Development of a Novel Compact Air-Driven Cold Plasma Instrument for Efficient Microbial Mutagenesis: A Case Study on Enhanced Pigment Production

Development of a Novel Compact Air-Driven Cold Plasma Instrument for Efficient Microbial Mutagenesis: A Case Study on Enhanced Pigment Production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of a Novel Compact Air-Driven Cold Plasma Instrument for Efficient Microbial Mutagenesis: A Case Study on Enhanced Pigment Production Naicai Zhong, yuan chen, Wenfeng PAN, Hailin Meng, kun Liang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8868583/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Low-temperature plasma provides a chemical-free method for random mutagenesis, however, conventional systems often require bulky equipment and rare gases, which limits their accessibility. Here, we present a Compact Plasma Mutagenesis Instrument (CPMI) that operates in ambient air at 2–8 W, reducing energy consumption while increasing the plasma–sample interaction volume. Mechanistic studies indicate that CPMI induces DNA lesions via reactive oxygen and nitrogen species, leading to base oxidation, strand breaks, and adduct formation. The Application of CPMI to Talaromyces albobiverticillus produced mutant strain CY110. This strain exhibited over 4.5-fold of the wild-type strain extracellular pigment production, significantly decreased citrinin levels, and enhanced antioxidant activity relative to the parental strain. Unlike chemical mutagens or UV irradiation, CPMI generates no toxic byproducts, and unlike genome-editing tools such as CRISPR/Cas, it offers a non-genetically modified organism (GMO) approach suitable for food-grade applications. Collectively, these results establish CPMI as an accessible, energy-efficient, and regulation-compliant mutagenesis platform that complements existing synthetic biology toolkits. Unlike traditional systems, the air-driven CPMI eliminates noble gas reliance, providing a cost-effective and portable platform for industrial microbial optimization. Compact Plasma Mutagenesis Instrument (CPMI) Plasma–DNA interactions Random mutagenesis Microbial pigment biosynthesis Synthetic biology toolbox Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Microbial fermentation offers a sustainable and efficient pathway for producing natural pigments and functional metabolites, serving as a vital alternative to synthetic dyes which face increasing regulatory bans and safety concerns globally ( 1 – 3 ). However, the low productivity of wild-type strains remains a significant bottleneck for large-scale biomanufacturing. While existing strain-improvement strategies such as chemical mutagenesis, radiation, and CRISPR-based genome editing are widely used, they are often hindered by hazardous waste production, regulatory constraints in food-grade applications, or the requirement for sophisticated genetic toolkits ( 4 – 6 ). Consequently, there is an urgent industrial demand for a non-GMO, efficient, and cost-effective mutagenesis platform.Low-temperature plasma, which generates abundant reactive oxygen and nitrogen species (RONS) at ambient temperature, has emerged as a promising physical mutagenesis approach for rapid selection of high-yield or phenotypically desirable mutants ( 6 ). Compared with chemical and conventional physical mutagens, plasma mutagenesis offers several advantages, including no chemical residues, rapid treatment of large cell populations, and a broad mutation spectrum. Importantly, mutants produced by random plasma mutagenesis are generally not classified as GMOs ( 7 ). Previous studies have demonstrated the effectiveness of atmospheric-pressure radio-frequency plasma (ARTP) technology as a representative platform ( 8 ). Despite these advantages, existing plasma mutagenesis systems have limitations that hinder their widespread application in food-grade microbial engineering and biomanufacturing. Commercial ARTP systems often operate at high power (~ 180 W), generating substantial heat that reduces cell viability and requires complex cooling or heat-dissipation setups. Many systems also rely on inert or rare gases (e.g., helium) to stabilize the discharge, increasing operational costs. In addition, the large device footprint complicates integration into sterile workspaces, raising the risk of contamination. These challenges underscore the need for a low-energy, low-temperature plasma mutagenesis platform capable of operating under standard laboratory conditions (without specialized gases), while satisfying safety, controllability, and cost-effectiveness requirements for food-grade strain improvement ( 9 – 12 ). To address these challenges, we developed a miniaturized, air-based, low-power cold plasma mutagenesis platform tailored for synthetic biology and other biofabrication applications. This system operates without inert gases, maintains high cell viability, and generates a multi-site DNA damage spectrum under controlled, low-energy conditions. Aligned with the "tool-driven" philosophy of synthetic biology, we systematically characterized its mutagenesis mechanism using an SOS–lacZ DNA damage reporter, molecular electrophoresis, and mass spectrometry analyses, This allowed us to elucidate the types of DNA lesions and their genetic consequences under defined plasma discharge conditions. To demonstrate practical utility, we applied this platform to fungal pigment production, showing that it can simultaneously enhance target polyketide pigment yields while suppressing safety-relevant secondary metabolites, e.g.,citrinin. This dual optimization highlights CPMI's potential as a non-GMO strategy for microbial production of food-grade natural products and exemplifies a complete synthetic biology "tool–mechanism–application" workflow. As detailed in this study, the CPMI achieved a 4.5-fold increase in pigment production while maintaining superior biosafety and energy efficiency (Section 2.1 ). 2. Results and Discussion 2.1 Development and Engineering Characterization of the Novel CPMI System We developed a novel, laboratory-scale Compact Plasma Mutagenesis Instrument (CPMI), functioning as a miniaturized long-gap glow discharge reactor (Fig. 1 ). A core engineering objective was to overcome the limitations of conventional Atmospheric and Room-Temperature Plasma (ARTP) systems, which typically rely on high-power inputs and rare gas logistics. As summarized in Table 1 , the CPMI system exhibits a significant advancement in energy efficiency and operational portability. While conventional ARTP systems (dimensions: 72 × 62 × 72 cm) generally require approximately 180 W and external gas supplies (Ar or N₂), the CPMI (30 × 20 × 30 cm) operates at a significantly lower power range of 2–8 W using ambient air as the discharge medium. This represents a > 95% reduction in power consumption and eliminates the need for complex gas flow and active cooling systems ( 13 ). Its compact footprint allows for direct integration into microbiological clean benches, effectively minimizing cross-contamination risks. The CPMI utilizes a high-voltage excitation source (30–100 kV, > 40 kHz) capable of maintaining a stable plasma plume across an 8-cm gap through insulated electrode surfacing, which enhances the plasma-sample interaction volume and discharge stability. Table 1 Comparative Engineering Parameters of CPMI and Conventional ARTP Systems. Parameter CPMI (This Study) Conventional ARTP Advantage Operational Power 2–8 W ≈ 180 W 95% Energy Saving Working Medium Ambient Air Argon / Nitrogen No gas logistics required Cooling Method Self-cooling (Ambient) Active Gas/Head Cooling Simplified maintenance Footprint 0.018 m³ (Compact) 0.32 m³ (Bulky) Benchtop/Clean bench compatible To ensure experimental traceability and interlaboratory reproducibility, we established a standardized operating procedure (SOP) for CPMI-mediated mutagenesis; the complete step-by-step protocol and parameter tables are provided in Supplementary Information (Section S1) and summarized in Fig. 1 d. Briefly, two to three morphologically uniform colonies or a calibrated spore suspension (~ 1 × 10^6 CFU·mL⁻¹) are loaded (10 µL per sterile carrier) and exposed under fixed geometry (standoff 2–6 cm, normal incidence) to CPMI at low power (2–8 W) for short durations (30–150 s). Samples are recovered immediately, serially diluted, and plated for CFU enumeration; putative phenotype-positive colonies are subjected to microplate screening, product quantification and ≥ 10-generation stability testing. We defined four key control points (KCPs) to minimize variation: (i) uniform sample spreading to avoid puddling and edge effects, (ii) maintenance of a stable standoff and normal incidence during exposure, (iii) rapid post-exposure recovery to limit drift, and (iv) standardized dilution and plating procedures for comparable survival/mutation estimates. Using this workflow, we derived survival and apparent-mutation curves (see next section) to identify an operational window that maximizes mutational activity while limiting cytotoxicity. Unlike ARTP systems, that rely on rare gases such as helium or argon, CPMI uses ambient air as the discharge medium. This eliminates the operational costs and chemical interferences associated with external gases and simplifies device configuration ( 12 ). The device generates minimal heat and operates stably at room temperature without additional cooling, preventing thermal damage to heat-sensitive strains. Growth inhibition assays with Salmonella typhimurium TA1535/pSK1002 showed that 2 W treatments did not significantly affect cell viability (G > 0.5), whereas 4–8 W treatments for 40–120 s markedly reduced survival (p < 0.05), and 6–8 W treatments for 100–120 s nearly caused complete cell death (G ≤ 0.10) (Fig. 2 ) ( 14 ). These results indicate that CPMI enables effective mutagenesis under low-energy conditions while providing a tunable operational window suitable for modular synthetic biology and other biofabrication applications. Mechanisms of DNA DamagePlasma discharge generates diverse reactive oxygen and nitrogen species (ROS/RNS), along with high-energy electrons and ion flux. These short-lived species can induce base oxidation, phosphodiester bond cleavage, depurination, and DNA strand breaks, triggering the cellular SOS repair response ( 15 – 17 ). Using a TA1535/pSK1002 strain harboring an SOS–lacZ reporter, even a mild 2 W, 2-min CPMI treatment significantly increased β-galactosidase activity (p < 0.05)-indicating detectable DNA damage under low-power conditions ( 14 ). Heatmap analysis (Fig. 4 C) revealed that increasing CPMI power from 4 to 8 W accelerated DNA damage, with higher β-galactosidase activity, earlier peak induction times (120 s → 60–40 s), and steeper slopes, These changes reflect enhanced damage accumulation and faster SOS response activation. At 6–8 W for 100–120 s, biomass (OD 600) approached that of the sterile control and G values ≤ 0.10, indicating near-total cell death. These observations suggest that high-power, long-duration CPMI treatment exerts bactericidal effects, likely via extensive DNA fragmentation, membrane disruption, or irreversible metabolic damage ( 17 , 18 ). Gel electrophoresis (Fig. 4 a-b) showed that 2–8 W treatments for 2 min caused minor single-strand breaks or recombination events, whereas 8 W for 10 min produced substantial fragmentation and smearing, indicative of severe structural DNA damage unsuitable for mutagenesis ( 15 ). Compared with conventional ARTP, CPMI induces more pronounced DNA damage at lower power, achieving higher chemical efficiency per unit energy. ARTP typically requires > 100 W and inert gases as the discharge medium, producing mainly ROS with limited reactive nitrogen species. In contrast, CPMI uses ambient air to generate both ROS (·OH, O₃) and RNS (·NO, ONOO⁻),, creating a synergistic ROS–RNS effect. Different species induce distinct DNA lesions: (·OH) primarily causes base oxidation (e.g., 8-oxo-dG), RNS promotes depurination and abasic site formation, and high-energy ions/electrons induce single- and double-strand breaks ( 19 ). Comet assays and electrospray ionization mass spectrometry(ESI–MS) analyses revealed a power- and time-dependent increase in DNA fragmentation (Figure S1 , Tables S1–S2). At 4–8 W for ≥ 40 s, comet tail moments were significantly elevated (p < 0.01), and oligodeoxynucleotides dA₈, dT₈, dG₈, and dC₈ exhibited multiple low m/z peaks indicative of fragmentation. Notably, dA₈ and dT₈ also showed high m/z peaks, suggesting polymerization or adduct formation. Base stability followed the order dC₈ > dG₈ > dT₈ > dA₈, reflecting differential sensitivity to oxidative and nitrosative stress. Mild CPMI conditions primarily caused repairable lesions, enhancing mutational diversity without lethality, whereas high-power, prolonged treatments induced extensive double-strand breaks and cell death, demonstrating a tunable “dual-effect” property. CPMI induced stronger degradation of dT₈ compared with ARTP, suggesting broader substrate reactivity and higher mutagenic potential. Overall, the air-based ROS–RNS synergistic mechanism underlies CPMI’s ability to generate a broader mutational spectrum under low-energy conditions.This provides a molecular rationale for its superior efficiency compared with conventional ARTP. Its low-energy, high-efficiency operation reduces costs and aligns with sustainable synthetic biology principles. Future studies integrating in situ plasma emission spectroscopy with high-resolution mass spectrometry may further clarify its chemical mechanisms. 2.2 Functional Applications: Enhancing Yield While Reducing Toxins Positioning CPMI within the broader synthetic biology toolbox highlights its complementary role. Unlike precise genome-editing tools such as CRISPR/Cas or multiplex automated genome engineering (MAGE), which face regulatory and market challenges in food and other biofabrication applications ( 6 , 9 , 21 ), CPMI generates diverse mutational spectra without introducing exogenous DNA, providing a non-GMO strategy. Its low-energy, modular design makes it particularly suitable for industrial and food biotechnology applications. CPMI complements existing tools by enabling rapid, low-cost random mutagenesis that can be integrated with high-throughput screening for strain optimization. 2.2 Talaromyces albobiverticillus Mutagenesis CPMI was applied to Talaromyces albobiverticillus CY-G (a fungus that produces pigments and antioxidants) spore suspensions at 6 W for 30–240 s. Post-treatment survival and mutation rates were assessed on potato dextrose agar(PDA) plates. Treatments of 90–150 s yielded the highest mutation rate (9.25% ± 0.63%) with 60–70\% survival, defining an optimal window for effective mutagenesis. Mutants were selected based on pigment diffusion and mycelial growth, cultured in 48-well plates, and screened for pigment production. Five genetically stable strains with pronounced phenotypic changes were isolated (Fig. 6 ). These results show that CPMI produces diverse mutants with distinct pigment phenotypes. Yield Enhancement Among the mutants, CY110 exhibited markedly increased extracellular pigment production, reaching 462.98 ± 2.08 mg/g in semi-solid fermentation, 4.5-fold of the wild-type strain the parental strain (102.50 ± 5.08 mg/g, p < 0.01) (Fig. 7 a–b). Pigment composition shifted from red:orange:yellow ratio of 2.373:1:1.541 in the parental strain to 1.285:1:1.270 in CY110, indicating altered flux through multiple biosynthetic branches. UV–Vis spectra (400–550 nm) confirmed that pigment structures were conserved, while peak areas reflected increased yield and modified ratios, likely result from differential enzyme activity or gene expression within pigment biosynthetic pathways ( 22 – 24 ). The results demonstrate that CPMI-derived mutants exhibit enhanced pigment yield and antioxidant capacity while maintaining safety. Biosafety Verification and Low-Toxin Profiling The biosafety of the high-yielding mutant CY110 was rigorously evaluated by quantifying its citrinin production. UV-Vis spectroscopic analysis at 331 nm indicated that CY110 produced only trace amounts of citrinin (0.21 ug/L), which is significantly below the most stringent international safety limit of 50 mg/L ( 25 – 27 ). Importantly, despite the 4.5-fold increase in extracellular pigment production, the citrinin level in CY110 remained consistent with the inherently low-toxin profile of the parental strain (0.25 ug/L), and represented a > 99.7% lower concentration compared to the commercial Monascus purpureus reference (72.19 ug/L). These findings confirm that CPMI-mediated mutagenesis successfully enhanced the metabolic flux toward pigment biosynthesis without triggering the activation of the citrinin PKS gene clusters. This "high-yield, low-toxin" characteristic underscores the industrial potential of CY110 as a safe microbial source for natural colorants, particularly in applications where regulatory compliance is paramount. Table 2 Citrinin content of different strains. Values are presented as mean ± standard deviation (µg/L) Strains Contention(µg/L) T.albobiverticillus CY110 0.21 T.albobiverticillus CY-G 0.25 M.purpureus 40805 72.19 Antioxidant Activity and Comparison with Commercial Antioxidants Crude pigment extracts from CY110 exhibited DPPH IC 50 of 106.72 µg/mL and ABTS IC 50 of 85.57 µg/mL, consistent with medium-to-high antioxidant activity among fungal pigments (35–205 µg/mL) ( 28 , 29 ). When 0.02% (w/w) CY110 pigment was incorporated into fish feed and subjected to a 10-day Schaal accelerated oxidation assay at 60°C yielded peroxide values comparable to 0.02% butylated hydroxyanisole(BHA) (p > 0.05) and significantly lower than the blank control (p < 0.01) (Fig. 7 d, Table 2 ). These results demonstrate the potential of CY110 pigments as natural antioxidants for food and feed applications ( 30 , 31 ). 4. "resulting peroxide values were comparable to 0.02% BHA" was revised to "resulting peroxide values were comparable to those of feed supplemented with 0.02% BHA" to avoid ambiguity (peroxide values, not the pigment, are being compared to BHA-supplemented feed).) In summary, CPMI enables efficient mutagenesis under low-energy conditions, enhancing both metabolite yield and safety. This non-GMO, low-cost, and tunable mutagenesis platform is suitable for industrial microbial strain development, supporting sustainable and safe production in food and feed industries ( 32 , 33 ). Table 3 Oxidative stability indices of fish feed supplemented with CY110 pigments or the commercial antioxidant (BHA). Feed Treatment Peroxide Value (mmol/kg) Anisidine Value Total Oxidation Value NC(Blank Control) 32.80 26.52 167.72 0.02% CY110 Pigment 16.27 15.09 80.17 0.02% BHA 18.92 16.18 91.86 3. Conclusion and Outlook In this study, we established a low-power, air-based plasma mutagenesis platform (CPMI) and demonstrated a complete workflow from tool development and mechanistic elucidation to application validation: Tool Innovation : CPMI operates stably using only ambient air, achieving controllable mutagenesis at 2–8 W. This design reduces operational costs and lowers experimental barriers. Mechanistic Insights : Using an SOS reporter system, comet assays, and ESI–MS analyses, we showed that CPMI induces diverse DNA lesions—including base oxidation and single- and double-strand breaks—via the synergistic action of ROS and RNS, providing a molecular basis for its high mutagenic efficiency. Application Validation : In Talaromyces albobiverticillus , CPMI-generated mutants exhibited a > 3.5-fold increase in extracellular pigment production and significantly reduced citrinin levels, demonstrating the platform’s dual advantage in enhancing yield and product safety. Complementary Role in the Synthetic Biology Toolbox : Compared with precise genome-editing tools such as CRISPR/Cas, CPMI offers a non-GMO, low-cost, and rapid random mutagenesis approach. It complements existing synthetic biology tools and expands the strategy options for microbial strain optimization. Overall, CPMI represents a mechanistically well-characterized and scalable physical mutagenesis tool, providing the synthetic biology community with a practical platform that combines operational flexibility with demonstrable application value. 4. Materials and Methods 4.1 Compact Plasma Mutagenesis Instrument (CPMI) and Operating Parameters A laboratory-designed compact plasma mutagenesis instrument (CPMI) was used in this study. The device employs a customized high-voltage pulsed power supply and a long-gap dielectric barrier discharge plasma source that operates in ambient air. The discharge voltage ranges from 30–100 kV at a frequency above 40 kHz, with adjustable power from 0–20 W. Standard experiments were conducted at 8 W, 40 kHz, with the plasma discharge zone positioned 3–4 cm above the sample for 10–150 s (Fig. 1 ). The compact design allows operation within a biosafety cabinet without an external inert gas supply, minimizing cross-contamination risks. The plasma source was coated with conductive silver paint and graphene-based insulating layers to prevent arcing and ensure uniform, stable discharge. The system enabled an 8 cm long-gap discharge, significantly enhancing the effective plasma volume and stability. 4.2 DNA Damage Assays To quantify DNA damage induced by reactive oxygen and nitrogen species during CPMI treatment, a microplate-based UMU assay was performed following the method described by Huang et al. ( 14 ). This assay uses an SOS-responsive reporter system to monitor β-galactosidase activity as an indirect measure of DNA damage. In addition, electrospray ionization mass spectrometry (ESI–MS) was used to analyze synthetic oligonucleotides (dA₈, dT₈, dG₈, dC₈), following the protocol of Wang et al. ( 28 ), This allowed detection of fragmentation and adduct formation, thereby elucidating the chemical mechanisms of plasma-induced mutagenesis. 4.3 Microbial Strain and Spore Suspension Preparation The wild-type strain Talaromyces albobiverticillus CY-G was isolated from aquaculture pond sediments in Nansha, Guangzhou, China. Species identity was confirmed by internal transcribed spacer(ITS) sequencing and BLAST analysis, showing 99% similarity to T. albobiverticillus (GenBank accession number: PP663690). Cultures were grown on potato dextrose agar (PDA) plates at 30°C for 72 h, after which spores were harvested with sterile water. Spore concentrations were adjusted to 1 × 10⁷ spores/mL using a hemocytometer. 4.4 CPMI Mutagenesis Procedure Aliquots of 10 µL spore suspension were placed on stainless steel discs (Φ10 mm) and positioned beneath the CPMI plasma discharge zone. Treatments were conducted at 8 W and 40 kHz, with exposure times of 10, 30, 60, 90, 120, and 150 s. After treatment, suspensions were serially diluted and spread onto PDA plates,which were then incubated at 30°C for 3 days. Survival and mutation rates were calculated as follows: Lethality (%) = [(N₀ – N) / N₀ ]× 100%, where N₀ is the initial spore count and N is the surviving spore count. Mutation frequency (%) = (Colonies with altered pigment production ÷ total surviving colonies) × 100. 4.5 Mutant Screening and Genetic Stability Evaluation Colonies with significantly higher pigment yields than the parental strain were selected and cultured in 48-well plates containing semi-solid potato dextrose agar (SPDA) medium at 30°C, 200 rpm for 3 days. Supernatants were harvested by centrifugation, and pigment concentrations were quantified spectrophotometrically. Citrinin content was determined using enzyme-linked immunosorbent assay(ELISA). The top three pigment-overproducing strains and one pigment-deficient mutant (negative control) were retained. Selected mutants were subjected to 15 successive subcultures to evaluate pigment yield and genetic stability across generations. 4.6 Semi-Solid Array Fermentation and Pigment Extraction A porous semi-solid medium was prepared containing potato extract (5 g/L), glucose (20 g/L), agar (20 g/L), and food-grade glycerol (5 g/L). Following sterilization, the medium was stirred to form a uniform porous structure, poured into sterile trays (20 × 30 cm), and allowed to solidify. Spore suspensions were evenly sprayed to ensure uniform inoculation, and trays were incubated in a modular fermentation array at 30°C for 10 days. Pigments were extracted continuously using 50% ethanol at a flow rate of 400 mL/min, with filtrates collected hourly until the pigment concentration stabilized. Extracts were filtered (0.22 µm), concentrated under reduced pressure, and lyophilized to obtain pigment powders. Pigment composition (red, orange, and yellow) was determined spectrophotometrically. 4.7 Antioxidant Activity Assays The antioxidant activities of pigment extracts were evaluated using DPPH and ABTS radical scavenging assays. Half-maximal inhibitory concentrations (IC₅₀) were calculated from dose–response curves to compare radical scavenging capacities. 4.8 Feed Antioxidant Performance Test Pigment powders were incorporated into commercial fishmeal-based feed at 0.02% (w/w). Negative controls contained no additives, and positive controls contained 0.02% butylated hydroxyanisole (BHA). Feed was prepared by extrusion, dried, and subjected to a Schaal accelerated oxidation test at 60 ± 2°C for 20 days with mixing every 5 days. At the endpoint, samples were stored at − 80°C until analysis. Peroxide value (POV), anisidine value (AV), and total oxidation value (Totox) were determined following Chinese national standards GB 5009.227—2016 and GB/T 24304—2009. 4.9 Statistical Analysis All experiments were performed in triplicate unless otherwise stated. Data are presented as mean ± standard deviation. Statistical significance was determined by using Student’s t-test, with p < 0.05 considered significant. Abbreviations Compact Plasma Mutagenesis Instrument (CPMI);non-genetically modified organism (GMO); reactive oxygen and nitrogen species (RONS);atmospheric-pressure radio-frequency plasma (ARTP);electrospray ionization mass spectrometry(ESI–MS);multiplex automated genome engineering (MAGE);potato dextrose agar(PDA);butylated hydroxyanisole(BHA);internal transcribed spacer(ITS);semi-solid potato dextrose agar (SPDA);enzyme-linked immunosorbent assay(ELISA);Peroxide value (POV);anisidine value (AV);total oxidation value (Totox) Declarations AUTHOR INFORMATION Corresponding Author Corresponding authors（first and major）: jinyi Zhong，Guangzhou Institute of Advanced Technology, Guangzhou 511458, National Innovation Center for Bio-Manufacturing Industry, Shenzhen 518107, China; Present Addresses：No. 1121, Haibin Road, Nansha District, Guangzhou City, Guangdong Province E-mail address: [email protected] . Author Contributions Naicai ZHONG：Writing – review & editing, Validation, Investigation, Data curation, Conceptualization. Yuan CHEN, Wenfeng PAN: Responsible for the data analysis and writing of the article Hailin Meng：Methodology, Investigation. Kun Liang, Jun Lu, Yanlin Jiang：Responsible for the review and confirmation of sampling sites. Chengwei Dong , Muzhi Yang Chenyou Zhong：Participate in sample collection, data sorting and related analysis coordination Yinglei Zhai：investigation, Formal analysis Jinyi ZHONG *:Project administration, Funding acquisition Funding Sources Any funds used to support the research of the manuscript should be placed here (per journal style). Notes The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ACKNOWLEDGMENT We acknowledge Guangzhou Key Research and Development Program [Grant No. 2023B03J1177] and The "Guangdong Province Synthetic Biology Manufacturing Pilot Platform" Project (Project No. 2405-440309-04-01-570346) for funding this project. References U.S. Department of Health and Human Services (HHS) and Food and Drug Administration (FDA) (2025) HHS, FDA to Phase Out Petroleum-Based Synthetic Dyes in Nation's Food Supply; https://www.fda.gov/news-events/press-announcements/hhs-fda-phase-out-petroleum-based-synthetic-dyes-nations-food-supply State Council of the People’s Republic of China. 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Food Control 2017, 73, 1025–1032. https://doi.org/10.1016/j.foodcont.2016.09.039 Venkatachalam M, Dufossé L, Fouillaud M (2019) Microbial Pigments in the Food Industry—Challenges and the Way Forward. Microorganisms 7:10. https://doi.org/10.3390/microorganisms7010010 Venkatachalam M, Mares G, Dufossé L, Fouillaud M (2023) Scale-Up of Pigment Production by the Marine-Derived Filamentous Fungus Talaromyces albobiverticillius 30548, from Shake Flask to Stirred Bioreactor. Fermentation 9:77. https://doi.org/10.3390/fermentation9010077 Shimizu T, Kinoshita H, Nihira T (2007) Identification and in vivo functional analysis by gene disruption of ctnA, an activator gene involved in citrinin biosynthesis in Monascus purpureus. Appl Environ Microbiol 73:5097–5105. https://doi.org/10.1128/AEM.01979-06 Wang Y, Ye F, Zhou B, Liang Y, Lin Q, Lu D, Zhou X, Liu J (2023) Comparative analysis of different rice substrates for solid-state fermentation by a citrinin-free Monascus purpureus mutant strain with high pigment production. Food Biosci 56:103245. https://doi.org/10.1016/j.fbio.2023.103245 Huang Y, Yang C, Molnar I, Chen S (2023) Comparative Transcriptomic Analysis of Key Genes Involved in Citrinin Biosynthesis in Monascus purpureus. J Fungi 9:200. https://doi.org/10.3390/jof9020200 Egea MB, Dantas LA, Sousa TL, Lima AG, Lemes AC (2023) The Potential, Strategies, and Challenges of Monascus Pigment for Food Application. Front Sustain Food Syst 7:1141644. https://doi.org/10.3389/fsufs.2023.1141644 Mohan-Kumari HP, Naidu KA, Vishwanatha S, Narasimhamurthy K, Vijayalakshmi G (2021) Bioactive Pigments of Monascus purpureus Attributed to Antioxidant and Anti-Inflammatory Activities. Front Sustain Food Syst 5:590427. https://doi.org/10.3389/fsufs.2021.590427 Umesh M, Suresh S, Santosh AS, Prasad S, Chinnathambi A, Obaid SA, Jhanani GK, Shanmugam S (2023) Valorization of pineapple peel waste for fungal pigment production using Talaromyces albobiverticillius: Insights into antibacterial, antioxidant and textile dyeing properties. Environ Res 229:115973. https://doi.org/10.1016/j.envres.2023.115973 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), Rychen G, Aquilina G, Azimonti G, Bampidis V, Bastos ML, Bories G, Chesson A, Cocconcelli PS, Flachowsky G, Kolar B, Kouba M, López-Alonso M, Puente SL, Mantovani A, Mayo B, Ramos F, Saarela M, Villa RE, Wallace RJ, Wester P, Lundebye AK, Nebbia C, Renshaw D, Innocenti ML, Gropp J (2018) Safety and efficacy of butylated hydroxyanisole (BHA) as a feed additive for all animal species. EFSA J 16:e05215. https://doi.org/10.2903/j.efsa.2018.5215 Manuel CR, Carlos QF, Carmen PC, Marisela VDL, Iván MA (2022) Fungal solid-state fermentation of food waste for biohydrogen production by dark fermentation. Int J Hydrogen Energy 47:30062–30073. https://doi.org/10.1016/j.ijhydene.2022.06.313 Zhang Y, Zhang W, Zhang X, Zhang Y, Zhang Y, Zhang Y, Zhang Y, Zhang Y, Zhang Y, Zhang Y (2021) Strategic Transgene-Free Approaches of CRISPR-Based Genome Editing in Crops. Front. Plant Sci 12:9958309. https://doi.org/10.3389/fpls.2021.995830 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials20251028.docx image1.png Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Mar, 2026 Reviews received at journal 21 Mar, 2026 Reviews received at journal 04 Mar, 2026 Reviewers agreed at journal 04 Mar, 2026 Reviewers agreed at journal 03 Mar, 2026 Reviewers invited by journal 03 Mar, 2026 Editor assigned by journal 19 Feb, 2026 Submission checks completed at journal 19 Feb, 2026 First submitted to journal 13 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8868583","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":601350412,"identity":"c94a3311-3b6b-49f4-a3d6-5bfd91665649","order_by":0,"name":"Naicai Zhong","email":"","orcid":"","institution":"Shenyang Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Naicai","middleName":"","lastName":"Zhong","suffix":""},{"id":601350413,"identity":"29a7b7de-5bc4-425b-b3e8-f68a29c55f7c","order_by":1,"name":"yuan chen","email":"","orcid":"","institution":"Guangzhou Institute of Advanced Technology","correspondingAuthor":false,"prefix":"","firstName":"yuan","middleName":"","lastName":"chen","suffix":""},{"id":601350415,"identity":"84e64449-9417-4eed-b395-5b5ee553b87a","order_by":2,"name":"Wenfeng PAN","email":"","orcid":"","institution":"Guangzhou Institute of Advanced Technology","correspondingAuthor":false,"prefix":"","firstName":"Wenfeng","middleName":"","lastName":"PAN","suffix":""},{"id":601350416,"identity":"4ef6a170-aad6-4674-9330-3945f10a2679","order_by":3,"name":"Hailin Meng","email":"","orcid":"","institution":"Guangzhou Institute of Advanced Technology","correspondingAuthor":false,"prefix":"","firstName":"Hailin","middleName":"","lastName":"Meng","suffix":""},{"id":601350420,"identity":"484ff85b-c1e5-4c38-adbb-7ec29e6b0125","order_by":4,"name":"kun Liang","email":"","orcid":"","institution":"Guangzhou Nansha Fishery Industry Park Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"kun","middleName":"","lastName":"Liang","suffix":""},{"id":601350423,"identity":"b5fa704f-8b11-42c3-b063-391c66d3cd96","order_by":5,"name":"jun Lu","email":"","orcid":"","institution":"Guangzhou Nansha Fishery Industry Park Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"jun","middleName":"","lastName":"Lu","suffix":""},{"id":601350424,"identity":"923556fe-e4b4-4be2-957d-141c51ea8c80","order_by":6,"name":"Yanlin Jiang","email":"","orcid":"","institution":"Guangzhou Nansha Fishery Industry Park Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Yanlin","middleName":"","lastName":"Jiang","suffix":""},{"id":601350426,"identity":"928dd5d7-04a7-41aa-9203-39cdc27488f8","order_by":7,"name":"chenyou Zhong","email":"","orcid":"","institution":"Guangzhou Liyuan Aquaculture Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"chenyou","middleName":"","lastName":"Zhong","suffix":""},{"id":601350428,"identity":"caf26e68-00ac-4899-8ff1-54087e9bc9ca","order_by":8,"name":"yinglei Zhai","email":"","orcid":"","institution":"Shenyang Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"yinglei","middleName":"","lastName":"Zhai","suffix":""},{"id":601350430,"identity":"3b01815c-23dd-4a93-a62d-0a2db4b67a15","order_by":9,"name":"jinyi zhong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIie3NMQuCQBTA8ScHuVzcapNfQQmiSb/Kya0RjUJDJ4ItQY239xGCZuUGF6PVoUWC5sClsVPaArWt4f7Le8P78QB0uj/M4ACoWUi7/kQmXK3p0E8tcZrzQQQJiupVePNO12NUP8Gzwbx0S0NQmIriwc5lFVspMJfjJe0lbJxINiszrgiiYGGnl8iGTEUWv1LYDCJGrIjnkChRX+QAsqtihAtJrdJI5oWTuwledBN3y2SNQ+mTfX4vw3BtE7PoIYeANzPgFlVDHY8671U2Sdvpw2fR6XQ63VdvfyhHLJn160kAAAAASUVORK5CYII=","orcid":"","institution":"Guangzhou Institute of Advanced Technology","correspondingAuthor":true,"prefix":"","firstName":"jinyi","middleName":"","lastName":"zhong","suffix":""}],"badges":[],"createdAt":"2026-02-13 07:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8868583/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8868583/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104205590,"identity":"10263a2e-9a19-4800-acc3-6d60ea08c3d1","added_by":"auto","created_at":"2026-03-09 06:42:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":660066,"visible":true,"origin":"","legend":"\u003cp\u003eCold Plasma Mutagenesis Instrument (CPMI), mechanism, and quantitative readouts.\u003c/p\u003e\n\u003cp\u003e(a) Benchtop CPMI unit (air-driven, operating at room temperature) equipped with an integrated reaction chamber and electrode assembly (dimensions:30 × 20 × 30 cm); The unit has an adjustable power 0–20 W;in this study,a power output of 2–8 W was utilized.\u003c/p\u003e\n\u003cp\u003e(b) Discharge geometry of the CPMI,showing a stabilized 8-cm discharge gap and a 2–6 cm sample standoff distance.\u003c/p\u003e\n\u003cp\u003e(c) tandardized seven-step workflow for CPMI-based mutagenesis: (1) streaking of microbial cultures and picking of single colonies; (2) loading of samples into the reaction chamber; (3) plasma exposure via the CPMI; (4) post-exposure recovery of microbes and serial dilution; (5) primary selection of potential mutants; (6) microplate-based screening for target phenotypes; (7) confirmation of mutant phenotypes. Extended mechanistic characterization is provided in Figures S1–S3 (Supporting Information, SI).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8868583/v1/14d08314771eb1480aa8ba76.png"},{"id":104205615,"identity":"f83b973c-ba28-4278-9970-13da1325fddc","added_by":"auto","created_at":"2026-03-09 06:43:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":86321,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of CPMI at different powers on the biomass of \u003cem\u003eSalmonella typhimurium\u003c/em\u003e TA1535/pSK1002 over 180 minutes, measured every 30 minutes. CPMI maintains cell viability at low power, supporting controlled mutagenesis\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8868583/v1/803fe6bc553cb14b7f613296.png"},{"id":104205606,"identity":"ba2db382-1487-4b67-bf6a-4f4302701a78","added_by":"auto","created_at":"2026-03-09 06:43:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75886,"visible":true,"origin":"","legend":"\u003cp\u003eDNA damage induction and SOS response in \u003cem\u003eS. typhimurium\u003c/em\u003e TA1535/pSK1002 using SOS–lacZ reporter. (a) β-Galactosidase activity at different CPMI powers and durations. (b) Temporal induction of the SOS response.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8868583/v1/05b28fd59670946ba2e085a2.png"},{"id":104205613,"identity":"9380427a-d426-4bf7-bcc8-bc7ae495b837","added_by":"auto","created_at":"2026-03-09 06:43:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":674285,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Agarose gel electrophoresis showing DNA band patterns after CPMI treatment at different powers.\u003cbr\u003e\n(b) DNA band modulation at different time intervals under 8 W CPMI treatment.\u003cbr\u003e\n(c) Power–time heatmap, where color intensity corresponds to ΔEmax (defined as A₄₂₀/OD₆₀₀; representing peak SOS-linked β-galactosidase activity, blank-corrected) on a logarithmic scale. “G” denotes the growth factor, calculated as OD₆₀₀ (sample)/OD₆₀₀ (control), which is used to differentiate microbial viability from cytotoxicity. Low-power exposures (2–8 W) induce the SOS response while sustaining microbial growth; in contrast, higher power or longer exposure duration increases ΔEmax and advances the timing of the SOS induction peak, with G decreasing under cytotoxic conditions.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8868583/v1/0abe3b257d3d48288527f06d.png"},{"id":104205611,"identity":"e5b86062-c6bb-4dbb-b7ea-58d9b11ea112","added_by":"auto","created_at":"2026-03-09 06:43:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":796477,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 6. (A) Colonies of mutant bacteria from different CPMI treatment groups, including screened strains CY70, CY90, CY110, CY130, and CY1301.\u003c/p\u003e\n\u003cp\u003e(B) Mortality and mutation rates of strains treated with CPMI for different times.\u003c/p\u003e\n\u003cp\u003e(C) Absorbance spectra of pigments from mutant strains.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8868583/v1/95ced95bc9415e826f475a37.png"},{"id":104205591,"identity":"99012879-ed66-4d42-8cb2-fe59e53e6a62","added_by":"auto","created_at":"2026-03-09 06:42:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":43537,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 7.(A) Total pigment content of Talaromyces albobiverticillus wild-type strain CY-G and mutant strain CY110.\u003c/p\u003e\n\u003cp\u003e(B) Distribution of different pigment fractions in CY-G and CY110.\u003c/p\u003e\n\u003cp\u003e(C) DPPH radical scavenging activity of pigments from CY110.\u003c/p\u003e\n\u003cp\u003e(D) ABTS radical scavenging activity of pigments from CY110.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8868583/v1/da1010dabf3d5b498bab6a97.png"},{"id":104408750,"identity":"4cc2a397-a217-49ca-a4af-ff102125ec6b","added_by":"auto","created_at":"2026-03-11 12:43:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2999909,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8868583/v1/91d6aad3-ce6c-4333-a119-9e2863272849.pdf"},{"id":104205592,"identity":"4fd81e0f-0b9e-451e-9ff3-393dc23c7c5b","added_by":"auto","created_at":"2026-03-09 06:42:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1053874,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials20251028.docx","url":"https://assets-eu.researchsquare.com/files/rs-8868583/v1/39cadee7a0dce0d1f63c15d8.docx"},{"id":104403707,"identity":"0ea2a177-16ca-4441-bcf9-8fe7bc45b89a","added_by":"auto","created_at":"2026-03-11 12:18:52","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":328514,"visible":true,"origin":"","legend":"","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8868583/v1/fd20ebc4546094ee2d1a155a.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of a Novel Compact Air-Driven Cold Plasma Instrument for Efficient Microbial Mutagenesis: A Case Study on Enhanced Pigment Production","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMicrobial fermentation offers a sustainable and efficient pathway for producing natural pigments and functional metabolites, serving as a vital alternative to synthetic dyes which face increasing regulatory bans and safety concerns globally (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). However, the low productivity of wild-type strains remains a significant bottleneck for large-scale biomanufacturing. While existing strain-improvement strategies such as chemical mutagenesis, radiation, and CRISPR-based genome editing are widely used, they are often hindered by hazardous waste production, regulatory constraints in food-grade applications, or the requirement for sophisticated genetic toolkits (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Consequently, there is an urgent industrial demand for a non-GMO, efficient, and cost-effective mutagenesis platform.Low-temperature plasma, which generates abundant reactive oxygen and nitrogen species (RONS) at ambient temperature, has emerged as a promising physical mutagenesis approach for rapid selection of high-yield or phenotypically desirable mutants (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Compared with chemical and conventional physical mutagens, plasma mutagenesis offers several advantages, including no chemical residues, rapid treatment of large cell populations, and a broad mutation spectrum. Importantly, mutants produced by random plasma mutagenesis are generally not classified as GMOs (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Previous studies have demonstrated the effectiveness of atmospheric-pressure radio-frequency plasma (ARTP) technology as a representative platform (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite these advantages, existing plasma mutagenesis systems have limitations that hinder their widespread application in food-grade microbial engineering and biomanufacturing. Commercial ARTP systems often operate at high power (~\u0026thinsp;180 W), generating substantial heat that reduces cell viability and requires complex cooling or heat-dissipation setups. Many systems also rely on inert or rare gases (e.g., helium) to stabilize the discharge, increasing operational costs. In addition, the large device footprint complicates integration into sterile workspaces, raising the risk of contamination. These challenges underscore the need for a low-energy, low-temperature plasma mutagenesis platform capable of operating under standard laboratory conditions (without specialized gases), while satisfying safety, controllability, and cost-effectiveness requirements for food-grade strain improvement (\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo address these challenges, we developed a miniaturized, air-based, low-power cold plasma mutagenesis platform tailored for synthetic biology and other biofabrication applications. This system operates without inert gases, maintains high cell viability, and generates a multi-site DNA damage spectrum under controlled, low-energy conditions. Aligned with the \"tool-driven\" philosophy of synthetic biology, we systematically characterized its mutagenesis mechanism using an SOS\u0026ndash;lacZ DNA damage reporter, molecular electrophoresis, and mass spectrometry analyses, This allowed us to elucidate the types of DNA lesions and their genetic consequences under defined plasma discharge conditions. To demonstrate practical utility, we applied this platform to fungal pigment production, showing that it can simultaneously enhance target polyketide pigment yields while suppressing safety-relevant secondary metabolites, e.g.,citrinin. This dual optimization highlights CPMI's potential as a non-GMO strategy for microbial production of food-grade natural products and exemplifies a complete synthetic biology \"tool\u0026ndash;mechanism\u0026ndash;application\" workflow. As detailed in this study, the CPMI achieved a 4.5-fold increase in pigment production while maintaining superior biosafety and energy efficiency (Section \u003cspan refid=\"Sec3\" class=\"InternalRef\"\u003e2.1\u003c/span\u003e).\u003c/p\u003e"},{"header":"2. Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Development and Engineering Characterization of the Novel CPMI System\u003c/h2\u003e \u003cp\u003eWe developed a novel, laboratory-scale Compact Plasma Mutagenesis Instrument (CPMI), functioning as a miniaturized long-gap glow discharge reactor (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A core engineering objective was to overcome the limitations of conventional Atmospheric and Room-Temperature Plasma (ARTP) systems, which typically rely on high-power inputs and rare gas logistics. As summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the CPMI system exhibits a significant advancement in energy efficiency and operational portability.\u003c/p\u003e \u003cp\u003eWhile conventional ARTP systems (dimensions: 72 \u0026times; 62 \u0026times; 72 cm) generally require approximately 180 W and external gas supplies (Ar or N₂), the CPMI (30 \u0026times; 20 \u0026times; 30 cm) operates at a significantly lower power range of 2\u0026ndash;8 W using ambient air as the discharge medium. This represents a\u0026thinsp;\u0026gt;\u0026thinsp;95% reduction in power consumption and eliminates the need for complex gas flow and active cooling systems (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Its compact footprint allows for direct integration into microbiological clean benches, effectively minimizing cross-contamination risks. The CPMI utilizes a high-voltage excitation source (30\u0026ndash;100 kV, \u0026gt;\u0026thinsp;40 kHz) capable of maintaining a stable plasma plume across an 8-cm gap through insulated electrode surfacing, which enhances the plasma-sample interaction volume and discharge stability.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparative Engineering Parameters of CPMI and Conventional ARTP Systems.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPMI (This Study)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConventional ARTP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAdvantage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOperational Power\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u0026ndash;8 W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026asymp;\u0026thinsp;180 W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95% Energy Saving\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWorking Medium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmbient Air\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eArgon / Nitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo gas logistics required\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCooling Method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSelf-cooling (Ambient)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eActive Gas/Head Cooling\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSimplified maintenance\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFootprint\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.018 m\u0026sup3; (Compact)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.32 m\u0026sup3; (Bulky)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBenchtop/Clean bench compatible\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003eTo ensure experimental traceability and interlaboratory reproducibility, we established a standardized operating procedure (SOP) for CPMI-mediated mutagenesis; the complete step-by-step protocol and parameter tables are provided in Supplementary Information (Section S1) and summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed. Briefly, two to three morphologically uniform colonies or a calibrated spore suspension (~\u0026thinsp;1 \u0026times; 10^6 CFU\u0026middot;mL⁻\u0026sup1;) are loaded (10 \u0026micro;L per sterile carrier) and exposed under fixed geometry (standoff 2\u0026ndash;6 cm, normal incidence) to CPMI at low power (2\u0026ndash;8 W) for short durations (30\u0026ndash;150 s). Samples are recovered immediately, serially diluted, and plated for CFU enumeration; putative phenotype-positive colonies are subjected to microplate screening, product quantification and \u0026ge;\u0026thinsp;10-generation stability testing.\u003c/p\u003e \u003cp\u003eWe defined four key control points (KCPs) to minimize variation: (i) uniform sample spreading to avoid puddling and edge effects, (ii) maintenance of a stable standoff and normal incidence during exposure, (iii) rapid post-exposure recovery to limit drift, and (iv) standardized dilution and plating procedures for comparable survival/mutation estimates. Using this workflow, we derived survival and apparent-mutation curves (see next section) to identify an operational window that maximizes mutational activity while limiting cytotoxicity.\u003c/p\u003e \u003cp\u003eUnlike ARTP systems, that rely on rare gases such as helium or argon, CPMI uses ambient air as the discharge medium. This eliminates the operational costs and chemical interferences associated with external gases and simplifies device configuration (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The device generates minimal heat and operates stably at room temperature without additional cooling, preventing thermal damage to heat-sensitive strains. Growth inhibition assays with \u003cem\u003eSalmonella typhimurium\u003c/em\u003e TA1535/pSK1002 showed that 2 W treatments did not significantly affect cell viability (G\u0026thinsp;\u0026gt;\u0026thinsp;0.5), whereas 4\u0026ndash;8 W treatments for 40\u0026ndash;120 s markedly reduced survival (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and 6\u0026ndash;8 W treatments for 100\u0026ndash;120 s nearly caused complete cell death (G\u0026thinsp;\u0026le;\u0026thinsp;0.10) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). These results indicate that CPMI enables effective mutagenesis under low-energy conditions while providing a tunable operational window suitable for modular synthetic biology and other biofabrication applications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMechanisms of DNA DamagePlasma discharge generates diverse reactive oxygen and nitrogen species (ROS/RNS), along with high-energy electrons and ion flux. These short-lived species can induce base oxidation, phosphodiester bond cleavage, depurination, and DNA strand breaks, triggering the cellular SOS repair response (\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Using a TA1535/pSK1002 strain harboring an SOS\u0026ndash;lacZ reporter, even a mild 2 W, 2-min CPMI treatment significantly increased β-galactosidase activity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)-indicating detectable DNA damage under low-power conditions (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Heatmap analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) revealed that increasing CPMI power from 4 to 8 W accelerated DNA damage, with higher β-galactosidase activity, earlier peak induction times (120 s \u0026rarr; 60\u0026ndash;40 s), and steeper slopes, These changes reflect enhanced damage accumulation and faster SOS response activation. At 6\u0026ndash;8 W for 100\u0026ndash;120 s, biomass (OD 600) approached that of the sterile control and G values\u0026thinsp;\u0026le;\u0026thinsp;0.10, indicating near-total cell death. These observations suggest that high-power, long-duration CPMI treatment exerts bactericidal effects, likely via extensive DNA fragmentation, membrane disruption, or irreversible metabolic damage (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-b) showed that 2\u0026ndash;8 W treatments for 2 min caused minor single-strand breaks or recombination events, whereas 8 W for 10 min produced substantial fragmentation and smearing, indicative of severe structural DNA damage unsuitable for mutagenesis (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared with conventional ARTP, CPMI induces more pronounced DNA damage at lower power, achieving higher chemical efficiency per unit energy. ARTP typically requires\u0026thinsp;\u0026gt;\u0026thinsp;100 W and inert gases as the discharge medium, producing mainly ROS with limited reactive nitrogen species. In contrast, CPMI uses ambient air to generate both ROS (\u0026middot;OH, O₃) and RNS (\u0026middot;NO, ONOO⁻),, creating a synergistic ROS\u0026ndash;RNS effect. Different species induce distinct DNA lesions: (\u0026middot;OH) primarily causes base oxidation (e.g., 8-oxo-dG), RNS promotes depurination and abasic site formation, and high-energy ions/electrons induce single- and double-strand breaks (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eComet assays and electrospray ionization mass spectrometry(ESI\u0026ndash;MS) analyses revealed a power- and time-dependent increase in DNA fragmentation (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Tables S1\u0026ndash;S2). At 4\u0026ndash;8 W for \u0026ge;\u0026thinsp;40 s, comet tail moments were significantly elevated (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and oligodeoxynucleotides dA₈, dT₈, dG₈, and dC₈ exhibited multiple low m/z peaks indicative of fragmentation. Notably, dA₈ and dT₈ also showed high m/z peaks, suggesting polymerization or adduct formation. Base stability followed the order dC₈ \u0026gt; dG₈ \u0026gt; dT₈ \u0026gt; dA₈, reflecting differential sensitivity to oxidative and nitrosative stress. Mild CPMI conditions primarily caused repairable lesions, enhancing mutational diversity without lethality, whereas high-power, prolonged treatments induced extensive double-strand breaks and cell death, demonstrating a tunable \u0026ldquo;dual-effect\u0026rdquo; property. CPMI induced stronger degradation of dT₈ compared with ARTP, suggesting broader substrate reactivity and higher mutagenic potential.\u003c/p\u003e \u003cp\u003eOverall, the air-based ROS\u0026ndash;RNS synergistic mechanism underlies CPMI\u0026rsquo;s ability to generate a broader mutational spectrum under low-energy conditions.This provides a molecular rationale for its superior efficiency compared with conventional ARTP. Its low-energy, high-efficiency operation reduces costs and aligns with sustainable synthetic biology principles. Future studies integrating \u003cem\u003ein situ\u003c/em\u003e plasma emission spectroscopy with high-resolution mass spectrometry may further clarify its chemical mechanisms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Functional Applications: Enhancing Yield While Reducing Toxins\u003c/h2\u003e \u003cp\u003ePositioning CPMI within the broader synthetic biology toolbox highlights its complementary role. Unlike precise genome-editing tools such as CRISPR/Cas or multiplex automated genome engineering (MAGE), which face regulatory and market challenges in food and other biofabrication applications (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), CPMI generates diverse mutational spectra without introducing exogenous DNA, providing a non-GMO strategy. Its low-energy, modular design makes it particularly suitable for industrial and food biotechnology applications. CPMI complements existing tools by enabling rapid, low-cost random mutagenesis that can be integrated with high-throughput screening for strain optimization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2 \u003cem\u003eTalaromyces albobiverticillus\u003c/em\u003e Mutagenesis\u003c/h2\u003e \u003cp\u003eCPMI was applied to \u003cem\u003eTalaromyces albobiverticillus\u003c/em\u003e CY-G (a fungus that produces pigments and antioxidants) spore suspensions at 6 W for 30\u0026ndash;240 s. Post-treatment survival and mutation rates were assessed on potato dextrose agar(PDA) plates. Treatments of 90\u0026ndash;150 s yielded the highest mutation rate (9.25% \u0026plusmn; 0.63%) with 60\u0026ndash;70\\% survival, defining an optimal window for effective mutagenesis. Mutants were selected based on pigment diffusion and mycelial growth, cultured in 48-well plates, and screened for pigment production. Five genetically stable strains with pronounced phenotypic changes were isolated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese results show that CPMI produces diverse mutants with distinct pigment phenotypes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eYield Enhancement\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAmong the mutants, CY110 exhibited markedly increased extracellular pigment production, reaching 462.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08 mg/g in semi-solid fermentation, 4.5-fold of the wild-type strain the parental strain (102.50\u0026thinsp;\u0026plusmn;\u0026thinsp;5.08 mg/g, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ea\u0026ndash;b). Pigment composition shifted from red:orange:yellow ratio of 2.373:1:1.541 in the parental strain to 1.285:1:1.270 in CY110, indicating altered flux through multiple biosynthetic branches. UV\u0026ndash;Vis spectra (400\u0026ndash;550 nm) confirmed that pigment structures were conserved, while peak areas reflected increased yield and modified ratios, likely result from differential enzyme activity or gene expression within pigment biosynthetic pathways (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe results demonstrate that CPMI-derived mutants exhibit enhanced pigment yield and antioxidant capacity while maintaining safety.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiosafety Verification and Low-Toxin Profiling\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe biosafety of the high-yielding mutant CY110 was rigorously evaluated by quantifying its citrinin production. UV-Vis spectroscopic analysis at 331 nm indicated that CY110 produced only trace amounts of citrinin (0.21 ug/L), which is significantly below the most stringent international safety limit of 50 mg/L (\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Importantly, despite the 4.5-fold increase in extracellular pigment production, the citrinin level in CY110 remained consistent with the inherently low-toxin profile of the parental strain (0.25 ug/L), and represented a\u0026thinsp;\u0026gt;\u0026thinsp;99.7% lower concentration compared to the commercial Monascus purpureus reference (72.19 ug/L). These findings confirm that CPMI-mediated mutagenesis successfully enhanced the metabolic flux toward pigment biosynthesis without triggering the activation of the citrinin PKS gene clusters. This \"high-yield, low-toxin\" characteristic underscores the industrial potential of CY110 as a safe microbial source for natural colorants, particularly in applications where regulatory compliance is paramount.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCitrinin content of different strains. Values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (\u0026micro;g/L)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eContention(\u0026micro;g/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eT.albobiverticillus CY110\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eT.albobiverticillus CY-G\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eM.purpureus 40805\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAntioxidant Activity and Comparison with Commercial Antioxidants\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCrude pigment extracts from CY110 exhibited DPPH IC\u003csub\u003e50\u003c/sub\u003e of 106.72 \u0026micro;g/mL and ABTS IC \u003csub\u003e50\u003c/sub\u003e of 85.57 \u0026micro;g/mL, consistent with medium-to-high antioxidant activity among fungal pigments (35\u0026ndash;205 \u0026micro;g/mL) (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). When 0.02% (w/w) CY110 pigment was incorporated into fish feed and subjected to a 10-day Schaal accelerated oxidation assay at 60\u0026deg;C yielded peroxide values comparable to 0.02% butylated hydroxyanisole(BHA) (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) and significantly lower than the blank control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ed, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results demonstrate the potential of CY110 pigments as natural antioxidants for food and feed applications (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e4. \"resulting peroxide values were comparable to 0.02% BHA\" was revised to \"resulting peroxide values were comparable to those of feed supplemented with 0.02% BHA\" to avoid ambiguity (peroxide values, not the pigment, are being compared to BHA-supplemented feed).)\u003c/p\u003e \u003cp\u003eIn summary, CPMI enables efficient mutagenesis under low-energy conditions, enhancing both metabolite yield and safety. This non-GMO, low-cost, and tunable mutagenesis platform is suitable for industrial microbial strain development, supporting sustainable and safe production in food and feed industries (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOxidative stability indices of fish feed supplemented with CY110 pigments or the commercial antioxidant (BHA).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeed Treatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePeroxide Value (mmol/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnisidine Value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal Oxidation Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC(Blank Control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e167.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.02% CY110 Pigment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e80.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.02% BHA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e91.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Conclusion and Outlook","content":"\u003cp\u003eIn this study, we established a low-power, air-based plasma mutagenesis platform (CPMI) and demonstrated a complete workflow from tool development and mechanistic elucidation to application validation:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eTool Innovation\u003c/b\u003e: CPMI operates stably using only ambient air, achieving controllable mutagenesis at 2\u0026ndash;8 W. This design reduces operational costs and lowers experimental barriers.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eMechanistic Insights\u003c/b\u003e: Using an SOS reporter system, comet assays, and ESI\u0026ndash;MS analyses, we showed that CPMI induces diverse DNA lesions\u0026mdash;including base oxidation and single- and double-strand breaks\u0026mdash;via the synergistic action of ROS and RNS, providing a molecular basis for its high mutagenic efficiency.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eApplication Validation\u003c/b\u003e: In \u003cem\u003eTalaromyces albobiverticillus\u003c/em\u003e, CPMI-generated mutants exhibited a\u0026thinsp;\u0026gt;\u0026thinsp;3.5-fold increase in extracellular pigment production and significantly reduced citrinin levels, demonstrating the platform\u0026rsquo;s dual advantage in enhancing yield and product safety.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eComplementary Role in the Synthetic Biology Toolbox\u003c/b\u003e: Compared with precise genome-editing tools such as CRISPR/Cas, CPMI offers a non-GMO, low-cost, and rapid random mutagenesis approach. It complements existing synthetic biology tools and expands the strategy options for microbial strain optimization.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eOverall, CPMI represents a mechanistically well-characterized and scalable physical mutagenesis tool, providing the synthetic biology community with a practical platform that combines operational flexibility with demonstrable application value.\u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Compact Plasma Mutagenesis Instrument (CPMI) and Operating Parameters\u003c/h2\u003e \u003cp\u003eA laboratory-designed compact plasma mutagenesis instrument (CPMI) was used in this study. The device employs a customized high-voltage pulsed power supply and a long-gap dielectric barrier discharge plasma source that operates in ambient air. The discharge voltage ranges from 30\u0026ndash;100 kV at a frequency above 40 kHz, with adjustable power from 0\u0026ndash;20 W. Standard experiments were conducted at 8 W, 40 kHz, with the plasma discharge zone positioned 3\u0026ndash;4 cm above the sample for 10\u0026ndash;150 s (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The compact design allows operation within a biosafety cabinet without an external inert gas supply, minimizing cross-contamination risks. The plasma source was coated with conductive silver paint and graphene-based insulating layers to prevent arcing and ensure uniform, stable discharge. The system enabled an 8 cm long-gap discharge, significantly enhancing the effective plasma volume and stability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.2 DNA Damage Assays\u003c/h2\u003e \u003cp\u003eTo quantify DNA damage induced by reactive oxygen and nitrogen species during CPMI treatment, a microplate-based UMU assay was performed following the method described by Huang et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). This assay uses an SOS-responsive reporter system to monitor β-galactosidase activity as an indirect measure of DNA damage. In addition, electrospray ionization mass spectrometry (ESI\u0026ndash;MS) was used to analyze synthetic oligonucleotides (dA₈, dT₈, dG₈, dC₈), following the protocol of Wang et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), This allowed detection of fragmentation and adduct formation, thereby elucidating the chemical mechanisms of plasma-induced mutagenesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Microbial Strain and Spore Suspension Preparation\u003c/h2\u003e \u003cp\u003eThe wild-type strain Talaromyces albobiverticillus CY-G was isolated from aquaculture pond sediments in Nansha, Guangzhou, China. Species identity was confirmed by internal transcribed spacer(ITS) sequencing and BLAST analysis, showing 99% similarity to T. albobiverticillus (GenBank accession number: PP663690). Cultures were grown on potato dextrose agar (PDA) plates at 30\u0026deg;C for 72 h, after which spores were harvested with sterile water. Spore concentrations were adjusted to 1 \u0026times; 10⁷ spores/mL using a hemocytometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.4 CPMI Mutagenesis Procedure\u003c/h2\u003e \u003cp\u003eAliquots of 10 \u0026micro;L spore suspension were placed on stainless steel discs (Φ10 mm) and positioned beneath the CPMI plasma discharge zone. Treatments were conducted at 8 W and 40 kHz, with exposure times of 10, 30, 60, 90, 120, and 150 s. After treatment, suspensions were serially diluted and spread onto PDA plates,which were then incubated at 30\u0026deg;C for 3 days. Survival and mutation rates were calculated as follows:\u003c/p\u003e \u003cp\u003eLethality (%) = [(N₀ \u0026ndash; N) / N₀ ]\u0026times; 100%, where N₀ is the initial spore count and N is the surviving spore count.\u003c/p\u003e \u003cp\u003eMutation frequency (%) = (Colonies with altered pigment production\u0026thinsp;\u0026divide;\u0026thinsp;total surviving colonies) \u0026times; 100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Mutant Screening and Genetic Stability Evaluation\u003c/h2\u003e \u003cp\u003eColonies with significantly higher pigment yields than the parental strain were selected and cultured in 48-well plates containing semi-solid potato dextrose agar (SPDA) medium at 30\u0026deg;C, 200 rpm for 3 days. Supernatants were harvested by centrifugation, and pigment concentrations were quantified spectrophotometrically. Citrinin content was determined using enzyme-linked immunosorbent assay(ELISA). The top three pigment-overproducing strains and one pigment-deficient mutant (negative control) were retained. Selected mutants were subjected to 15 successive subcultures to evaluate pigment yield and genetic stability across generations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Semi-Solid Array Fermentation and Pigment Extraction\u003c/h2\u003e \u003cp\u003eA porous semi-solid medium was prepared containing potato extract (5 g/L), glucose (20 g/L), agar (20 g/L), and food-grade glycerol (5 g/L). Following sterilization, the medium was stirred to form a uniform porous structure, poured into sterile trays (20 \u0026times; 30 cm), and allowed to solidify. Spore suspensions were evenly sprayed to ensure uniform inoculation, and trays were incubated in a modular fermentation array at 30\u0026deg;C for 10 days. Pigments were extracted continuously using 50% ethanol at a flow rate of 400 mL/min, with filtrates collected hourly until the pigment concentration stabilized. Extracts were filtered (0.22 \u0026micro;m), concentrated under reduced pressure, and lyophilized to obtain pigment powders. Pigment composition (red, orange, and yellow) was determined spectrophotometrically.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.7 Antioxidant Activity Assays\u003c/h2\u003e \u003cp\u003eThe antioxidant activities of pigment extracts were evaluated using DPPH and ABTS radical scavenging assays. Half-maximal inhibitory concentrations (IC₅₀) were calculated from dose\u0026ndash;response curves to compare radical scavenging capacities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.8 Feed Antioxidant Performance Test\u003c/h2\u003e \u003cp\u003ePigment powders were incorporated into commercial fishmeal-based feed at 0.02% (w/w). Negative controls contained no additives, and positive controls contained 0.02% butylated hydroxyanisole (BHA). Feed was prepared by extrusion, dried, and subjected to a Schaal accelerated oxidation test at 60\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 20 days with mixing every 5 days. At the endpoint, samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis. Peroxide value (POV), anisidine value (AV), and total oxidation value (Totox) were determined following Chinese national standards GB 5009.227\u0026mdash;2016 and GB/T 24304\u0026mdash;2009.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.9 Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed in triplicate unless otherwise stated. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical significance was determined by using Student\u0026rsquo;s t-test, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCompact Plasma Mutagenesis Instrument (CPMI);non-genetically modified organism (GMO); reactive oxygen and nitrogen species (RONS);atmospheric-pressure radio-frequency plasma (ARTP);electrospray ionization mass spectrometry(ESI\u0026ndash;MS);multiplex automated genome engineering (MAGE);potato dextrose agar(PDA);butylated hydroxyanisole(BHA);internal transcribed spacer(ITS);semi-solid potato dextrose agar (SPDA);enzyme-linked immunosorbent assay(ELISA);Peroxide value (POV);anisidine value (AV);total oxidation value (Totox)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorresponding Author\u003c/p\u003e\n\u003cp\u003eCorresponding authors（first and major）: jinyi Zhong，Guangzhou Institute of Advanced Technology, Guangzhou 511458, National Innovation Center for Bio-Manufacturing Industry, Shenzhen 518107, China;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePresent Addresses：No. 1121, Haibin Road, Nansha District, Guangzhou City, Guangdong Province\u003c/p\u003e\n\u003cp\u003eE-mail address: [email protected].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNaicai ZHONG：Writing\u0026nbsp;\u0026ndash;\u0026nbsp;review \u0026amp; editing, Validation, Investigation, Data curation, Conceptualization.\u003c/p\u003e\n\u003cp\u003eYuan CHEN, Wenfeng PAN: Responsible for the data analysis and writing of the article\u003c/p\u003e\n\u003cp\u003eHailin Meng：Methodology, Investigation.\u003c/p\u003e\n\u003cp\u003eKun Liang, Jun Lu, Yanlin Jiang：Responsible for the review and confirmation of sampling sites.\u003c/p\u003e\n\u003cp\u003eChengwei Dong , Muzhi Yang \u0026nbsp;Chenyou Zhong：Participate in sample collection, data sorting and related analysis coordination\u003c/p\u003e\n\u003cp\u003eYinglei Zhai：investigation, Formal analysis\u003c/p\u003e\n\u003cp\u003eJinyi ZHONG *:Project administration, Funding acquisition\u0026nbsp;Funding Sources\u003c/p\u003e\n\u003cp\u003eAny funds used to support the research of the manuscript should be placed here (per journal style).\u003c/p\u003e\n\u003cp\u003eNotes\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge Guangzhou Key Research and Development Program [Grant No. 2023B03J1177] and The \u0026quot;Guangdong Province Synthetic Biology Manufacturing Pilot Platform\u0026quot; Project (Project No. 2405-440309-04-01-570346) for funding this project.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eU.S. Department of Health and Human Services (HHS) and Food and Drug Administration (FDA) (2025) HHS, FDA to Phase Out Petroleum-Based Synthetic Dyes in Nation's Food Supply; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.fda.gov/news-events/press-announcements/hhs-fda-phase-out-petroleum-based-synthetic-dyes-nations-food-supply\u003c/span\u003e\u003cspan address=\"https://www.fda.gov/news-events/press-announcements/hhs-fda-phase-out-petroleum-based-synthetic-dyes-nations-food-supply\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eState Council of the People\u0026rsquo;s Republic of China. 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Plant Sci 12:9958309. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2021.995830\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2021.995830\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioprocess-and-biosystems-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioprocess and Biosystems Engineering](https://www.springer.com/journal/449)","snPcode":"449","submissionUrl":"https://submission.nature.com/new-submission/449/3","title":"Bioprocess and Biosystems Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Compact Plasma Mutagenesis Instrument (CPMI), Plasma–DNA interactions, Random mutagenesis, Microbial pigment biosynthesis, Synthetic biology toolbox","lastPublishedDoi":"10.21203/rs.3.rs-8868583/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8868583/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLow-temperature plasma provides a chemical-free method for random mutagenesis, however, conventional systems often require bulky equipment and rare gases, which limits their accessibility. Here, we present a Compact Plasma Mutagenesis Instrument (CPMI) that operates in ambient air at 2\u0026ndash;8 W, reducing energy consumption while increasing the plasma\u0026ndash;sample interaction volume. Mechanistic studies indicate that CPMI induces DNA lesions via reactive oxygen and nitrogen species, leading to base oxidation, strand breaks, and adduct formation. The Application of CPMI to Talaromyces albobiverticillus produced mutant strain CY110. This strain exhibited over 4.5-fold of the wild-type strain extracellular pigment production, significantly decreased citrinin levels, and enhanced antioxidant activity relative to the parental strain. Unlike chemical mutagens or UV irradiation, CPMI generates no toxic byproducts, and unlike genome-editing tools such as CRISPR/Cas, it offers a non-genetically modified organism (GMO) approach suitable for food-grade applications. Collectively, these results establish CPMI as an accessible, energy-efficient, and regulation-compliant mutagenesis platform that complements existing synthetic biology toolkits. Unlike traditional systems, the air-driven CPMI eliminates noble gas reliance, providing a cost-effective and portable platform for industrial microbial optimization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Development of a Novel Compact Air-Driven Cold Plasma Instrument for Efficient Microbial Mutagenesis: A Case Study on Enhanced Pigment Production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-09 06:42:07","doi":"10.21203/rs.3.rs-8868583/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-23T05:11:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-21T16:51:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-04T09:30:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194275436779521499556682432048920369273","date":"2026-03-04T08:47:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"190944617061834035324317532703562971826","date":"2026-03-04T01:37:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-03T13:43:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-19T09:50:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-19T09:08:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioprocess and Biosystems Engineering","date":"2026-02-13T07:14:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioprocess-and-biosystems-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioprocess and Biosystems Engineering](https://www.springer.com/journal/449)","snPcode":"449","submissionUrl":"https://submission.nature.com/new-submission/449/3","title":"Bioprocess and Biosystems Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"87395a68-18c3-4fed-8f88-c4e238ff76bf","owner":[],"postedDate":"March 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T03:23:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-09 06:42:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8868583","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8868583","identity":"rs-8868583","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}