities of glycerol dehydrogenase (GDH), glycerol dehydratase (GDHt) and 1,3-propanediol oxidoreductase (PDOR) reached 0.16 U/mg,10.8 U/mg and 0.61 U/mg, respectively. The 4-min of plasma inducement was chosen as an optimized exposure time. In batch fermentation at 6% of glycerol concentration, DBD plasma-treated cells had a 59% increase in both 1,3-PD concentration (23.8 g/L versus 15 g/L) and productivity (1.19 g/(L·h) versus 0.75 g/(L·h)) over untreated cells, while the yield of 1,3-PD was the same as untreated cells (0.53 versus 0.52 mol/mol). In fed-batch fermentation at 4% of initial glycerol concentration, the final concentration, productivity and molar yield of 1,3-PD were 46.8 g/L, 0.49 mol/mol and 1.51 g/(L·h), respectively. The volumetric productivity of 1,3-PD under plasma inducement increased 40% more than that of the control (1.08 g/(L·h)).Finally, cells of K. pneumoniae CGMCC 2028 were mutagenized by DBD plasma at air atmospheric pressure. The optimized conditions of plasma exposure were 3 mm of discharge gap,47.5 W of output power and 120 s of exposure time. The highest positive mutation rate was obtained when cells of K. pneumoniae were irradiated by plasma under the optimized conditions. A stable mutant (Kp-M2) with a enhanced level of 1,3-PD concentration was obtained by separate selections in different seed media with different concentrations of glycerol and 1,3-PD. The final concentration, yield and productivity in batch fermentation by Kp-M2 were 19.9 g/L,0.63 mol/mol and 2.84 g/(L·h), respectively, increasing 23%,26% and 58% compared with the control. The specific activities of GDH, GDHt and PDOR at the exponential phase during batch fermentation were 0.65 U/mg,14.4 U/mg and 1.61 U/mg, respectively, increasing 6-fold,0.57-fold and 11-fold, compared to those of wild type. The metabolic flux of glycerol in Kp-M2 was redistributed toward the reductive branch. In fed-batch fermentation, the final concentration, yield and productivity in Kp-M2 were 76.7 g/L,0.58 mol/mol and 2.13 g/(L·h), respectively, increasing 56%,55% and 35%.Kinetics model of 1,3-PD production from glycerol was investigated in batch fermentation by the mutant Kp-M2. The results showed that the model could provide a reasonable description for the batch fermentation of 1,3-PD. Culture parameters of the mutant Kp-M2 in batch fermentation were optimized by Response Surface Methodology (RSM). The optimum conditions for 1,3-PD production were 57 g/L of initial glycerol concentration,7.3 of initial pH value,10 h of inoculation time,37℃of culture temperature,150 rpm of rotation speed. The final concentration of 1,3-PD was 29.5 g/L with a productivity of 2.11 g/(L·h), corresponding to a yield of 0.65 mol/mol in batch fermentation under the optimum conditions. Additionally, the concentration of 1,3-PD was 92.0 g/L with a productivity of 2.56 g/(L·h), corresponding to a yield of 0.56 mol/mol in fed-batch fermentation.