Proteomic and Mechanistic Insights into the Efficiency of Atmospheric and Room-Temperature Plasma Mutagenesis-Driven Bioconversion of Corn Stover by Trichoderma longibrachiatum

Abstract

The valorization of agricultural residues, particularly corn stover, represents a sustainable approach for resource utilization and protein production in which high-performing microbial strains are essential. This study systematically evaluated fungal lignocellulolytic capabilities during corn stover solid-state fermentation and employed atmospheric and room-temperature plasma (ARTP) mutagenesis to enhance the degradative capacity of Trichoderma longibrachiatum. Comparative screening revealed that T. longibrachiatum exhibited superior comprehensive degradation of the major lignocellulosic components compared to other tested strains. ARTP mutagenesis yielded mutant strain TL-MU07, which displayed significantly enhanced enzymatic capabilities with improvements in FPase (22.1%), CMCase (10.1%), and xylanase (16.1%) activities, resulting in increased cellulose degradation (14.6%) and protein accumulation (14.7%). Proteomic analysis revealed 289 significantly differentially expressed proteins, with pathway enrichment demonstrating enhancement of glycosaminoglycan degradation, amino sugar metabolism, and membrane remodeling. Key mechanistic adaptations included downregulation of Zn(2)-C6 transcriptional repressors, upregulation of detoxification enzymes (ALDH-like proteins), and enhanced secretory pathway components. The ARTP-derived mutant strain TL-MU07 represents a valuable microbial resource for agricultural waste bioconversion, offering enhanced lignocellulolytic capabilities for industrial applications while elucidating specific proteomic changes associated with improved biomass degradation efficiency for sustainable protein production in the circular bioeconomy.

1. Introduction

Agricultural residues, particularly corn stover, accumulate globally in staggering quantities exceeding 1 billion metric tons annually, presenting both environmental challenges and untapped resource potential [1]. The improper disposal of these residues through field burning contributes significantly to atmospheric pollution and greenhouse gas emissions while simultaneously wasting valuable biomass resources [2]. Concurrently, escalating demand for protein-rich animal feed, driven by expanding livestock production to satisfy global nutritional requirements, creates a compelling imperative to develop innovative bioconversion strategies that transform these agricultural byproducts into high-value nutritional products [3]. This convergence of environmental sustainability and resource valorization has catalyzed substantial research interest in biological processes that effectively harness agricultural residues. Recent advances have demonstrated the strategic integration of chemical and biological catalysis in cascade processes for complete valorization of agricultural residues within modern biorefinery frameworks. For instance, enzymatic hydrolysis of defatted wheat bran coupled with optimized fermentation has produced high-value carotenoids and lipids, achieving unprecedented yields through Rhodosporidium toruloides fermentation [4]. Similarly, innovative pretreatment approaches utilizing high-pressure CO₂ with catalytic additives have achieved remarkable xylan conversion rates (82%) and enhanced enzymatic hydrolysis efficiency of giant reed biomass, resulting in total glucose yields of 67.8% [5]. These developments underscore the transformative potential of enzymatic bioconversion within integrated biorefinery systems for maximizing resource recovery while minimizing environmental impact. The recalcitrant nature of lignocellulosic biomass presents formidable challenges for bioconversion processes, primarily due to the complex architectural arrangement of its three principal components—cellulose (40–50%), hemicellulose (25–35%), and lignin (15–20%)—creating a naturally resistant matrix that has evolved to withstand biological degradation [6,7]. In modern biorefinery frameworks, strategic combinations of pretreatment technologies with fungal enzymatic hydrolysis effectively overcome lignocellulosic recalcitrance. Recent research on Cynara cardunculus demonstrated that combining steam explosion with mild organosolv treatment and subsequent enzymatic treatment achieved efficient fractionation and isolation of lignin and cellulose components [8]. While these integrated physicochemical–enzymatic approaches offer significant advantages, biological pretreatment methods present complementary sustainable alternatives. Solid-state fermentation (SSF) has emerged as a particularly promising approach for overcoming these challenges, offering numerous advantages, including reduced water requirements, minimized bacterial contamination risk, enhanced enzyme production, and closer simulation of the natural habitats of lignocellulolytic microorganisms [9,10]. Despite these advantages, SSF processes face several notable limitations that present ongoing challenges. These include heterogeneous heat and mass transfer, difficulty in pH and temperature control, challenges in scale-up to industrial levels, limited real-time monitoring capabilities, and complex substrate–microorganism interactions that affect process reproducibility. Additionally, downstream processing of SSF products often requires optimization due to the solid nature of the fermentation matrix [11,12].

The identification and development of efficient lignocellulolytic microorganisms constitute a critical determinant of successful bioconversion processes [9]. Among cellulolytic fungi, Trichoderma species have garnered particular attention due to their robust production of diverse hydrolytic enzymes, rapid colonization capabilities, and potential antagonistic properties against plant pathogens [10,13]. Particularly, T. longibrachiatum demonstrates exceptional potential for lignocellulose degradation due to its robust cellulase production, thermotolerance, and environmental adaptability [14,15]. Despite these natural capabilities, wild-type strains often exhibit suboptimal performance in industrial applications due to limitations in enzyme production levels, substrate specificity, or sensitivity to inhibitory compounds—limitations particularly evident under challenging SSF conditions with heterogeneous substrate distribution and fluctuating moisture gradients [10,16].

Conventional mutation breeding approaches, including ultraviolet irradiation and chemical mutagenesis, have been widely employed to enhance fungal lignocellulolytic capabilities but frequently suffer from low mutation efficiencies and limited genomic coverage [17]. The atmospheric and room-temperature plasma (ARTP) mutagenesis system has emerged as a revolutionary technology for microbial strain improvement, generating a rich mixture of reactive chemical species that induce diverse DNA modifications with higher frequency and broader genomic distribution compared to conventional methods [18,19,20]. The ARTP technology produces various reactive species—including electrons, ions, free radicals, and excited molecules—that create extensive genetic diversity for subsequent screening [18]. The application of ARTP mutagenesis to improve lignocellulolytic fungi has demonstrated remarkable success, with studies reporting a 2.21-fold increase in filter paper activity and a 34% enhancement in endoglucanase activity in Trichoderma reesei [21,22]. Moreover, when ARTP was combined with other technologies in Trichoderma afroharzianum, FPase, CMCase, pNPCase, and pNPGase activities increased by 4.33-, 6.37-, 4.92-, and 4.15-fold, respectively [23]. Despite these promising results, most investigations have focused predominantly on enzyme production under submerged conditions, with limited exploration of comprehensive degradative capabilities during SSF of agricultural residues [24]. Furthermore, systematic analysis of the molecular mechanisms underlying improved cellulolytic performance in ARTP-induced mutants remains insufficient, impeding the development of more targeted strain improvement strategies.

The present study addresses these knowledge gaps through systematic screening and enhancement of fungal strains for corn stover bioconversion through (1) comparative evaluation of various fungal species to identify promising candidates with complementary degradative capabilities; (2) application of ARTP mutagenesis to generate improved strains with enhanced lignocellulolytic efficiency; and (3) proteomic elucidation of molecular mechanisms underlying enhanced degradative capabilities in mutant strains. The innovations of this research lie in its comprehensive integration of advanced strain improvement technology with molecular characterization, evaluating overall bioconversion efficiency based on multiple performance indicators, including degradation rates of principal lignocellulosic components, protein accumulation, and microbial colonization patterns. The application of high-resolution proteomic analysis to elucidate mutation-induced changes in protein expression profiles provides unprecedented insights into the molecular adaptations facilitating enhanced lignocellulolytic performance [25,26]. These insights not only contribute to fundamental scientific understanding but also inform future rational strain design strategies for agricultural waste valorization, ultimately establishing an efficient, environmentally sustainable approach for transforming abundant agricultural residues into valuable protein resources, thereby contributing to both environmental sustainability and global food security objectives [24].

2. Materials and Methods

2.1. Microorganisms and Cultivation Conditions

2.1.1. Fungal Strains and Maintenance

Four filamentous fungi with documented lignocellulolytic capabilities were selected for this study: Trichoderma longibrachiatum (T. longibrachiatum), Trichoderma reesei (T. reesei), Aspergillus niger (A. niger), and Aspergillus oryzae (A. oryzae). All strains were soil-derived isolates maintained in our laboratory, originally isolated from agricultural soils through selective enrichment techniques. The fungal cultures were maintained as conidial suspensions (1 × 10⁷ spores/mL) in 30% (v/v) glycerol solution at −80 °C. Mutant derivatives of T. longibrachiatum (designated as TL-RS00 for the parental strain) generated via ARTP mutagenesis were preserved using identical protocols.

2.1.2. Inoculum Preparation

For experimental inoculations, fungal spores were harvested from 4-day-old potato dextrose agar (PDA) plates by with 10 mL of sterile 0.10% (v/v) Tween-80 solution. The resulting suspensions were gently agitated with a sterile glass rod to dislodge conidia, followed by filtration through four layers of sterile cheesecloth to remove mycelial fragments. Spore concentrations were determined using a hemocytometer and standardized to 1 × 10⁷ spores/mL through appropriate dilution with sterile 0.10% Tween-80 solution.

2.2. Solid-State Fermentation

2.2.1. Substrate Preparation and Pretreatment

Corn stover was harvested from Hebei Province, China (39°37′52″ N 118°10′44″ E), air-dried naturally, and processed to achieve particle sizes below 2 mm. Structural component analysis followed the National Renewable Energy Laboratory (NREL) analytical procedures [27], revealing compositional values of 30.30% cellulose, 24.91% hemicellulose, and 19.45% lignin (dry basis). High-performance liquid chromatography (HPLC), utilizing an Agilent 1260 system with a Hi-Plex H column (Agilent, Santa Clara, CA, USA), was employed to determine the concentrations of glucose, xylose, and arabinose. Nitrogen content was determined at 10.22 g/kg using standard Kjeldahl methodology with an automatic Kjeldahl analyzer (Kjeltec 8400, FOSS, Hillerød, Denmark).

2.2.2. Fermentation Setup and Parameters

SSF was conducted in 250 mL Erlenmeyer flasks containing 10 g dry substrate. Moisture content was adjusted to 67% using a nutrient solution (g/L: KH₂PO₄, 2.0; (NH₄)₂SO₄, 1.4; MgSO₄·7H₂O, 0.3; CaCl₂, 0.3; NaCl, 0.5; FeSO₄·7H₂O, 0.005; MnSO₄, 0.0016; ZnSO₄, 0.0014; CoCl₂, 0.002; pH 5.5). Following sterilization (121 °C, 30 min), flasks were inoculated with 1 × 10⁶ spores/g substrate and incubated at 30 °C. Periodic gentle agitation ensured uniform colonization without disrupting mycelial networks. Comparative studies between fungal species extended to 12 days, while mutant evaluations were conducted over 5 days based on established enzymatic profiles.

2.3. ARTP Mutagenesis

2.3.1. ARTP Apparatus Settings and Operation

Mutagenesis was performed using an ARTP mutagenic apparatus (Wuxi Yuanqing Tianmu Biological Technology Co., Ltd., Wuxi, China).

2.3.2. Spore Preparation for Mutagenesis

Prior to ARTP treatment, the suspension as previously described was subjected to a washing procedure whereby aliquots were centrifuged at 6000× g for 5 min (Eppendorf 5417R, Hamburg, Germany), the supernatant discarded, and the pellet resuspended in sterile saline solution (0.9% NaCl). This washing process was repeated three times to eliminate residual culture components that might interfere with plasma-mediated mutagenesis. The final spore suspension was prepared at 1 × 10⁷ spores/mL in sterile saline solution and maintained on ice until treatment.

2.3.3. Mutagenesis Procedure and Survival Rate Determination

For each mutagenesis treatment, 10 μL of the prepared spore suspension was pipetted onto a sterile stainless-steel plate positioned on the sliding sample stage of the ARTP system. Exposure times ranging from 0 to 360 s (at 40 s intervals) were evaluated to establish the optimal treatment duration. Following plasma exposure, each treated sample was immediately transferred to 990 μL sterile saline solution and serially diluted to appropriate concentrations. Aliquots (100 μL) of each dilution were spread on PDA plates and incubated at 30 °C for 48 h. Colony-forming units (CFUs) were enumerated, and survival rates were calculated using the formula

$$\mathrm{S}\mathrm{u}\mathrm{r}\mathrm{v}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{C}\mathrm{F}\mathrm{U}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{C}\mathrm{F}\mathrm{U}\mathrm{o}\mathrm{f}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\times 100$$

(1)

The lethality curve was plotted as survival rate versus exposure time, and the treatment duration corresponding to approximately 80–90% lethality (10–20% survival) was selected for subsequent mutagenesis, as this range has been empirically associated with optimal mutation efficiency [28]. The optimized exposure time (320 s) was utilized for generating the mutant library, with 1505 individual colonies isolated following treatment.

2.3.4. Mutant Preservation and Stability Assessment

The genetic stability of selected mutants was assessed through sequential subculturing on non-selective media for five generations. At each generation, phenotypic characteristics, including growth rate, sporulation patterns, and colony morphology were documented. Additionally, cellulolytic activity was quantified to detect any reversion toward wild-type enzyme production levels. Only mutants demonstrating consistent phenotypic traits and stable enzymatic performance across all generations were maintained for subsequent screening procedures.

2.4. Screening of Mutants

2.4.1. Primary Screening on CMC-Na Plates

Initial screening of the mutant library was conducted using carboxymethylcellulose sodium (CMC-Na) agar plates to rapidly identify colonies with enhanced cellulolytic capacity. The screening medium contained (g/L) CMC-Na, 10.0; NaNO₃, 2.0; K₂HPO₄, 1.0; MgSO₄·7H₂O, 0.5; KCl, 0.5; FeSO₄·7H₂O, 0.01; agar, 20.0; pH 5.0. Spores from individual mutants were point-inoculated onto the center of CMC-Na plates and incubated at 28 °C for 72 h to allow colony development and enzyme secretion.

Following incubation, plates were flooded with 0.1% (w/v) Congo red solution for 15 min, followed by destaining with 1 M NaCl for 30 min to visualize hydrolysis zones. The hydrolytic capacity (HC) was quantified as the ratio of the hydrolysis zone diameter to colony diameter [29], with measurements obtained using a digital caliper (Mitutoyo 500-196-30, Mitutoyo Corporation, Kawasaki, Japan). The cellulolytic index was calculated as (HC-1), representing the proportion by which the clearing zone exceeded the colony diameter. Mutants exhibiting HC values exceeding the parental strain by more than 35% were selected for secondary screening.

2.4.2. Secondary Screening in Shake Flask Cultures

Promising mutants identified through primary screening were subsequently evaluated in submerged fermentation to assess their cellulolytic enzyme production capacity under standardized conditions. The fermentation medium contained (g/L) microcrystalline cellulose, 20.0; peptone, 1.0; (NH₄)₂SO₄, 5.0; KH₂PO₄, 2.0; CaCl₂, 0.3; MgSO₄·7H₂O, 0.3; FeSO₄·7H₂O, 0.005; MnSO₄, 0.0016; ZnSO₄, 0.0014; CoCl₂, 0.002; pH 5.0.

Erlenmeyer flasks (250 mL) containing 50 mL medium were inoculated with standardized spore suspensions (1 × 10⁶ spores/mL final concentration) and incubated at 30 °C with orbital agitation (180 rpm) for 96 h. Following fermentation, crude enzyme extracts were obtained by centrifugation at 10,000× g for 15 min at 4 °C, and the supernatants were assessed for total cellulase activity (filter paper activity, FPase), water-soluble protein content, and peptide concentration. Mutants demonstrating superior performance across multiple parameters compared to the parental strain were selected for tertiary screening.

2.4.3. Tertiary Screening Under SSF

The final screening phase evaluated selected mutants under SSF conditions to assess their performance in an environment closely resembling industrial applications. SSF was conducted as described in Section 2.2.2, with corn stover serving as the substrate. After 5 days of incubation, samples were collected and analyzed for enzyme activities (FPase, endoglucanase or CMCase, and xylanase), substrate degradation rates (cellulose, hemicellulose, and lignin), and protein accumulation parameters (true protein content and peptide content). Additionally, spore production capacity was quantified as an indicator of robust colonization and reproductive fitness. Data from all measured parameters were integrated to identify the optimal mutant strain for subsequent proteomic analysis.

2.5. Analytical Methods

2.5.1. Enzyme Extraction

For enzyme extraction, a modified protocol based on the method of El-Shishtawy et al. [30] was implemented. Fermented substrate was extracted with 0.05 M sodium citrate buffer (pH 4.8) at a ratio of 1:10 (w/v), with continuous agitation at ambient temperature (22–25 °C) for 60 min. The resulting suspension was first strained through multiple layers of sterile gauze to remove coarse particulates, followed by centrifugation at 4000× g for 10 min at 4 °C to clarify the extract. The clear supernatant served as the crude enzyme preparation for all subsequent activity measurements. Each enzymatic determination was performed in triplicate.

To accurately represent catalytic efficiency in relation to the solid substrate mass, all enzyme activities were converted from volumetric measurements (U/mL) to substrate-specific activities (U/g d.s., where d.s. denotes dry substrate) using the following calculation:

$$\mathrm{E}\mathrm{n}\mathrm{z}\mathrm{y}\mathrm{m}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{U}/\mathrm{g}\mathrm{d}.\mathrm{s}.\right)={\displaystyle \frac{\mathrm{A}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\mathrm{U}/\mathrm{m}\mathrm{L}\right)\times \mathrm{E}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}\left(\mathrm{m}\mathrm{L}\right)}{\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{d}\mathrm{r}\mathrm{y}\mathrm{s}\mathrm{u}\mathrm{b}\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\left(\mathrm{g}\right)}}$$

(2)

2.5.2. Enzyme Activity Assays

Cellulolytic, hemicellulolytic, and ligninolytic enzyme activities were determined using standard spectrophotometric methods with a microplate spectrophotometer (Epoch, Bio-Tek Instruments, Winooski, VT, USA). Total cellulase activity (FPase) activity was measured according to the IUPAC standardized filter paper assay using Whatman No. 1 filter paper (Whatman International Ltd., Maidstone, UK), while endoglucanase (CMCase) activity utilized 1% (w/v) carboxymethylcellulose in 50 mM sodium citrate buffer (pH 4.8) [31,32]. Xylanase activity was determined using the 3,5-dinitrosalicylic acid (DNS) colorimetric method with DNS reagent (Sigma-Aldrich, St. Louis, MO, USA), and β-xylosidase activity was measured using the p-nitrophenol (pNP) assay with p-nitrophenyl-β-D-xylopyranoside (Sigma-Aldrich) [33,34]. Ligninolytic enzyme activities (laccase, lignin peroxidase, and manganese peroxidase) were evaluated following previously established protocols [35]. For all assays, one enzyme unit (U) was defined as the amount of enzyme releasing 1 μmol of product (reducing sugar, pNP, or corresponding substrate) per minute under the specified conditions.

2.5.3. Compositional Analysis of Lignocellulosic Substrate

Degradation rates were calculated accounting for dry matter loss during fermentation using established formulas [36]. The degradation of cellulose, hemicellulose, and lignin was determined by comparing their contents before and after fermentation, following the NREL analytical procedures mentioned in Section 2.2.1.

2.5.4. Protein and Peptide Quantification

Total water-soluble protein content was quantified using the Bradford method with a Bradford protein assay kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) as the standard. True protein determination followed a modified TCA precipitation protocol as described by Elgar et al. [37]., where fermented samples were treated with 15% TCA solution at 4 °C, followed by nitrogen quantification of the precipitate using Kjeldahl methodology and conversion to protein content using a factor of 6.25. Peptide content in the TCA-soluble fraction was measured using the Pierce Quantitative Peptide Assay Kit (Thermo Scientific, Waltham, MA, USA, catalog number: 23290) according to manufacturer specifications.

2.5.5. Morphological Analysis of Fungal Colonies

Colony morphology on CMC-Na plates was assessed after 4 days of incubation at 30 °C. Parameters evaluated included colony diameter, texture, pigmentation, zonation patterns, and sporulation intensity.

Spore production was quantified by suspending fermented material (1 g) in 0.10% (v/v) Tween-80 solution (19 mL), vortexing (60 s) using a vortex mixer (Vortex-Genie 2, Scientific Industries, Bohemia, NY, USA), and filtering through sterile cheesecloth. Spore concentration was determined using a hemocytometer (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) under light microscopy (Olympus CX43, Olympus Corporation, Tokyo, Japan) and expressed as spores per gram dry substrate.

2.6. Proteomic Analysis

For proteome analysis, extracellular proteins were extracted from day 5 fermentation samples by adding deionized water, shaking at 180 rpm for 1 h using a shaking incubator (HZQ-X300, Hangzhou Huier Instrument Equipment Co., Ltd., Hangzhou, China), followed by centrifugation using an ultracentrifuge (Optima XPN-100, Beckman Coulter, Brea, CA, USA) and filtration through a 0.22 µm membrane (Millipore, Burlington, MA, USA). The supernatants were concentrated by lyophilization using a freeze dryer (FreeZone 2.5, Labconco, Kansas City, MO, USA) and stored at −80 °C (Forma 900, Thermo Fisher Scientific, Waltham, MA, USA).

Protein samples (100 μg) underwent tryptic digestion at 37 °C (HeraTherm Incubator, Thermo Scientific) overnight using trypsin (Promega, Madison, WI, USA), followed by C18 purification (Waters Corporation, Milford, MA, USA) and vacuum concentration (Savant SPD131DDA, Thermo Fisher Scientific). LC-MS/MS analysis was performed on an AB SCIEX Triple TOF 5600 plus system using sequential window acquisition of all theoretical mass spectra (SWATH). The protocol employed initial data-dependent acquisition (DDA) to establish a spectral library, followed by SWATH acquisition for quantitative analysis [38].

Spectral library generation and SWATH data processing utilized Skyline version 3.5 software following established criteria: exclusion of modified peptides and those shared between protein entries, minimum 95% confidence threshold, selection of up to five fragment ions ranked by intensity, and application of a mass-to-charge tolerance of 10 ppm. The mProphet algorithm was implemented for sample normalization and data confidence assessment [39].

CAZyme annotation was conducted using the hmmscan tool from HMMER software (version 3.2.1) against the dbCAN database [40], with outputs processed using the hmmscan-parser.sh script (https://github.com/carden24/Bioinformatics_scripts/blob/master/hmmscan-parser.sh, accessed on 20 January 2025). KEGG pathway mapping was performed via KOBAS 2.0, while secretion signals were predicted using SignalP 4.0. Data visualization and statistical analyses were accomplished using Circos (version 0.69.8), Cytoscape (version 3.7.1), and the R package vioplot (version 4.3.1) for comprehensive interpretation of the proteomic landscape [41].

2.7. Statistical Analysis

All experiments were conducted with three independent biological replicates, with results presented as mean ± standard error. Statistical significance was determined using univariate analysis of variance followed by Duncan’s multiple range test in IBM SPSS Statistics v.22.0. Differences were considered significant at p < 0.05 and highly significant at p < 0.01. Graphical representations were prepared using GraphPad Prism 10.

3. Results and Discussion

3.1. Comparative Evaluation of Lignocellulolytic Capabilities Among Selected Fungal Genera During Corn Stover SSF

The highly recalcitrant architecture of lignocellulosic biomass in corn stover presents a significant barrier to efficient bioconversion. This inherent recalcitrance is rooted in the intricate cross-linked structure of cellulose, hemicellulose, and lignin, which have evolved as natural defense mechanisms against microbial degradation [6,42]. To surmount these structural barriers, the concerted action of multiple enzyme systems is essential. Each of these enzyme systems is specifically targeted at particular components of the lignocellulosic matrix. Our experimental approach encompassed comprehensive monitoring of seven key enzyme activities: cellulase (FPase and CMCase), hemicellulase (xylanase and β-xylosidase), and lignin-degrading enzymes (MnP, LiP, and Lac). The initial screening focused on comparing two genera renowned for lignocellulolytic potential: Trichoderma (T. longibrachiatum and T. reesei) and Aspergillus (A. niger and A. oryzae). These strains were chosen based on their well-documented capacity for producing diverse hydrolytic enzymes. However, their relative efficiency in degrading corn stover still awaited systematic evaluation. The experimental design incorporated multiple analytical approaches: enzyme activity profiling, substrate composition analysis, and protein accumulation assessment, enabling a multifaceted comparison of strain performance.

3.1.1. Temporal Dynamics and Strain-Specific Patterns of Enzyme Production

The temporal evolution of enzymatic activities during SSF of corn stover unveiled a distinct triphasic pattern across all tested fungal strains. A comprehensive surveillance of seven key lignocellulolytic enzymes showed characteristic growth-associated kinetic profiles, which materialized in three distinct phases: an initial rapid ascent (0–3 days), a peak plateau (4–7 days), and a subsequent decline (8–12 days) (Figure 1). In the initial phase, the exponential surges in enzyme activities were concurrent with vigorous hyphal colonization. During the plateau phase, each strain displayed its unique enzyme production patterns. The final decline phase reflected the combined impacts of substrate transformation and the physiological adaptation of the fungi to the changing nutritional conditions. The observed non-linear enzyme production dynamics can be attributed to three principal main factors that unfold throughout the fermentation process. First, progressive transformation modifies the accessibility of degradable components, transitioning from readily available fractions to more recalcitrant structures. Secondly, the accumulation of metabolic end-products, including mono- and oligosaccharides, phenolic compounds, and organic acids, exerts feedback inhibition on enzyme synthesis pathways through sophisticated carbon catabolite repression mechanisms. Thirdly, the initiation of autolytic processes in the later fermentation stages triggers the release of intracellular proteases. These proteases degrade extracellular enzymes, thereby further contributing to the decline in enzymatic activities [43].

Strain-specific variations in enzyme production were evident across the three enzyme systems. While T. longibrachiatum excelled in cellulolytic enzyme production, Aspergillus strains, particularly A. niger, demonstrated superior hemicellulolytic capabilities with consistently high xylanase and β-xylosidase activities. Notably, A. niger exhibited exceptionally high β-xylosidase activity, suggesting a specialized capacity for final stages of hemicellulose degradation. On the other hand, Trichoderma strains dominated lignin degradation, with T. reesei maintaining high MnP activity (up to 41.48 U/g d.s.) during days 5–7, while T. longibrachiatum achieved peak LiP and Lac activities (693.78 U/g d.s. and 106.31 U/g d.s., respectively) on 6d (Figure 1e–g). These distinct enzymatic profiles reflect genus-specific evolutionary adaptations that shape their lignocellulose degradation strategies and ecological niches.

3.1.2. Strain-Specific Enzymatic Profiles and Comparative Analysis of Lignocellulose Degradation

A systematic evaluation of lignocellulolytic enzyme production on the fifth day of fermentation across the four fungal strains uncovered distinctive enzymatic profiles that reflect their evolutionary adaptations to diverse ecological niches.

T. longibrachiatum demonstrated remarkable superiority in cellulolytic enzyme production, with FPase and CMCase reaching 5.485 U/g d.s. and 10.523 U/g d.s., respectively, significantly exceeding levels observed in other tested strains (p < 0.0001) (Figure 2a,b). Particularly noteworthy was the maintenance of peak activities during the plateau phase, indicating a sustained production capacity that confers considerable advantages in comprehensive cellulose hydrolysis (Figure 1a,b).

The kinetics of hemicellulolytic enzyme production revealed intriguing taxonomic preferences (Figure 1d and Figure 2c). Xylanase activity exhibited considerable strain variability, with A. oryzae producing the highest levels (2.80 U/g d.s.), closely followed by T. longibrachiatum (2.59 U/g d.s.), while A. niger and T. reesei demonstrated comparatively lower activities (2.18 and 2.07 U/g d.s., respectively). Statistical analysis confirmed significant differences between high and low producers (p < 0.0001). Conversely, in the case of β-xylosidase production, A. niger emerged as the outstanding producer, with an activity of 2.91 U/g d.s., which was substantially higher than that of all other tested fungi (p < 0.0001). This high β-xylosidase activity indicates A. niger’s specialized capacity for hemicellulose depolymerization, especially in hydrolyzing xylo-oligosaccharides to xylose monomers [44,45]. Notably, T. longibrachiatum exhibited intermediate yet significant β-xylosidase activity (1.306 U/g d.s.), which, combined with its substantial xylanase production, suggests considerable hemicellulose degradation capability despite not matching A. niger’s specialized β-xylosidase production.

Analysis of ligninolytic enzyme systems unveiled intricate strain-specific specialization patterns (Figure 2e–g). T. longibrachiatum demonstrated extraordinary production of LiP and Lac, reaching peak activities of 693.78 U/g d.s. and 106.31 U/g d.s., respectively, substantially exceeded those of other tested strains (p < 0.0001). Concurrently, T. reesei exhibited distinctive specialization in MnP production, achieving activities up to 41.48 U/g d.s., significantly higher than observed in any other tested strain (p < 0.0001). The elevated ligninolytic capacity of Trichoderma strains compared to Aspergillus species indicates their enhanced adaptation to woody substrates where lignin modification is crucial for accessing cellulose and hemicellulose components.

3.1.3. Quantitative Assessment of Structural Component Degradation and Strain-Specific Deconstruction Patterns

To elucidate the effectiveness of different fungal strains in lignocellulose deconstruction, compositional analysis of corn stover was performed before and after 5 days of SSF using the NREL standard protocol. This allowed for the accurate quantification of the degradation rates of cellulose, hemicellulose, and lignin under standardized conditions.

A systematic assessment of the lignocellulosic degradation patterns during SSF revealed distinct strain-specific efficacies in transforming corn stover structural constituents (

Table 1. Effects of fungal pretreatment on compositional profiles of corn stover after 5 days of SSF (Mean ± SD).

Sample	Dry Matter Loss (%)	Cellulose (%)	Hemicellulose (%)	Lignin (%)
Corn stover material	——	30.30 ± 1.41	24.91 ± 0.80	19.45 ± 0.42
A. niger	13.58 ± 0.69	29.71 ± 1.13	23.75 ± 0.95	19.80 ± 0.49
A. oryzae	11.06 ± 1.38	29.63 ± 0.71	24.11 ± 0.62	20.46 ± 0.10
T. longibrachiatum	15.97 ± 0.76	29.78 ± 1.31	24.56 ± 0.90	19.67 ± 0.41
T. reesei	13.95 ± 0.23	30.34 ± 1.52	24.68 ± 0.90	19.62 ± 0.48

Note: Values represent the relative content (% w/w) of cellulose, hemicellulose, and total lignin (sum of acid-insoluble lignin and acid-soluble lignin), along with dry matter loss during fermentation. Data are presented as mean ± standard deviation (n = 3). Control values (unfermented corn stover) are shown in the first row for comparison.

and

Table 2. Degradation rates of major lignocellulosic components in corn stover following 5 days of SSF with different fungal strains (Mean ± SD).

Sample	Cellulose (%)	Hemicellulose (%)	Lignin (%)
A. niger	15.26 ± 1.45 ab	17.62 ± 0.65 a	12.04 ± 1.37 b
A. oryzae	12.96 ± 2.21 c	13.88 ± 2.16 c	6.43 ± 2.34 c
T. longibrachiatum	17.23 ± 1.65 a	16.92 ± 3.18 ab	14.84 ± 0.45 a
T. reesei	13.86 ± 0.74 bc	14.74 ± 0.91 bc	13.23 ± 0.59 ab

Note: Values represent the percentage of each original component degraded, calculated based on dry matter loss and changes in relative component content. The degradation rate of each component was determined according to the formula [Initial content × 100% − (100% − Dry matter loss%) × Final relative content%]/(Initial content × 100%) × 100%. For example, cellulose degradation by A. niger was calculated as [1 × 30.30% − (1 − 13.58%) × 29.71%]/(1 × 30.30%) × 100% = 15.26%. The same calculation method was applied to determine the degradation rates of hemicellulose and lignin. Data are presented as mean ± standard deviation (n = 3). Different superscript letters within the same column indicate significant differences (p < 0.05) according to one-way ANOVA followed by Duncan’s multiple range test, while shared letters indicate no significant difference (p > 0.05).

). Quantitative analysis of substrate compositional changes demonstrated that T. longibrachiatum exhibited exceptional comprehensive degradation capacity across all three major structural components. This organism achieved the highest cellulose degradation rate (17.23 ± 1.65%), significantly outperforming A. oryzae (12.96 ± 2.21%) and T. reesei (13.86 ± 0.74%), while showing statistical equivalence to A. niger (15.26 ± 1.45%). Similarly, T. longibrachiatum demonstrated substantial hemicellulose degradation (16.92 ± 3.18%), approaching the efficiency of A. niger (17.62 ± 0.65%) and significantly exceeding that of A. oryzae (13.88 ± 2.16%) and T. reesei (14.74 ± 0.91%). Most remarkably, T. longibrachiatum exhibited superior lignin degradation capacity (14.84 ± 0.45%), significantly surpassing all other tested strains, particularly A. oryzae, which showed the lowest lignin degradation (6.43 ± 2.34%).

The impressive degradation profile of T. longibrachiatum was correlated with its exceptional enzymatic machinery. In particular, the synchronized production of cellulolytic and ligninolytic enzymes, as evidenced by the previously mentioned elevated activities of FPase, CMCase, LiP, and Lac, likely plays a crucial role. This synergistic enzyme expression probably facilitates the comprehensive disruption of the recalcitrant lignocellulosic matrix, thereby enabling more efficient access to, and utilization of, the interconnected structural polymers [46,47]. The coordinated degradation of all three major components suggests sophisticated regulatory mechanisms governing the expression of diverse catalytic systems in response to the complex substrate composition.

The initial composition of corn stover in our study is comparable to values reported in the literature, which typically range from 30–35% cellulose, 20–25% hemicellulose, and 18–22% lignin [48]. Comparative analysis of our degradation data with the relevant literature provides valuable insights into the efficiency of our selected fungal strains. After 5 days of SSF, T. longibrachiatum achieved 17.23% cellulose degradation, surpassing the 11.86% reported for Lentinula edodes after 9 days [49] and the 10.5% observed for Trametes versicolor after 21 days [50]. The hemicellulose degradation rates in our study are somewhat lower than those reported in longer fermentation periods with L. edodes (19.33%) after 9 days [49], and with T. versicolor (21.9%) after 21 days [50]. Our lignin degradation results are comparable to those achieved by other fungal species in longer fermentation periods, though lower than the 43.36% using a combined bacterial consortium over 20 days [51]. Interestingly, our A. oryzae results differ from those reported by Guo et al. [6], who observed higher degradation rates (cellulose 30.67%, hemicellulose 20.67%) after 6 days, suggesting that specific cultivation conditions significantly influence degradation performance. The relatively high degradation rates achieved in our short fermentation period compared to longer durations in other studies highlight the potential time-efficiency advantages of our selected strains, particularly T. longibrachiatum, with its balanced degradation profile across all three major lignocellulosic components.

3.1.4. Preliminary Determination of T. longibrachiatum as the Preferred Candidate

A comprehensive evaluation of enzymatic capabilities and substrate degradation efficiencies has unambiguously identified T. longibrachiatum as the optimal candidate for corn stover bioconversion. Quantitative analysis revealed significantly elevated levels of key hydrolytic enzymes, including FPase, CMCase, lignin peroxidase, and laccase, exceeding activities measured in other tested strains (p < 0.0001). The exceptional enzymatic capabilities of T. longibrachiatum translated directly into superior substrate transformation, achieving the highest degradation rates for cellulose, hemicellulose, and lignin. This balanced deconstruction of all major structural components distinguishes T. longibrachiatum from other tested fungi, which exhibited more selective degradation patterns.

The exceptional performance of T. longibrachiatum likely originates from intricate metabolic synergies among its diverse enzymatic systems. Key synergistic interactions occur between ligninolytic and cellulolytic enzyme systems, where high lignin peroxidase and laccase activities facilitate disruption of the protective lignin matrix, enhancing accessibility of carbohydrate polymers to hydrolytic enzymes [52]. Complementary interactions between endoglucanases and the entire cellulase complex enable coordinated attack on amorphous and crystalline cellulose regions, while potential synergies between cellulases and hemicellulases contribute to enhanced substrate degradation by sequential polymer removal.

3.2. Parametric Optimization of ARTP Mutagenesis and Screening for Enhanced Lignocellulolytic Mutants

3.2.1. Dose–Response Kinetics of Spore Viability Under ARTP Plasma Treatment

Determining the optimal exposure time for ARTP mutagenesis constitutes a crucial prerequisite for maximizing positive mutation frequency while maintaining sufficient viable cells for subsequent screening. The relationship between exposure duration and spore survival in T. longibrachiatum was established through standard plate-counting methods, revealing a characteristic survival curve with important implications for mutation efficiency (Figure 3).

The survival curve demonstrates an overall negative correlation between exposure time and spore viability, with survival rates declining steadily as exposure duration increased. This trend is a consequence of the cumulative DNA damage inflicted by the reactive species generated within the ARTP plasma jet. These reactive species gradually undermine cellular viability by causing lethal mutations to accumulate and by damaging essential biomolecules structurally [19,20]. Interestingly, an anomalous pattern emerged within the 200–280 s exposure range, where the survival rate temporarily increased despite longer exposure durations. This unexpected phenomenon likely reflects the activation of cellular repair mechanisms capable of mitigating plasma-induced damage, thereby transiently enhancing survival rates through DNA repair pathway upregulation and stress-response mechanisms [53,54]. Beyond the 280 s mark, survival rates resumed their decline, with the 320 s exposure point corresponding to approximately 85% lethality. Empirical evidence from previous studies has shown that this specific lethality range (80–90%) is associated with optimal positive mutation frequencies [28]. The mechanistic basis for this optimization window likely reflects a balance between mutation load and selection—exposure conditions generating lethality below 80% typically produce insufficient mutational diversity, while conditions exceeding 90% lethality disproportionately increase negative mutations that compromise essential cellular functions, thereby diminishing the pool of beneficial variants.

Based on these findings, the 320 s exposure time was selected for subsequent mutagenesis experiments, providing optimal conditions for generating the mutant library while maintaining adequate viable cell populations for efficient screening of enhanced lignocellulolytic variants.

3.2.2. Primary Screening of Enhanced Cellulolytic Mutants Through Hydrolytic Zone Analysis

After conducting ARTP mutagenesis at the optimized exposure duration of 320 s, a hierarchical screening strategy was employed to isolate mutant strains with enhanced cellulolytic capacity. The initial screening was carried out on CMC-Na plates, offering an efficient high-throughput approach for the preliminary assessment of mutants [55].

The parental T. longibrachiatum strain exhibited a baseline HC value of 2.081 ± 0.262, corresponding to a cellulolytic index of 1.081 ± 0.262, which reflects its inherent capacity for cellulase production. A comprehensive analysis of 1505 colonies revealed a distinctly asymmetric distribution of mutational outcomes (

Table 3. Primary screening results of ARTP-mutated strains based on CMC hydrolysis capacity.

Strain Category	HC (D/d)	Cellulolytic Index	Number of Strains
T. longibrachiatum parental strain	2.081 ± 0.262	1.081 ± 0.262	659
Positive mutants (HC > 20%)	>2.500	>1.30	92
Selected mutants for secondary screening(HC > 35%)	>2.800	>1.50	19
Negative mutants (HC < 20%)	<1.665	<0.865	754
Total strains screened			1505

Note: HC represents the hydrolysis capacity, calculated as the ratio of clearing zone diameter (D) to colony diameter (d) on CMC-Na plates after Congo red staining. Cellulolytic index is calculated as HC-1, representing the proportion of the clearing zone extending beyond the colony edge.

). Approximately 43.8% (659) of the examined strains displayed HC values comparable to the parental strain, suggesting that these variants either experienced no significant mutations affecting cellulase expression or underwent compensatory mutations that preserved the wild-type phenotype. Positive mutations were detected in only 6.1% (92) of the strains. Among these positive mutants, 19 strains (1.3% of the total population) showed particularly notable improvements in cellulolytic activity. Their HC values exceeded the baseline by more than 35% (HC > 2.800). In contrast, negative mutations that led to a reduction in cellulolytic capacity (HC < 1.665) were predominant, accounting for 50.1% (754) of the colonies examined.

The fact that negative mutations are much more prevalent than positive ones is in line with evolutionary expectations. Random mutagenic events are more likely to disrupt rather than enhance functional pathways [56,57]. The significantly higher frequency of deleterious mutations likely reflects the intricate nature of cellulase expression and secretion systems, which involve complex regulatory networks, multiple structural genes, and sophisticated secretory mechanisms—all of which present numerous potential targets for disruptive mutations. On the other hand, beneficial mutations enhancing cellulolytic capacity likely require specific modifications to regulatory elements, secretory efficiency, or enzyme structure, representing a comparatively restricted mutational space [58,59,60].

3.2.3. Secondary Screening and Comprehensive Analysis of Selected Mutant Strains

Based on the primary screening results, the 19 mutants exhibiting exceptional cellulolytic potential (HC > 2.800) were chosen for secondary screening. This was to comprehensively assess their lignocellulolytic capabilities under conditions that more closely mimic industrial applications. Secondary screening in shake-flask fermentations revealed distinctive enzymatic and protein production profiles among the 19 selected mutants (Figure 4).

FPase exhibited notable enhancement in several mutant strains. Specifically, strains MU_07 (4.735 U/mL), MU_06 (4.603 U/mL), and MU_15 (4.597 U/mL) demonstrated significantly elevated FPase activities compared to the parental strain and other mutants. The observed increases of approximately 45–49% in cellulolytic capacity suggest successful modification of regulatory elements controlling cellulase gene expression or potential improvements in enzyme stability and catalytic efficiency [61]. These enhancements may result from mutations affecting transcription factor binding sites, mRNA stability, or post-translational modifications, all of which optimize enzyme’s functionality under fermentation conditions [62,63,64].

Analysis of water-soluble protein content provided further differentiation among the high-performing mutants. Strains MU_07 and MU_15 exhibited exceptional secretory capacity, secreting 20.532 mg/mL and 19.589 mg/mL of extracellular protein, respectively. This significant enhancement in protein secretion efficiency probably results from beneficial modifications in the cellular secretory machinery, potentially involving alterations in signal peptide processing, endoplasmic reticulum quality control mechanisms, or vesicular transport pathways [65,66]. The simultaneous improvement in both enzyme activity and protein secretion in these strains suggests synergistic mutations affecting multiple aspects of protein production and secretion.

Peptide content, which provides insights into protein metabolism and degradation dynamics, varied significantly across the mutant library. Strains MU_07 (1.229 mg/mL), MU_06 (1.227 mg/mL), and MU_17 (1.250 mg/mL) demonstrated superior performance in this parameter. The elevated peptide levels may indicate enhanced proteolytic processing of secreted proteins or altered amino acid metabolism that contributes to improved protein quality and stability [67,68]. This parameter offers complementary information to the total protein measurements, potentially reflecting differences in protein turnover rates and extracellular proteolytic activities among the mutant strains.

Collectively, the data from secondary screening data revealed distinct biochemical phenotypes among the mutants. Several strains showed improvements across multiple parameters. Notably, strain MU_07 demonstrated consistent superiority across all three evaluated metrics, suggesting comprehensive enhancement of its lignocellulolytic potential. However, to definitively identify the optimal strain, subsequent validation through fermentation performance assessment under conditions more closely mimicking industrial applications is necessary. The observed differences in parameter correlations among various mutants also imply that diverse molecular mechanisms underlie their improved phenotypes. This potentially presents multiple opportunities for strain improvement in future research endeavors.

3.2.4. Colony Morphology Differentiation of T. longibrachiatum Cellulolytic Mutants

Based on the comprehensive enzymatic and protein analyses from the secondary screening, nine representative mutant strains with varying levels of performance were selected for further morphological characterization alongside the parental strain. Morphological characteristics of microbial colonies provide valuable insights into their physiological states and metabolic capacities, particularly when visualized on substrate-specific media [69]. The comparative analysis of colony morphology on CMC-Na plates revealed remarkable phenotypic differences between the parental T. longibrachiatum strain and various ARTP-induced mutants, with distinctive patterns correlating with cellulolytic performance (Figure 5).

The parental strain (Figure 5A) showed moderate growth, presenting a relatively concentrated colony structure. Its peripheral expansion was limited, and the sporulation was modest, as indicated by the moderate yellowish-green pigmentation. In contrast, the high-performing mutants demonstrated significantly enhanced growth features. Strain TL-MU07 (Figure 5B) developed robust colonies characterized by extensive mycelial networks, uniform and dense sporulation, and significantly increased colonization diameter, along with intense yellow-green pigmentation, indicators of enhanced metabolic efficiency, and substrate utilization [69,70]. Similarly, TL-MU06 (Figure 5F) and TL-MU15 (Figure 5D) displayed vigorous radial expansion with abundant sporulation; they had subtle architectural differences compared to TL-MU07.

Conversely, several mutants exhibited compromised morphologies. TL-MU10 (Figure 5C) displayed reduced colony density and sporulation intensity with irregular peripheral patterning, while TL-MU08 (Figure 5G) manifested highly dispersed, fragmented colony architecture with scattered sporulation clusters, reflecting potential disruptions in hyphal networking or intercellular communication [70]. Intermediate performers TL-MU16 (Figure 5H) and TL-MU19 (Figure 5I) developed moderately expanded colonies with non-uniform sporulation patterns and sectored regions of varying developmental intensity.

Enhanced growth on cellulose-specific media serves visual confirmation of improved cellulolytic capacity, as efficient cellulose hydrolysis generates accessible carbon sources that support accelerated growth and development. The wide range of morphological phenotypes witnessed in the mutant library demonstrate the pleiotropic effects of ARTP mutagenesis, affecting not only specific enzymatic activities but also broader aspects of fungal physiology, intercellular organization, and developmental programming [71,72]. These morphological evaluations provide a valuable supplementary approach to biochemical characterization, offering immediate visual indication of cellulolytic potential that corresponds with quantitative measurements of enzyme activities and protein production profiles.

3.3. Comparative Evaluation of Mutant Strains Under SSF

Following the identification of promising mutants through plate screening and liquid culture evaluation, a comprehensive assessment of their lignocellulolytic capabilities under SSF conditions was conducted. SSF represents a critical industrial parameter due to its superior enzyme production efficiency and closer resemblance to natural fungal growth environments [73,74,75]. The selected strains (RS_00, MU_06, 07, and 15) were subjected to SSF using corn stover as the primary substrate, a widely available agricultural residue with significant potential for bioconversion in lignocellulosic biorefineries. Enzymatic activities and degradation parameters were quantified following a standardized 5-day incubation period.

MU_07 exhibited superior enzymatic performance under SSF conditions, with significantly elevated FPase (22.1%), CMCase (10.1%), and xylanase (16.1%) activities compared to the parental strain (Figure 6a–c). This concurrent enhancement of both cellulolytic and hemicellulolytic enzymes suggests that beneficial mutations affected higher-order regulatory mechanisms governing the broader lignocellulolytic system rather than enzyme-specific factors. Substrate degradation analysis confirmed that increased enzyme production translated into improved practical bioconversion capabilities, with MU_07 demonstrating enhanced cellulose degradation (14.6%) and hemicellulose utilization rates (12.9%) (Figure 6d,e). Interestingly, lignin degradation remained relatively stable across all strains (Figure 6f), indicating mutations primarily affected cellulolytic and hemicellulolytic systems while leaving lignin-modifying capabilities largely unaltered. Protein production parameters further differentiated mutant performance, with MU_07 showing significantly elevated true protein content (14.7%) compared to the parental strain (Figure 6h). Peptide content was also significantly higher in MU_07 (Figure 6i). Additionally, sporulation capacity was markedly enhanced in MU_07, which produced significantly higher spore counts than both the parental strain and other mutants (Figure 6g), indicating superior colonization efficiency and reproductive fitness under SSF conditions. Collectively, these improvements establish MU_07 as the most promising strain for industrial applications requiring enhanced lignocellulose degradation.

Based on this comprehensive evaluation cascade, strain MU_07 was unequivocally identified as the most promising candidate for further development and application. Its consistent superiority across multiple assessment platforms suggests beneficial mutations affecting central regulatory mechanisms governing the cellulolytic system rather than isolated enzymatic components, potentially enhancing transcriptional regulation, secretory efficiency, or enzyme stability [58,59,60]. The exceptional performance of MU_07 under industrial-mimicking conditions particularly underscores its practical utility for lignocellulose bioconversion applications and validates the effectiveness of the ARTP mutagenesis and hierarchical screening strategy employed in this study. Consequently, strain MU_07 was selected as the optimal candidate for subsequent investigations aimed at elucidating the molecular basis of its enhanced lignocellulolytic capacity and further optimizing its industrial application potential.

3.4. Proteomic Analysis Reveals Molecular Basis for Enhanced Cellulolytic Performance in ARTP-Generated Mutant

3.4.1. Characterization of Proteome Architecture and Strain-Specific Expression Patterns

To elucidate the molecular mechanisms underlying the enhanced cellulolytic performance of the ARTP-induced mutant, a comprehensive proteomic analysis was conducted comparing strain MU_07 (designated as TL_M4) with the parental strain (designated as TL_S4) during SSF. Samples were collected after 5 days of cultivation and subjected to high-resolution mass spectrometry-based proteomics.

Venn diagram analysis revealed substantial conservation of the core proteome between strains, with 4940 proteins (98.33%) shared by both (Figure 7a). This conservation suggests ARTP mutagenesis induced targeted modifications rather than extensive genomic disruptions. Nevertheless, strain-specific expression patterns were evident, with 58 proteins (1.15%) uniquely detected in MU_07 and 26 proteins (0.52%) exclusively in the parental strain, potentially representing critical determinants of observed phenotypic differences [76].

Sample correlation analysis demonstrated exceptional reproducibility among biological replicates while clearly distinguishing between strains (Figure 7b). Correlation coefficients confirmed strong technical consistency and biological stability. Contrastingly, inter-strain correlations (0.677–0.689) quantitatively indicated substantial proteomic divergence. Hierarchical clustering analysis corroborated this pattern by grouping samples into two distinct strain-specific clades, offering compelling molecular evidence of significant physiological reconfiguration in MU_07 post-mutagenesis. Principal component analysis further substantiated these findings, with samples distinctly separated along PC1 (37.30% of total variance), while PC2 (19.80%) primarily reflected comparable intra-group variation (Figure 7c). The minimal overlap in confidence ellipses statistically confirmed the distinct proteomic signatures between MU_07 and the parental strain.

Collectively, these multivariate analyses of protein expression profiles provide compelling evidence that ARTP mutagenesis induced systematic reconfiguration of the T. longibrachiatum proteome. While preserving the majority of the core proteome essential for cellular viability, the mutations appear to have strategically altered specific protein expression patterns, including both quantitative modulation of shared proteins and qualitative changes manifested as strain-specific proteins. We next sought to dissect the specific protein expression changes and functional categories that might directly contribute to the enhanced cellulolytic phenotype observed in the mutant strain.

3.4.2. Differential Proteome Analysis Reveals Coordinated Metabolic and Regulatory Adaptations Underlying Enhanced Lignocellulolytic Capacity

Having established distinct proteomic profiles between the parental strain and MU_07, a detailed analysis of differential protein expression patterns and their functional implications was undertaken to elucidate the molecular mechanisms underlying the enhanced cellulolytic performance of the mutant strain.

Quantitative analysis of protein abundance revealed substantial reconfiguration of the MU_07 proteome compared to the parental strain. As visualized in the volcano plot (Figure 8a), 289 proteins exhibited significant differential expression (p < 0.05), with 110 proteins upregulated and 179 proteins downregulated in MU_07 relative to the parental strain. Among the most significantly upregulated proteins in MU_07 were an ALDH-like protein, a microtubule-associated protein, an MPN domain-containing protein, and a DUF3818 domain-containing protein. The substantial upregulation of ALDH-like protein is particularly noteworthy, as aldehyde dehydrogenases play critical roles in detoxification of reactive aldehydes generated during lignin degradation, potentially enhancing the strain’s tolerance to lignocellulose breakdown products that can inhibit fungal growth and enzyme activity [77]. Similarly, the increased abundance of microtubule-associated and MPN domain-containing proteins suggests enhanced intracellular trafficking and protein turnover systems, which may facilitate efficient secretion of cellulolytic enzymes and recycling of cellular components during nutrient-limited SSF [78]. Conversely, the most dramatically downregulated proteins included a Zn(2)-C6 fungal-type domain-containing protein, protein BNI4, and several uncharacterized proteins. The pronounced decrease in Zn(2)-C6 fungal-type domain-containing protein is particularly intriguing, as these proteins regulate carbon utilization and gluconeogenesis [79,80]. Some C6 proteins act as transcription activators in secondary metabolite production [79], so their downregulation may reduce inhibitory metabolites. Furthermore, certain C6 zinc proteins negatively regulate conidiation [81], suggesting that their decreased expression in our mutants likely contributed to the enhanced spore formation observed on CMC-Na plates and during SSF. The reduced abundance of protein BNI4, involved in cell wall biogenesis and chitin deposition, suggests altered cell wall architecture in MU_07, potentially facilitating more efficient secretion of cellulolytic enzymes across the fungal cell membrane [82,83].

To gain functional insights into these proteome-wide alterations, KEGG pathway annotation was performed on the differentially expressed proteins (Figure 8b). This analysis revealed that carbohydrate metabolism pathways were most prominently represented, with 23 differentially expressed proteins involved in these processes. This substantial reconfiguration of carbohydrate metabolism provides direct molecular evidence for enhanced capacity to utilize complex polysaccharides in the mutant strain. Additional metabolic pathways significantly affected included global and overview maps (13 proteins), amino acid metabolism (8 proteins), lipid metabolism (8 proteins), and metabolism of cofactors and vitamins (8 proteins), indicating metabolic reprogramming beyond the immediate cellulolytic machinery. Notably, transcription (7 proteins) and protein folding, sorting, and degradation (6 proteins) were among the most significantly altered genetic information processing pathways, suggesting coordinated modulation of gene expression and protein secretion systems. The substantial representation of transport and catabolism (9 proteins) among cellular processes further supports enhanced capacity for substrate acquisition and utilization in MU_07. Collectively, this pathway distribution analysis indicates that ARTP mutagenesis induced multifaceted physiological reconfiguration affecting not only carbohydrate metabolism but also complementary cellular systems that support efficient lignocellulose degradation under SSF conditions.

KEGG enrichment analysis (Figure 8c) provided statistical validation of significantly altered metabolic pathways in MU_07. Glycosphingolipid biosynthesis–ganglio series exhibited the highest enrichment factor. This significant remodeling of membrane composition in the mutant strain likely enhances secretory capacity and cellular resistance to fermentation-induced stresses [84,85]. The glycosaminoglycan degradation pathway demonstrated the most statistically significant enrichment (lowest p-value), highlighting its relevance to MU_07’s enhanced degradative capabilities. This pathway encompasses enzymes that systematically dismantle complex polysaccharide structures, and its preferential enhancement strategically reinforces the strain’s capacity to access and process recalcitrant structural carbohydrates within lignocellulosic biomass [86]. The amino sugar and nucleotide sugar metabolism pathway contained the largest number of differentially expressed proteins among enriched pathways (represented by the largest circle diameter). This pathway serves as a central hub connecting primary carbohydrate metabolism with cell wall biosynthesis and remodeling, while also processing oligosaccharides released during lignocellulose degradation. Its substantial reconfiguration in MU_07 indicates comprehensive enhancement of the strain’s capacity to both degrade complex substrates and efficiently assimilate the resulting breakdown products [87]. Galactose metabolism also demonstrated significant enrichment, reflecting enhanced capacity for metabolism of hemicellulose-derived sugars.

The molecular mechanisms underlying MU_07’s enhanced cellulolytic activity integrate multiple coordinated physiological adaptations. The downregulation of Zn(2)-C6 fungal-type domain-containing proteins likely contributes to enhanced cellulolytic activity through reduced production of potentially inhibitory secondary metabolites and improved spore formation. Simultaneously, the upregulation of detoxification systems (ALDH-like proteins) and enhanced membrane transport capabilities empower MU_07 to maintain robust enzyme production despite the accumulation of potentially inhibitory degradation products during SSF. The strategic augmentation of complementary carbohydrate utilization pathways, especially amino sugar metabolism, glycosaminoglycan degradation, and galactose metabolism, indicates an improved ability to both liberate and subsequently metabolize the diverse sugars derived from lignocellulosic biomass. This comprehensive enhancement of both lignocellulose degradation and sugar utilization pathways represents a highly valuable industrial phenotype. It allows for the efficient conversion of complex substrates into fungal biomass and metabolic products [88]. Furthermore, the reconfiguration of protein folding, sorting, and degradation systems suggests enhanced capacity for protein secretion and quality control, potentially alleviating bottlenecks in the production and export of cellulolytic enzymes. This integrated enhancement of regulatory, metabolic, and secretory systems revealed by proteomic analysis provides compelling molecular evidence for the mechanistic basis of MU_07’s superior cellulolytic performance under SSF conditions.

3.5. Comparative Characterization of TL-MU07

TL-MU07 exhibits several distinctive characteristics compared to previously reported strains, positioning it as a particularly promising candidate for industrial bioconversion applications.

The enzyme production profile of TL-MU07 represents a substantial improvement over previously characterized strains under comparable conditions. While T. longibrachiatum MDU-6 produced 9.9 U/g of CMCase after 10 days of SSF [89], TL-MU07 achieved 11.08 U/g within 5 days (Figure 6b). Similarly, in shake flask culture, wild-type T. longibrachiatum typically exhibit FPase activities of 2.04–3.07 U/mL after 7–8 days [90,91], whereas TL-MU07 produced 4.735 U/mL after 96 h (Figure 4a), demonstrating both enhanced activity and accelerated production kinetics. Protein secretion capacity is notably amplified in TL-MU07, which achieved extracellular protein concentrations of 20.532 mg/mL within 96 h of cultivation (Figure 4b). This represents a significant improvement compared to previous reports of T. longibrachiatum that typically reach 2.0–2.19 mg/mL protein concentrations after 192 h [91,92]. Furthermore, TL-MU07 demonstrated exceptional adaptation to SSF conditions, producing 2.28 × 10¹⁰ spores/g after 5 days (Figure 6g), which substantially exceeds the previously reported value for industrial T. longibrachiatum (3.75 × 10⁹ spores/g after 6 days of cultivation) [14].

These distinctive characteristics collectively position TL-MU07 as a highly promising candidate for industrial bioconversion applications, offering enhanced process efficiency, reduced operational time requirements, and improved tolerance to industrial fermentation conditions compared to previously reported strains.

4. Conclusions

This study demonstrates the comparative lignocellulolytic capabilities of the evaluated fungal strains during corn stover bioconversion, revealing distinct enzymatic profiles and degradation patterns. Notably, T. longibrachiatum exhibited a consistently superior and balanced performance by combining the highest cellulose degradation (17.23%) with exceptional lignin breakdown (14.84%) and robust cellulolytic enzyme activities. In contrast, A. niger excelled in hemicellulose degradation (17.62%) and demonstrated outstanding β-xylosidase activity, while A. oryzae showed moderate performance across the measured parameters, including a relatively lower lignin degradation capacity (6.43%). T. reesei performed at an intermediate level with a notable manganese peroxidase activity (41.48 U/g d.s.). Building upon these comparative findings, ARTP mutagenesis was employed to further enhance T. longibrachiatum, yielding mutant strain TL-MU07 with remarkable improvements in cellulolytic enzyme production, substrate degradation efficiency, and protein accumulation. Specifically, TL-MU07 achieved 14.6% higher cellulose degradation and 14.7% greater protein accumulation compared to the parental strain under SSF conditions. Proteomic analysis revealed strategic reconfiguration of multiple cellular systems in the mutant, including downregulation of transcriptional repressors, enhancement of detoxification mechanisms, and optimization of carbohydrate metabolism pathways. This multifaceted physiological adaptation provides a molecular basis for the superior performance observed in practical applications. The comprehensive enhancement of both lignocellulose degradation capability and stress tolerance represents valuable industrial traits for agricultural waste valorization. These findings not only contribute to fundamental understanding of fungal lignocellulose degradation mechanisms but also provide an efficient strategy for developing high-performance microbial strains for sustainable biomass utilization in biorefinery applications and protein-enriched feed production.