Elicitation-Induced Enhancement of Lovastatin and Pigment Production in Monascus purpureus C322

Abstract

Monascus purpureus is a filamentous fungus renowned for producing bioactive secondary metabolites, including lovastatin and azaphilone pigments. Lovastatin is valued for its cholesterol-lowering properties and cardiovascular benefits, while Monascus pigments exhibit anti-cancer, anti-inflammatory, and antimicrobial activities, underscoring their pharmaceutical and biotechnological relevance. This study evaluated the impact of carbohydrate-derived elicitors—mannan oligosaccharides, oligoguluronate, and oligomannuronate—on the enhancement of pigment and lovastatin production in M. purpureus C322 under submerged fermentation. Elicitors were added at 48 h in shake flasks and 24 h in 2.5 L stirred-tank fermenters. All treatments increased the production of yellow, orange, and red pigments and lovastatin compared to the control, with higher titres upon scale-up. OG led to the highest orange pigment yield (1.2 AU/g CDW in flasks; 1.67 AU/g CDW in fermenters), representing 2.3- and 3.0-fold increases. OM yielded the highest yellow and red pigments (1.24 and 1.35 AU/g CDW in flasks; 1.58 and 1.80 AU/g CDW in fermenters) and the highest lovastatin levels (10.46 and 12.6 mg/g CDW), corresponding to 2.03–3.03-fold improvements. These results highlight the potential of carbohydrate elicitors to stimulate metabolite biosynthesis and facilitate scalable optimisation of fungal fermentation.

1. Introduction

Microbial secondary metabolites such as alkaloids, statins, and pigments have garnered great industrial interest due to their therapeutic and commercial value. However, the natural production levels of these metabolites are often suboptimal [1,2,3,4,5]. Various strategies have been developed to enhance metabolite yields, including cell immobilisation, genetic engineering, and elicitation techniques [6,7,8,9,10]. Among these, elicitation has emerged as a powerful biotechnological tool, employing biotic and abiotic agents to induce stress responses and activate metabolic pathways that lead to increased metabolite production [11,12].

Elicitation is a biotechnological approach that involves applying biotic or abiotic agents to stimulate the production of secondary metabolites by activating specific stress or defence responses within microorganisms [11,12]. These agents, termed elicitors, modulate cellular responses to external stimuli, leading to enhanced biosynthesis of bioactive compounds with pharmaceutical and industrial relevance. In fungi like A. terreus and M. purpureus, elicitors such as carbohydrate oligomers have been reported to enhance secondary metabolite biosynthesis, a process attributed to the upregulation of key metabolic pathways [13,14,15,16]. The efficacy of carbohydrate-based elicitors, including mannan oligosaccharides (MOs), oligoguluronate (OG), and oligomannuronate (OM), has been demonstrated in multiple microbial systems, yet their role in Monascus fermentations remains relatively underexplored [17,18,19,20,21,22,23,24,25]. By applying elicitation strategies, secondary metabolite production can be optimised without the need for genetic modifications, offering a safe, sustainable, and scalable approach for industrial fermentation processes.

Monascus spp. are filamentous fungi that have been used in East Asian food and traditional medicine for centuries, particularly in the production of red yeast rice. Recognised for their Generally Recognised As Safe (GRAS) status, these fungi are valued for their ability to naturally synthesise bioactive and therapeutic secondary metabolites during fermentation [26,27,28]. Among these, lovastatin—a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase—is widely used for cholesterol reduction, obesity management, and triglyceride regulation, making it essential for cardiovascular health [29,30,31]. In addition, studies have suggested that lovastatin may possess anti-cancer potential, particularly in inhibiting tumour cell proliferation and inducing apoptosis [11,32]. Meanwhile, Monascus pigments, which include red, yellow, and orange compounds, have drawn interest beyond their role as natural food colorants. These pigments possess anti-cancer, anti-inflammatory, and antimicrobial properties, making them valuable for pharmaceutical and nutraceutical applications [33,34]. Their potential as functional food additives and therapeutic agents further underscores their significance in biotechnology and healthcare [6,35,36].

This study investigates the effects of carbohydrate elicitors on the production of pigments and lovastatin in M. purpureus C322, comparing their influence in both shake flask cultures and stirred-tank fermenters. By assessing metabolite yield improvements under different fermentation conditions, this work provides insight into the potential of carbohydrate-based elicitors as biotechnological tools for process optimisation. The findings contribute to a deeper understanding of elicitor-driven metabolic regulation and present a scalable strategy for enhancing secondary metabolite production in industrial fermentation applications.

In addition to elicitor-based strategies, several other approaches have been employed to improve secondary metabolite production in Monascus species. These include genetic engineering techniques targeting key biosynthetic or regulatory genes to increase pathway flux and process-based modifications such as fed-batch fermentation, which can enhance metabolite yields by optimising nutrient supply and reducing stress accumulation [20,21,23,24,37,38,39]. While the present study focuses on elicitation, these alternative strategies represent promising directions for further yield optimisation.

2. Materials and Methods

2.1. Microorganism Maintenance

The Monascus purpureus C322 used in this study was sourced from the University of Westminster’s culture collection. The strain was preserved on yeast malt extract medium and was routinely subcultured every two weeks onto Hiroi Potato Dextrose Agar, following the methodology outlined in [40]. Each agar plate was inoculated with 10⁶ spores/mL and incubated at 28 °C for a period of 10 to 15 days. After incubation, the plates were stored at 4 °C for future use.

2.2. Fermentation Setup and Culture Conditions

For the submerged fermentation experiments, the strain was cultivated in a chemically defined growth medium (pH 6.5), based on the protocol described in [41]. Two types of fermentation systems were utilised in the study: 500 mL Erlenmeyer flasks and 2.5 L jacketed glass stirred-tank bioreactors. The fermenters (Fermac 310/60; Electrolab Biotech, Tewkesbury, UK) were equipped with two impellers mounted on a central shaft. The upper impeller, a pitched-blade (mixed-flow) design, was positioned just below the liquid surface to facilitate foam disruption. The lower impeller was a standard Rushton turbine (radial-blade, axial-flow) mounted near the base of the vessel. The impellers were vertically spaced at approximately H/2, where H represents the working liquid height. This configuration was selected to optimise mixing throughout the vessel and minimise foam formation during fermentation. For the flask cultures, the incubation was carried out at 25 °C with a shaking speed of 120 rpm. The fermenters were incubated at 32 °C, with an agitation rate of 300 rpm. To initiate the fermentation process, a pre-culture was prepared in the shake flasks and fermenters by inoculating the medium with M. purpureus C322 and incubating it at 25 °C with shaking at 120 rpm for a period of 7 days. During the fermentation process, key parameters such as pH, carbohydrate concentration, and pigment production were monitored daily, while the biomass and lovastatin concentrations were measured at the end of the fermentation cycle.

2.3. Elicitor Preparation and Application

Three carbohydrate-based elicitors were used in this study: mannan oligosaccharides (MOs), oligoguluronate (OG), and oligomannuronate (OM). These elicitors were derived from natural sources by hydrolysis. MOs were extracted from locust bean gum, and OG and OM were obtained from sodium alginate, utilising a combination of enzyme-mediated and acid hydrolysis protocols, as described in [23,25,42]. The elicitors were added at a concentration of 150 mg/L after 48 h in shake flasks and 24 h in stirred-tank fermenters, respectively, based on previous in-house studies [17,18,19,20,21,22,23,24,25,42]. To ensure consistency across experiments, all elicitors were prepared from high-purity starting materials using standardised protocols developed during prior optimisation studies [17,18,19,20,21,22,23,24,25,42]. These protocols consistently yielded oligosaccharides with a degree of polymerisation ranging between 7 and 9 and standardised the concentration and timing of elicitor addition. All reagents used were of research-grade quality to maintain reproducibility and accuracy throughout the study. The addition of these elicitors was aimed at enhancing metabolite production, particularly pigment and lovastatin biosynthesis, by triggering specific pathways in the microorganism.

2.4. Analytical Techniques for Biomass, Carbohydrate, Pigment, and Lovastatin Quantification

Key fermentation parameters, including pH, residual carbohydrate concentration, and pigment production, were monitored every 24 h throughout the fermentation. Biomass accumulation and lovastatin concentration were measured at the end of the fermentation process to evaluate the effects of elicitors on metabolite production. Biomass quantification was carried out using a gravimetric approach [43]. The total biomass was collected through vacuum filtration using a Merck™ (Dorset, United Kingdom) Vacuum Flask fitted with Büchner Funnels and a Fischerbrand FB70155 pump. The filtered biomass was transferred onto Whatman No. 1 filter paper, which was then oven-dried at 100 °C for 12 h before the dry cell weight (CDW) was determined. Pigment production was assessed through spectrophotometric analysis, with absorbance measured at 400 nm for yellow pigments, 470 nm for orange pigments, and 510 nm for red pigments, following the method described in [30,44]. Carbohydrate consumption was determined using the phenol–sulfuric acid assay, a widely used method for quantifying residual sugars in fermentation media [31,45]. Lovastatin production was analysed using High-Performance Liquid Chromatography (HPLC), employing a Lichrospher RP-18 Endcapped column at 25 °C. The analysis was performed under isocratic conditions, using a mobile phase consisting of 55:45 (vol/vol) HPLC-grade acetonitrile and 0.1% phosphoric acid. The injection volume was 25 μL, and the flow rate was 1 mL/min. Detection was carried out at 238 nm, following the methodology described in [46,47].

2.5. Statistical Analysis

All experiments were performed in triplicate, and data were expressed as mean ± standard deviation. Statistical significance between experimental groups was assessed using one-way ANOVA followed by Tukey’s post hoc test, with a significance level set at p < 0.05. GraphPad Prism version 10 and OriginPro version 2025 software was used for statistical analysis and data visualisation.

3. Results

3.1. Impact of Elicitation on Biomass Accumulation

Biomass accumulation, measured as cell dry weight (CDW), exhibited differences between shake flasks and 2.5 L stirred-tank bioreactors. Across all conditions, fermenters yielded comparatively higher biomass concentrations than shake flasks, suggesting that controlled environmental conditions such as improved aeration and mixing provide growth advantage. In shake flasks, CDW values ranged from 6.3 g/L to 6.7 g/L across different treatment groups. Fermenters achieved CDW values ranging from 6.97 g/L to 8.03 g/L. The control group (C), with no added elicitor, showed a 1.12-fold increase in CDW between the fermenters and the shake flasks. Likewise, the groups supplemented with elicitors—OG, OM, and MO—displayed increased CDW by 1.12, 1.22, and 1.08 folds, respectively, upon transitioning from shake flasks to fermenters (Figure 1). One-way ANOVA (p > 0.05) confirmed that elicitor treatments did not significantly alter biomass accumulation compared with the control.

3.2. Elicitor-Driven Enhancement of Pigment Production

Pigment production by Monascus purpureus C322 varied across different elicitor treatments, showing distinct trends in concentration (OD values) and yield (AU/g CDW) between shake flasks and fermenters (Figure 2,

Table 1. Average pigment yields (AU/g CDW) * in shake flasks (SF) and fermenters (F).

Group	Yellow		Orange		Red
	SF	F	SF	F	SF	F
C	0.61	0.71	0.54	0.55	0.52	0.59
OG	1.03	1.36	1.22	1.67	1.13	1.60
OM	1.24	1.60	1.16	1.42	1.35	1.80
MO	0.87	1.04	0.89	1.12	0.98	1.16

* Formula: Absorbance units (AU)/Cell dry weight in grams (g CDW).

). The addition of elicitors (OG, OM, MO) increased yellow, orange, and red pigment biosynthesis compared to the control (C). Statistical analysis (ANOVA, p < 0.01) confirmed that all elicitor treatments significantly influenced pigment production, with Tukey’s post hoc test identifying significant differences among treatment groups. For yellow pigment (OD₄₀₀), OM-treated cultures exhibited the highest concentrations (OD₄₀₀ 8.3 in the shake flasks, OD₄₀₀ 13.4 in the fermenters), followed by OG (OD₄₀₀ 7.2, OD₄₀₀ 10.8) and MO (OD₄₀₀ 6.0, OD₄₀₀ 8.0) (Figure 2a,b). The lowest yellow pigment concentration was observed in the control group, with values of OD₄₀₀ 5.0 in the shake flasks and OD₄₀₀ 6.2 in the fermenters. Tukey’s analysis revealed that yellow pigment levels in OM-treated cultures were significantly higher than the control (p < 0.01), OG (p < 0.01), and MO (p < 0.01).

Orange pigment production (OD₄₇₀) was highest with OG supplementation, reaching OD₄₇₀ 9.2 in the shake flasks and OD₄₇₀ 12.4 in the fermenters, followed by OM (OD₄₇₀ 8.5, OD₄₇₀ 11.2) and MO (OD₄₇₀ 6.3, OD₄₇₀ 8.1) (Figure 2a,b). The control group exhibited the lowest orange pigment concentration, with OD₄₇₀ 3.4 in the shake flasks and OD₄₇₀ 4.6 in the fermenters. Tukey’s multiple comparisons test confirmed that OG and OM significantly increased orange pigment concentration compared to MO and the control (p < 0.01). Red pigment production (OD₅₁₀) also increased with elicitor supplementation, with the highest concentrations observed in OM-treated cultures (OD₅₁₀ 9.1 in the shake flasks, OD₅₁₀ 14.6 in the fermenters), followed by OG (OD₅₁₀ 8.1, OD₅₁₀ 13.0) and MO (OD₅₁₀ 6.8, OD₅₁₀ 9.7). Similar to yellow and orange pigment production, the control group exhibited the lowest red pigment concentration, measuring OD₅₁₀ 2.9 in the shake flasks and OD₅₁₀ 4.2 in the fermenters (Figure 2a,b). Tukey’s analysis confirmed that OM-treated cultures produced significantly more red pigment than the control (p < 0.01), OG (p < 0.01) and MO (p < 0.01). All pigment concentrations followed a comparable pattern across all conditions, with higher values recorded in fermenters compared to shake flasks.

Scaling up from the shake flasks to fermenters resulted in increased pigment production in M. purpureus C322 across all elicitor conditions, indicating enhanced metabolic activity under controlled bioreactor conditions. The control group exhibited the lowest pigment concentrations, whereas elicitor-treated cultures displayed a more predictable and enhanced pigment profile. Pigment accumulation followed different kinetics between the two systems. In shake flasks, production increased gradually over the cultivation period, while in fermenters, peak concentrations were attained more rapidly, consistent with the accelerated kinetics typically observed under optimised and controlled conditions. Tukey’s multiple comparisons confirmed that these variations were statistically significant (p < 0.01) across all conditions. The pigment yield data (AU/g CDW) provided in Table 1 and Figure 3 aligned with the concentration-based trends. Figure 3a–c illustrate the 3D surface plots of yellow, orange, and red pigment yields, respectively, highlighting the variation between elicitor treatments. OG-treated cultures exhibited the highest orange pigment yield (1.67 AU/g CDW) (Figure 3b), while OM-treated cultures showed the highest red pigment yield (1.80 AU/g CDW) (Figure 3c), reinforcing the distinct roles of OG and OM in stimulating orange and red pigment biosynthesis, respectively (Table 1, Figure 3).

When comparing elicitor-treated groups to the control, OG increased yellow pigment by 1.7- and 1.9-fold in shake flasks and fermenters; orange pigment by 2.2- and 3.0-fold; and red pigment by 2.2- and 2.7-fold, respectively. OM supplementation increased yellow by 2.0- and 2.2-fold; orange by 2.1- and 2.6-fold; and red by 2.6- and 3.0-fold, respectively. MO exhibited fold increases of 1.4 and 1.5 for yellow; 1.6 and 2.0 for orange; and 1.9 and 2.0 for red in both systems (shake flasks and fermenters), respectively. As far as the scale-up is concerned, in terms of the fold increase in pigment production from the shake flasks to fermenters across different treatments, the highest orange pigment enhancement was achieved by OG supplementation (1.4-fold; 1.22 → 1.67 AU/g CDW), the highest yellow pigment increase by both OG and OM (1.3-fold; 1.03 → 1.36 AU/g CDW for OG and 1.24 → 1.58 AU/g CDW for OM), and the highest red pigment enhancement by OM supplementation (1.3-fold; 1.35 → 1.80 AU/g CDW). Overall, OG produced the highest enhancement across all pigment categories during scale-up (1.3-fold for yellow, 1.4-fold for orange, and 1.4-fold for red), followed by OM (1.3-fold increase for red, 1.3-fold for yellow, and 1.2-fold for orange) and MO (1.2-fold increase for red, 1.2-fold for yellow, and 1.2-fold for orange), whereas the control group exhibited the lowest fold increase (1.1-fold for yellow, 1.0-fold for orange, and 1.1-fold for red).

3.3. Lovastatin Production in Shake Flasks and Fermenters

Lovastatin production varied across different elicitor treatments in both shake flasks and fermenters (Figure 4). ANOVA analysis (p < 0.01) confirmed significant differences among groups, with elicitor-treated cultures producing higher lovastatin yields compared to the control (C). In shake flasks, the control group exhibited a lovastatin yield of 5.72 mg/g CDW (31.82 mg/L), whereas elicitor-treated cultures showed increased production. OM-treated cultures produced the highest lovastatin yield (12.11 mg/g CDW; 79.54 mg/L), followed by OG (10.46 mg/g CDW; 70.08 mg/L) and MO (9.41 mg/g CDW; 58.33 mg/L). Tukey’s post hoc test confirmed significant differences among the groups, with OM supplementation resulting in significantly higher lovastatin production than MO (p < 0.01) and OG (p = 0.03). OG-treated cultures also exhibited significantly greater lovastatin production than MO (p = 0.0112), while all elicitor treatments were significantly different from the control (p < 0.01) (Figure 3d and Figure 4).

In fermenters, an increase in lovastatin yield was observed across all conditions (Figure 4). The control group in fermenters produced 5.28 mg/g CDW (36.93 mg/L), whereas elicitor-treated cultures exhibited increased yields, with OM-treated fermenters producing the highest yield (12.6 mg/g CDW; 98.29 mg/L), followed by OG (11.29 mg/g CDW; 84.66 mg/L) and MO (9.58 mg/g CDW; 67.04 mg/L). Tukey’s test confirmed statistically significant differences, with OM-treated cultures producing significantly higher lovastatin yields than MO (p < 0.01) and OG (p = 0.01). OG-treated cultures also exhibited significantly greater lovastatin production than MO (p < 0.01), while all elicitor-treated cultures showed a statistically significant increase compared to the control (p < 0.01) (Figure 3d and Figure 4). The relative fold increase in fermenters compared to shake flasks was 1.2-fold for OM (12.6 mg/g CDW in fermenters vs. 12.11 mg/g CDW in shake flasks), 1.2-fold for OG (11.29 mg/g CDW vs. 10.46 mg/g CDW), and 1.1-fold for MO (9.58 mg/g CDW vs. 9.41 mg/g CDW). Compared to the control group, OM supplementation resulted in the highest lovastatin production, with a 2.1-fold increase in shake flasks and a 2.4-fold increase in fermenters. OG supplementation yielded 1.8-fold and 2.1-fold increases in shake flasks and fermenters, respectively, while MO led to 1.6-fold and 1.8-fold increases. All elicitor-treated cultures produced higher lovastatin titres than the control, and production levels were consistently maintained or improved during scale-up, with OM and OG showing the greatest increases.

3.4. Effect of Elicitors on pH Variation and Carbohydrate Consumption

pH variation and carbohydrate consumption were monitored in both shake flasks and 2.5 L stirred-tank bioreactors. Similar trends were observed across all conditions. An initial decline in pH was recorded across all cultures, followed by a gradual increase during the later stages of fermentation. The initial pH was set to 6.5 in both shake flasks and fermenters. In the shake flasks, the pH decreased to a minimum of 5.8–5.9 during the mid-phase of fermentation, before increasing towards the end, ranging between 6.8 and 7.1. A comparable trend was observed in fermenters, where the pH dropped to approximately 5.7–5.8 before increasing to around 6.1–6.3. Statistical analysis showed no significant differences among the control and elicitor-treated groups (p > 0.05).

Carbohydrate consumption patterns indicated a progressive depletion in both shake flasks and fermenters. An initial rapid consumption phase was followed by a slower depletion rate at later stages. Statistical analysis confirmed no significant variation among experimental groups (p > 0.05), indicating that elicitor supplementation did not significantly alter carbohydrate utilisation rates.

These findings are consistent with previous studies involving elicitation with OG, OM, and MO, which demonstrated enhanced secondary metabolite production without altering pH or sugar utilisation patterns significantly [17,18,19,20,21,22,23,24,25].

4. Discussion

The elicitor-based strategies demonstrated significant potential in enhancing the biosynthesis of secondary metabolites in fungi, particularly in M. purpureus C322. The use of carbohydrate-derived elicitors, such as OG, OM, and MO, resulted in substantial increases in pigment and lovastatin production in M. purpureus C322 without a notable change in biomass concentration, reinforcing previous findings that suggest the role of elicitors in metabolic regulation [13,14,17,18,19,20,21,22,23,24,25]. These results support reports that carbohydrate elicitors can modulate cellular stress responses, redirecting metabolic flux towards enhanced secondary metabolite accumulation. The observed enhancement in pigment biosynthesis following elicitor supplementation suggests a direct influence on the azaphilone biosynthetic pathway, which governs the production of yellow, orange, and red pigments in M. purpureus [28]. OG supplementation was the most effective in promoting orange pigment production, while OM led to the highest red pigment concentrations. This supports previous studies that indicate different elicitors can activate distinct metabolic pathways by modulating gene expression and enzymatic activity [17,18,19,20,21,22,23,24,25]. Furthermore, OM and OG showed a statistically significant increase in pigment production compared to MO and the control, confirming that specific oligosaccharide structures may interact differently with microbial regulatory networks [48,49]. Notably, pigment production was higher in fermenters compared to shake flasks, highlighting the role of optimised bioprocess conditions in maximising fungal metabolism. The more rapid accumulation of pigments in fermenters suggests that the controlled aeration, agitation, and homogeneity in fermenters provides a more favourable environment for mass transfer and metabolic activity [50,51,52]. Additionally, the ability of elicitors to sustain high pigment yields in both systems suggests that they can be reliably incorporated into large-scale fermentation processes to improve pigment production efficiency [17,18,19,20,21,22,23,24,25].

Moreover, the results demonstrate a strong elicitor-induced enhancement of lovastatin biosynthesis, with OM-treated cultures exhibiting the highest yield. Previous studies suggest that polyketide biosynthesis pathways, including lovastatin production, are responsive to elicitors due to their regulatory effects on transcription factors involved in secondary metabolism [53,54,55,56]. The upregulation of genes encoding polyketide synthases in response to elicitor supplementation has been reported in other microbial and plant species, further supporting the hypothesis that OM exerts its effect by enhancing precursor availability and enzyme activity in the lovastatin biosynthetic pathway [53,54,55,56]. The significant increase in lovastatin yield in fermenters, compared to shake flasks, further confirms the role of bioprocess optimisation in maximising secondary metabolite production. A notable correlation between pigment and lovastatin biosynthesis trends suggests a potential overlap in their regulatory pathways. Given that both pigment and lovastatin biosynthesis utilise acetyl-CoA and malonyl-CoA as precursor molecules, it is plausible that elicitors function by enhancing the availability of these intermediates, thereby promoting simultaneous upregulation of multiple secondary metabolites [35,57,58,59]. The metabolic interplay between these pathways provides an opportunity for co-optimisation strategies to enhance industrial production of both pigments and lovastatin using targeted elicitor selection.

The patterns of pH and carbohydrate utilisation remained consistent across all treatments, indicating that elicitor addition did not disrupt primary metabolic processes. The initial decline in pH observed during early fermentation likely reflects organic acid accumulation associated with primary metabolic activity [60,61,62,63,64], while the gradual increase in pH during the stationary phase may be attributed to the production of alkaline by-products such as ammonia, a trend previously reported in fungal fermentations [35,65,66] Similarly, sugar utilisation followed a biphasic trend—characterised by rapid depletion during the log phase, followed by slower consumption in the stationary phase—consistent with established metabolic transitions in filamentous fungi [35,67,68,69]. These findings support the view that elicitor-mediated enhancement of secondary metabolism occurs without significant alteration of the basal metabolic framework.

Building on these metabolic and regulatory insights, industrial applicability remains a crucial factor in evaluating the effectiveness of elicitor-based strategies. The results indicate that elicitor supplementation can be integrated into large-scale fermentation without compromising process efficiency, with fermenters providing superior control over key parameters such as pH, dissolved oxygen, and nutrient supply, which are critical for secondary metabolism [50,51,52,70]. Moreover, the ability to enhance specific metabolite profiles through different elicitor treatments provides flexibility in industrial applications, where varying pigment-to-lovastatin ratios may be desirable. The findings of this study reinforce the utility of carbohydrate-based elicitors in enhancing fungal secondary metabolism, with OG and OM demonstrating distinct and complementary effects on pigment and lovastatin biosynthesis. Although mechanistic elucidation was beyond the experimental scope of the present study, previous research has shown that strategies such as genetic engineering of key biosynthetic or regulatory genes and fed-batch fermentation processes can also enhance pigment and lovastatin production by modulating intracellular signalling and improving physiological conditions. These approaches represent complementary avenues that may be explored alongside elicitor treatments to maximise yields and optimise metabolic fluxes [20,21,23,24,37,38,39].

Future research should focus on elucidating the precise molecular mechanisms underlying elicitor-induced metabolic changes, particularly in relation to quorum sensing and transcriptional regulation. Understanding the regulatory interactions between elicitor-induced pathways could further refine bioprocess strategies, leading to more efficient industrial-scale production of high-value fungal metabolites. An additional avenue worth investigating is the effect of elicitor addition to washed cells, which could help confirm whether the observed enhancements in metabolite production are due to direct pathway modulation rather than nutritional contribution. Although the elicitors used in this study were applied at concentrations too low to serve as significant carbon sources, and no notable increase in biomass was observed, experiments using washed biomass could offer more conclusive evidence of their role as metabolic triggers.

5. Conclusions

This study extends the knowledge supporting carbohydrate-based elicitation as a viable strategy for enhancing secondary metabolite production. The significant improvements in pigment and lovastatin yields across different fermentation systems using M. purpureus C322 demonstrates the scalability potential of elicitor supplementation for industrial bioprocesses. The ability to enhance production of lovastatin and pigments, without an increase in biomass, through strategic elicitor selection provides a valuable tool for optimising large-scale production. The results further reinforce that fermentation scale, when coupled with elicitor supplementation, can influence fungal secondary metabolism. This strategy not only enhances productivity but also lays the groundwork for future metabolic engineering efforts in industrial biotechnology.

Overall, this work demonstrates that elicitor-driven fermentation strategies hold significant promise for the large-scale production of valuable fungal metabolites. The integration of elicitor supplementation into biotechnological applications presents an innovative and practical approach for improving yields, reducing production costs, and expanding the commercial viability of fungal secondary metabolites in diverse industrial sectors.