Non-thermal atmospheric-pressure plasma promotes cellulase production in Neurospora crassa

Microorganisms, particularly filamentous fungi, have become the dominant platforms for industrial enzyme production due to their rapid growth, low cost, and adaptability. However, current production technologies face limitations in yield and cost-efficiency, prompting the need for innovative enhancement strategies. Non-thermal atmospheric-pressure plasma has emerged as a promising tool for stimulating microbial enzyme production. In this study, we have employed micro-surface dielectric barrier discharge (MS-DBD) plasma, which operates in a completely different manner from jet plasma, and evaluated its potential for enhancing the production of cellulolytic enzymes in Neurospora crassa. The extracellular activity of cellulases increased (maximum 10.41 ± 3.44% increase) after MS-DBD plasma treatment. The transcription levels of the four cellulase genes were significantly elevated (highest in the 120 s treatment). The fungal hyphal membrane was depolarized and chemically altered after plasma treatment. The levels of intracellular Ca²⁺ and nitric oxide (NO) were elevated, and a high-affinity Ca²⁺ influx system was activated after plasma treatment. Ca²⁺ channel inhibitors reduced fungal cellulase production by downregulating intracellular NO levels. Plasma-mediated enhancement of enzyme production seemed to occur at plasma energies below 500-600 J. However, the combination of the plasma source type and treatment time can affect the efficiency of enzyme production. We also observed the promotion of fungal cellulase production when jet plasma was applied to larger volume of fungal hyphae. Our results suggest that plasma may be a genetically and environmentally safe tool for fungal enzyme production on an industrial scale and can be applied to bioreactors.