## 2 Materials and Methods
### ARTP Mutagenesis and Screening  
The general workflow involved the preparation of _S. fradiae_ spore suspensions followed by ARTP treatment (Si Qing Yuan Biotechnology Co., Ltd./now Tmax Tree Co., Ltd.; Wuxi, China) and pre- and rescreening (Figure 1). The ARTP system (model: ARTP-IIS; weight: 95 kg; voltage: 220 V-50/60 Hz 500 VA; size: 73 cm \(\times\) 65 cm \(\times\) 69 cm; Figure 2) needs high-purity helium (>99.99%) as a working gas and a typical electric socket as a power source. Inside the ARTP operation chamber, the helium flowing through the discharge region between the two electrodes is ionized by the radio frequency electric field and then acts on the microbial sample fixed on the metal plate sheet on a regulating platform via the nozzle. Since the breakdown voltage is not high (100-200 V), the plasma maintains discharge consistency, derives little ultraviolet radiation, and combines with the cooling of the cathode to maintain a biocompatible gas temperature. The continuously flowing gas seldom mixes with the surrounding air, thereby minimizing the production of germicidal ozone. It has been reported that the generation of reactive chemical species (He*, He\({}_{2}\)*, He+, He\({}_{2}\)+, and N\({}_{2}\)+) was considered as the biggest cause of physical plasma mutagenesis [28; 29], and therefore requires careful adjustment of the plasma-generating parameters. The ARTP manufacturer provides a standard value for each adjustable parameter to meet the conditions of biocompatibility and produce enough active chemical substances [9]. Firstly, it is recommended to use a gas flow rate of 10 SLPM (standard liters per minute) or above to prevent the gas from combining with the surrounding air to produce germicidal ozone. Further research showed that when the gas flow rate is between 5-30 SLPM, the production of active materials is proportional to the gas flow rate [8]. Secondly, the suggested value of the distance between the sample and nozzle is 2 mm, which was used in almost all reports. In line with increasing distance (2-10 mm), the active chemical substances generate decreased sharply and not enough to cause damage to the cells [8]. Thirdly, it was found that the temperature was within a biologically compatible range between 36 \({}^{\circ}\)C and 57 \({}^{\circ}\)C when the energy was between 40 W and 200 W [9]. In the early study, the radio frequency power input of 40 W was applied in obtaining high-yield butanol _Clostridium acetobutylicum_ strain [30]. Finally, for different species, the recommended values for the treatment time are different, including bacteria (15-120 s), _Actinomycetes_ (30-180 s), fungi (60-360 s), yeast (30-240 s), and microalgae (5-150 s) [8]. Early research showed that along with the increasing time (0.5-10 min), more DNA damages in cells was produced under the same conditions as other parameters [28]. rate = 10 SLPM, 2 mm = distance between sample and nozzle, radiofrequency power input = 40 W, and ARTP treatment times = 0-210 s. Untreated spores were used as controls. Several constants were used to evaluate the effects of multiple ARTP mutagenesis rounds, and were calculated based on Equations (1)-(5).