Effect of Plasma-Activated Water on the Cellulase-Producing Strain Aspergillus niger A32

To investigate the effect and mechanism of plasma-activated water (PAW) on Aspergillus niger, PAW was prepared using a needle array–plate dielectric barrier discharge plasma system. The concentrations of long-lived reactive oxygen and nitrogen species (RONS), namely, H2O2, NO2−, and NO3−, in the PAW were 48.76 mg/L, 0.046 mg/L, and 172.36 mg/L, respectively. Chemically activated water (CAW) with the same concentration of long-lived RONS was also prepared for comparison. A. niger A32 was treated with PAW and CAW. After treatment, the treated strains were observed and analyzed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to screen probable mutants. The results indicated that the pH, conductivity, and ORP values of PAW were 2.42, 1935 μS/cm, and 517.07 mV, respectively. In contrast, the pH and ORP values of CAW were 6.15 and 301.73 mV, respectively, which differed significantly from those of PAW. In addition, the conductivity of CAW showed no change. SEM and TEM analyses revealed that A. niger A32 treated with CAW exhibited less damage compared with the control. In contrast, A. niger A32 treated with PAW showed significant shrinkage, deformation, and exudate attachment over time. Following PAW treatment, after four passages, a high cellulase-producing stable mutant strain A-WW5 was screened, exhibiting a filter paper enzyme activity of 29.66 U/mL, a cellulose endonuclease activity of 13.79 U/mL, and a β-glucosidase activity of 27.13 U/mL. These values were found to be 33%, 38%, and 2.1% higher than those of the original fungus sample, respectively. In total, 116 SNPs and 61 InDels were present in the genome of the mutant strain A-WW5. The above findings indicate that the impact of PAW on A. niger is not only attributed to long-lasting H2O2, NO2−, and NO3− particles but also to other short-lived active particles; PAW is expected to become a new microbial breeding mutagen.