Microbial Bioactives

1. Introduction

Environmental degradation has increasingly become one of the defining challenges of the modern era. Rapid urban expansion, industrial intensification, and large-scale agricultural practices have collectively altered natural ecosystems in ways that are difficult to reverse. These anthropogenic pressures have disrupted ecological stability, reduced habitat connectivity, and accelerated biodiversity loss across terrestrial and aquatic systems (Hanski, 1999). At the same time, climatic shifts associated with global warming are further amplifying environmental stressors, creating complex interactions between ecological disturbances and anthropogenic activities (Quereda Sala et al., 2000). Taken together, these pressures have intensified the search for sustainable technologies capable of mitigating environmental damage while maintaining economic productivity.

One particularly concerning issue involves the widespread contamination of natural environments by emerging pollutants. Pharmaceuticals and personal care products (PPCPs), for instance, have been detected in aquatic ecosystems worldwide, often at trace concentrations yet with potentially significant ecological consequences (Patel et al., 2019). Conventional wastewater treatment processes frequently fail to completely remove these compounds, allowing them to accumulate in rivers, soils, and sediments (Wang & Wang, 2016). This persistence raises questions about long-term ecological effects and underscores the need for environmentally friendly remediation strategies capable of degrading or transforming such contaminants.

In response to these challenges, global research and industrial communities have increasingly turned toward sustainability-oriented frameworks. Among these, Circular Supply Chain Management (CSCM) has emerged as a promising paradigm that promotes resource efficiency, waste minimization, and regenerative production systems (Farooque et al., 2019). Rather than treating industrial by-products as waste streams, circular approaches emphasize their transformation into valuable resources within closed-loop production systems. Such strategies are especially relevant in biotechnology, where organic residues can be converted into high-value biomolecules through microbial processes.

Parallel to these developments, advances in green technologies are reshaping the landscape of sustainable agriculture and environmental management. Nanotechnology, for example, has introduced innovative tools such as nano-biofertilizers that improve nutrient delivery and enhance crop resilience while reducing the ecological footprint of conventional fertilizers (Akhtar et al., 2022). These nanoscale materials possess high surface area and enhanced solubility, enabling more efficient nutrient uptake by plants and minimizing nutrient runoff into surrounding ecosystems (Nasrollahzadeh et al., 2019). Such approaches align closely with precision agriculture strategies aimed at optimizing productivity while preserving environmental integrity (Duhan et al., 2017). Within the broader context of sustainable agricultural policies, these technologies are increasingly being integrated into green development frameworks designed to reduce chemical inputs and environmental risks (Ginter, 2022).

Beyond agricultural applications, biotechnology offers powerful tools for transforming biomass into useful products. Microbial enzymes play a central role in this transformation, acting as biocatalysts capable of replacing energy-intensive or hazardous chemical processes. Enzymes such as cellulases and related lignocellulolytic enzymes facilitate the degradation of complex plant biomass into fermentable sugars, thereby supporting diverse industrial applications ranging from biofuel production to waste management (Kuhad et al., 2011). Their versatility has made them increasingly important across multiple sectors, including pharmaceuticals, food processing, and environmental remediation (Ejaz et al., 2021). The economic significance of industrial enzymes continues to grow as industries seek sustainable alternatives to conventional chemical treatments (Guerrand, 2018).

Among these enzymes, fungal laccases have attracted considerable attention due to their broad substrate specificity and ability to catalyze oxidation reactions involving phenolic and non-phenolic compounds. These multicopper oxidases are naturally produced by a variety of fungi, particularly white-rot species, and play an essential role in lignin degradation within forest ecosystems. Because of their oxidative capacity, laccases have been explored for numerous industrial applications including bioremediation, pulp and paper processing, textile treatment, and biosensor development. The challenge, however, lies in producing these enzymes efficiently and sustainably at industrial scale.

Solid-State Fermentation (SSF) has emerged as one of the most promising strategies for achieving this goal. Unlike submerged fermentation, SSF involves the cultivation of microorganisms on moist solid substrates with little or no free water, thereby closely mimicking the natural habitats of many fungi. This cultivation mode often leads to higher enzyme yields, improved stability, and lower energy requirements compared with liquid fermentation systems (Rehman et al., 2014). Moreover, SSF enables the direct utilization of agricultural residues as both physical support and nutrient source for microbial growth.

The use of agro-industrial waste materials as substrates represents a particularly attractive feature of SSF. Large quantities of lignocellulosic residues are generated globally from agricultural and food-processing industries, including rice straw, wheat straw, corn stover, sugarcane bagasse, and fruit processing by-products. Traditionally, these materials have been underutilized or disposed of through environmentally harmful practices such as open burning. However, their rich composition of cellulose, hemicellulose, and lignin makes them excellent substrates for microbial fermentation processes (Mussatto et al., 2012). Recent research has increasingly focused on converting these residues into valuable biomolecules, thereby contributing to sustainable waste management strategies (Šelo et al., 2021).

In the context of fungal fermentation, lignocellulosic substrates provide not only structural support but also act as natural inducers for enzyme production. Lignin and other aromatic compounds present in plant biomass can stimulate laccase synthesis by white-rot fungi, which utilize these enzymes to break down complex lignin polymers. Several studies have demonstrated that agricultural residues such as sugarcane bagasse, rice husk, and sunflower residues can effectively support fungal growth and enhance laccase production during SSF processes (Karp et al., 2015). Similarly, pilot-scale investigations have shown that rice straw and sunflower residues can be successfully converted into both medicinal mushrooms and laccase enzymes through controlled fermentation processes (Postemsky et al., 2017). In addition, the cultivation of edible and medicinal mushrooms on agro-industrial waste has been widely reported as an efficient strategy for producing lignocellulolytic enzymes while simultaneously generating valuable fungal biomass (Kumla et al., 2020).

In addition to enzyme production, the bioconversion of agro-industrial residues can generate a wide range of other valuable biomaterials. For example, lignocellulosic biomass can serve as a feedstock for biopolymers that are increasingly used in textile, packaging, and biomedical industries (Dias et al., 2025). Natural fibers derived from plant biomass are also gaining attention as sustainable alternatives to synthetic materials, particularly in industrial applications requiring biodegradable or renewable resources (Manaia et al., 2019). At the molecular level, microbial and plant-derived polymers are being explored for applications in advanced materials and biotechnology, further demonstrating the versatility of biomass-derived products (Rudin & Phillip, 2013).

Recent technological advances have expanded these possibilities even further. Electrospinning technologies, for instance, enable the fabrication of nanofibrous biomaterials capable of delivering therapeutic compounds in biomedical applications (Bonakdar & Rodrigue, 2024). Such nanofiber systems are increasingly investigated for controlled drug delivery and tissue engineering purposes (Luraghi et al., 2021). Similarly, bioactive compounds recovered from agricultural or winery by-products are being explored for their antimicrobial and antioxidant properties (Teixeira et al., 2014). Essential oils and plant-derived compounds have demonstrated promising antibacterial and antibiofilm activities, suggesting potential applications in food safety and medical biotechnology (Firmino et al., 2018).

Another emerging area involves microbial exopolysaccharides, which are extracellular polymers produced by bacteria and fungi. These biomolecules exhibit diverse functional properties, including emulsifying, antioxidant, and immunomodulatory activities, making them valuable in biomedical and food applications (Kaur & Dey, 2023). Advances in microbial biotechnology have revealed numerous exopolysaccharide-producing microorganisms capable of generating these materials under controlled fermentation conditions (Netrusov et al., 2023). In parallel, advances in biomaterial engineering and encapsulation technologies have enabled the controlled release of bioactive compounds from packaging and polymer matrices, highlighting new applications of fermentation-derived biomolecules in food preservation and biomedical systems (Siddiqui et al., 2023).

Given the rapidly expanding body of research in this field, systematic synthesis of available evidence has become increasingly important. Systematic reviews and meta-analyses provide structured frameworks for evaluating diverse studies and identifying consistent trends across experimental systems. The PRISMA guidelines, widely adopted in scientific literature, offer standardized procedures for reporting such reviews and ensuring transparency in study selection and data analysis (Urrútia & Bonfill, 2010). These methodological approaches are particularly valuable for assessing complex biotechnological systems, where multiple variables—such as substrate composition, fungal strain selection, and environmental conditions—can significantly influence outcomes.

The economic relevance of biomass-derived materials and enzymes further reinforces the need for integrated research efforts. Market analyses suggest that demand for cellulose-based fibers and related biomaterials is expected to grow substantially as industries transition toward renewable and biodegradable materials (Grand View Research, 2016). Similarly, enzyme markets continue to expand as biotechnology becomes increasingly central to sustainable industrial production.

Despite these advances, several challenges remain in optimizing laccase production through SSF. Variability in substrate composition, differences among fungal strains, and limitations in large-scale fermentation systems can all affect enzyme yields and process stability. Consequently, a comprehensive understanding of these variables is essential for developing efficient and scalable biotechnological processes.

In this context, the present systematic review aims to synthesize current knowledge regarding sustainable fungal laccase production through solid-state fermentation using agro-industrial residues. By examining substrate selection, fermentation conditions, fungal strains, and process optimization strategies, this review seeks to clarify key factors influencing enzyme production and highlight opportunities for integrating SSF technologies into circular bioeconomy frameworks. Through this analysis, the study contributes to the broader goal of developing sustainable biotechnological approaches that simultaneously address environmental challenges and industrial resource demands.

2. Materials and Methods

2.1 Study Design and Review Framework

This study was designed as a systematic review and meta-analysis to evaluate the methodologies and outcomes associated with fungal laccase production through Solid-State Fermentation (SSF), with particular emphasis on the valorization of agro-industrial residues. The review aimed to synthesize current experimental evidence regarding fungal strain selection, substrate utilization, fermentation optimization, and enzyme productivity. The overall methodological framework followed internationally recognized guidelines for systematic reviews and meta-analyses to ensure transparency, methodological rigor, and reproducibility of the study selection and analytical process. Specifically, the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were applied throughout the review process to standardize literature identification, screening, eligibility assessment, and final inclusion of studies (Page et al., 2021; Urrútia & Bonfill, 2010). The general approach and analytical workflow were also guided by established meta-analytic principles widely used in evidence synthesis research (Higgins et al., 2022). The literature selection procedure and study inclusion steps are summarized in Figure 1.

Figure 1: PRISMA flow diagram illustrating literature search, screening, and study inclusion. The diagram illustrates the systematic screening process used to identify eligible studies evaluating fungal laccase production through solid-state fermentation using agro-industrial residues. From 520 initially retrieved records, 13 studies met the inclusion criteria and were incorporated into the quantitative meta-analysis.

2.2 Literature Search Strategy

A structured literature search was conducted across multiple international scientific databases to identify relevant studies published between 2000 and 2025. The databases included PubMed, Scopus, Web of Science, and Google Scholar to ensure broad coverage of peer-reviewed literature related to fungal enzyme biotechnology and fermentation processes. The search strategy incorporated combinations of keywords and Boolean operators to maximize retrieval of relevant studies. Search terms included “fungal laccase production,” “solid-state fermentation,” “agro-industrial residues,” “lignocellulosic waste,” “enzyme induction,” “bioreactor design,” and “SSF optimization.” The search strategy was iteratively refined to capture both experimental fermentation studies and process optimization research related to laccase production. Only peer-reviewed journal articles written in English were considered for inclusion to maintain methodological consistency and ensure data reliability. Similar systematic search strategies have been widely used in recent reviews addressing complex biological and biotechnological research domains (Amin et al., 2025; Setu et al., 2025).

2.3 Study Screening and Eligibility Criteria

Following database retrieval, all identified records were exported into reference management software, and duplicate entries were removed prior to screening. The screening process was conducted in three sequential stages, including title screening, abstract evaluation, and full-text assessment. Two independent reviewers evaluated the retrieved studies to minimize selection bias and ensure objectivity in study inclusion. In cases of disagreement between reviewers, discrepancies were resolved through discussion and consensus.

Studies were included in the review if they met the following criteria: (i) investigation of fungal laccase production using Solid-State Fermentation systems, (ii) utilization of lignocellulosic or agro-industrial residues as fermentation substrates, (iii) quantitative reporting of laccase activity or enzyme yield, and (iv) description of process optimization strategies such as nutrient supplementation, inducer application, or environmental parameter adjustment. Studies were excluded if they lacked quantitative enzyme production data, were review articles rather than original research, or were not written in English. This structured eligibility framework ensured that only experimentally robust and methodologically comparable studies were included in the final dataset.

2.4 Data Extraction and Standardization

Relevant data were extracted systematically from the eligible studies using a predefined data collection framework. Extracted variables included fungal species and strains, substrate types and chemical composition, nutritional supplements, inducer compounds, fermentation conditions (temperature, pH, moisture content, and incubation duration), and reported laccase activity expressed as enzyme units per gram of substrate (U/g). Additional information regarding reactor configuration, fermentation scale, and enzyme characterization methods was also recorded where available.

The extracted data were organized into standardized tables to facilitate cross-study comparison and quantitative synthesis. To ensure comparability among studies that used different reporting formats or experimental conditions, enzyme activity values were normalized when necessary. This normalization allowed the calculation of comparable outcome measures and effect sizes across different experimental systems. Standardization procedures followed commonly accepted meta-analytic practices used in biological and experimental research (Borenstein et al., 2009).

2.5 Substrate Preparation and Characterization

The studies included in this review primarily employed lignocellulosic agro-industrial residues as fermentation substrates. These residues included rice straw, wheat straw, corn straw, sugarcane bagasse, apple pomace, banana peels, sunflower seed hulls, and other crop-derived by-products commonly generated in agricultural processing systems. Such materials are widely recognized as suitable substrates for SSF due to their high content of cellulose, hemicellulose, and lignin, which provide both structural support and nutrient sources for fungal growth (Šelo et al., 2021).

In most studies, substrates were pretreated prior to fermentation to improve microbial accessibility and remove contaminants. Typical pretreatment procedures involved washing the biomass with distilled water to remove dust and soil particles, followed by drying at temperatures between 60 and 70°C until constant weight was achieved. After drying, substrates were milled or chopped to particle sizes ranging from 1 to 5 mm to enhance surface area and facilitate fungal colonization. The chemical composition of the substrates, including cellulose, hemicellulose, and lignin content, was obtained either from the original experimental studies or from standardized characterization procedures described in the literature. Particular attention was given to lignin content because lignin and related aromatic compounds can function as natural inducers of laccase synthesis in white rot fungi (Wang et al., 2019). The moisture content of substrates was adjusted to approximately 50–70% using sterile distilled water to ensure optimal hydration while maintaining adequate porosity for oxygen diffusion within the fermentation matrix.

2.6 Fungal Strain Selection and Inoculum Preparation

White rot fungi belonging primarily to the Basidiomycota phylum were the most commonly reported microorganisms in SSF-based laccase production studies. Representative species included Trametes versicolor, Pleurotus ostreatus, Marasmiellus palmivorus, and Coriolopsis caperata. These fungi possess strong ligninolytic systems capable of producing extracellular oxidative enzymes such as laccase, lignin peroxidase, and manganese peroxidase, making them highly suitable for lignocellulosic biomass degradation (Postemsky et al., 2017).

Fungal strains were generally obtained from established microbial culture collections or isolated from natural lignocellulosic environments such as forest soils and decaying wood. Inocula were prepared by cultivating the fungi on potato dextrose agar (PDA) or malt extract agar plates until adequate mycelial growth was achieved. Mycelial plugs or spore suspensions were then transferred to the prepared substrates to initiate fermentation. The typical inoculum size ranged from 5 to 10% (w/w), which allowed rapid fungal colonization while preventing excessive substrate depletion during early fermentation stages.

2.7 Nutritional Supplementation and Inducer Application

Although lignocellulosic residues provide baseline nutrients for fungal growth, many studies incorporated additional carbon and nitrogen sources to enhance fungal metabolism and enzyme production. Common carbon supplements included glucose, sucrose, and maltose, while nitrogen supplementation frequently involved yeast extract, peptone, or ammonium salts. The Carbon-to-Nitrogen (C/N) ratio was carefully adjusted depending on the fungal strain and substrate composition, with typical values ranging from 20:1 to 40:1.

Metal ions and aromatic compounds were frequently used as inducers to stimulate laccase gene expression. Copper sulfate (CuSO4) was the most commonly used metal inducer because laccase enzymes belong to the multicopper oxidase family. Additionally, phenolic compounds and flavonoid-rich agro-industrial residues were applied as environmentally friendly inducers to enhance enzyme production. Inducer concentrations were optimized in each experimental study to achieve maximal enzyme activity while avoiding inhibitory effects on fungal growth.

2.8 Solid-State Fermentation Conditions and Reactor Systems

SSF experiments were typically conducted using small-scale laboratory systems, including Erlenmeyer flasks, tray bioreactors, and static fermentation containers. In these setups, substrates were distributed evenly to a thickness of approximately 2–5 cm to ensure adequate aeration and uniform fungal colonization. Fermentation temperatures were generally maintained between 25°C and 30°C, and incubation periods ranged from 7 to 21 days depending on the fungal strain and substrate composition.

Relative humidity was maintained above 70% to prevent substrate desiccation during fermentation. Moisture levels were periodically monitored, and sterile distilled water was added when necessary to maintain optimal hydration conditions. For larger-scale experimental systems, specialized reactor configurations such as rotary drum bioreactors and immersion fermentation systems were utilized to improve oxygen transfer and reduce heat accumulation during microbial metabolism. These reactor designs help address mass transfer limitations and thermal gradients that may arise during large-scale SSF operations.

2.9 Enzyme Extraction and Activity Assays

Following fermentation, laccase enzymes were extracted from the solid fermentation matrix using buffered extraction solutions. Typically, fermented substrates were mixed with citrate or acetate buffer (50 mM, pH 4.5–5.5) at ratios ranging from 1:5 to 1:10 (w/v). The mixture was agitated at 100–200 rpm for approximately 30–60 minutes to facilitate enzyme release into the solution phase. The resulting suspension was then filtered through cheesecloth or centrifuged at 5,000–10,000 × g for 10–15 minutes to remove solid particles and obtain the crude enzyme extract.

Laccase activity was measured using standard spectrophotometric assays based on the oxidation of substrates such as ABTS or guaiacol. Enzyme activity was determined by monitoring changes in absorbance at 420 nm for ABTS oxidation or 465 nm for guaiacol oxidation. The resulting enzyme activity values were expressed as units per gram of dry substrate (U/g), enabling consistent comparison across studies (Karp et al., 2015).

2.10 Meta-Analysis and Statistical Evaluation

Quantitative data extracted from the included studies were analyzed using established meta-analytic techniques. Weighted mean effect sizes were calculated for different experimental variables, including substrate types, fungal strains, and inducer treatments. When sufficient data were available, pooled estimates were generated using random-effects models to account for variability among independent studies (DerSimonian & Laird, 1986).

Heterogeneity among studies was evaluated using the I² statistic, which quantifies the proportion of total variation attributable to differences between studies rather than random error (Higgins et al., 2003). Subgroup analyses were conducted to assess the influence of substrate type, fermentation duration, and nutritional supplementation on laccase production. Forest plots were generated to visualize effect size distributions, while funnel plots and regression tests were used to assess potential publication bias within the dataset (Egger et al., 1997). All statistical analyses were performed using R statistical software (version 4.3.2) and appropriate meta-analysis packages.

2.11 Quality Assessment and Risk-of-Bias Evaluation

The methodological quality of the included studies was evaluated using modified risk-of-bias criteria adapted from the Cochrane framework for systematic reviews (Higgins et al., 2022). Key evaluation parameters included reproducibility of inoculum preparation, adequacy of substrate characterization, clarity of fermentation parameters, and reliability of enzyme assay methodologies. Studies that lacked sufficient methodological detail or failed to report quantitative enzyme production data were excluded from the meta-analysis to maintain analytical reliability and reduce potential bias in the final results.

3. Results

3.1 Fungal Laccase Production from Agro-Industrial Residues and Dye Decolorization Performance under Solid-State Fermentation

The systematic review synthesized experimental evidence describing fungal laccase production through solid-state fermentation (SSF) and the associated application of these enzymes in dye decolorization. The studies included in the analysis collectively demonstrate that agro-industrial residues serve as highly effective substrates for fungal growth and enzyme expression. A clear pattern emerges from the compiled dataset: the interaction between fungal species and substrate composition strongly influences enzyme yield and downstream environmental applications. The PRISMA screening framework ensured that only studies reporting quantitative enzyme activity or dye removal efficiency were included in the analysis (Page et al., 2021; Urrútia & Bonfill, 2010).

The dataset summarized in Table 1 highlights substantial variability in laccase activity across fungal strains and substrate types. Enzyme yields ranged from relatively low activity values, such as 14.19 U/g reported for Daedalea flavida cultivated on cotton stalk residues, to extremely high activity levels exceeding 10,000 U/g for Trametes versicolor grown on brewer’s spent grain (Šelo et al., 2021; Saldarriaga-Hernández et al., 2020). Such variation illustrates how substrate composition, pretreatment methods, and fungal metabolic characteristics collectively determine fermentation outcomes.

Table 1: Laccase Activity Across Fungal Strains and Agro-Waste Substrates. This table extracts studies that provided both mean activity and standard deviation (SD), allowing for the calculation of weighted mean differences in enzyme yield across different substrates.

Among the studies examined, Trametes versicolor appeared repeatedly as a highly efficient laccase producer. When cultivated on olive leaves, this species generated a mean activity of 276.62 U/g, demonstrating strong enzymatic capability on lignin-rich substrates. Even more striking results were observed when brewer’s spent grain was used as the fermentation substrate, yielding enzyme activities of approximately 10,108 U/g. These values significantly exceed those observed for other substrates in the dataset, suggesting that brewery by-products may provide an optimal combination of carbon sources, lignin derivatives, and micronutrients for fungal metabolism (Šelo et al., 2021). Steam-exploded cornstalk substrates also supported substantial enzyme production, reaching mean activities of approximately 2,600 U/g, while untreated cornstalk produced somewhat lower yields of around 1,241 U/g. This contrast highlights the role of substrate pretreatment in improving enzymatic accessibility to lignocellulosic components.

Other fungal species included in the dataset also demonstrated notable enzyme productivity. For example, Coriolus versicolor cultivated on sweet sorghum bagasse generated mean laccase activity of approximately 205 U/g, suggesting that this substrate can support moderate enzyme synthesis under SSF conditions (Mishra & Jana, 2019). Similarly, Pycnoporus sanguineus grown on a mixture of wheat bran and corncob residues produced enzyme activity of about 138.6 U/g (Orlikowska et al., 2018). Although these values are lower than those observed with brewer’s spent grain, they nonetheless confirm that a variety of lignocellulosic agricultural residues can function as effective fermentation matrices.

An additional example of substrate versatility appears in the case of Marasmiellus palmivorus, which produced laccase activity of approximately 667 U/g when cultivated on pineapple leaf residues (Schneider et al., 2018). This observation reinforces the broader conclusion that plant-derived agricultural wastes—particularly those containing significant lignin fractions—can support fungal ligninolytic enzyme production. The findings align with earlier research suggesting that lignocellulosic substrates act not only as nutrient sources but also as natural inducers of laccase expression in white-rot fungi (Karp et al., 2015; Šelo et al., 2021).

Beyond enzyme yield, the systematic review also examined the functional applications of these laccases in dye degradation processes. Table 2 summarizes the decolorization efficiency of several fungal species against various industrial dyes. The results reveal consistently high removal efficiencies across different fungal systems, although the specific performance varied depending on the dye structure and reaction time.

Table 2: Study Precision and Performance in Dye Decolorization by Laccases. This table focuses on Dye Decolorization Efficiency by laccases produced via SSF. For a Funnel plot, the "Decolorization Rate" serves as the effect size, while the "Standard Error" or "Precision" can be derived from the variance in experimental conditions or the scale of the trial.

One of the most effective examples was observed for Ganoderma species, which achieved complete decolorization (100%) of malachite green within 16 hours under SSF-derived laccase treatment. Similarly strong performance was observed for P. acaciicola, which removed approximately 97.2% of Violet P3P dye within 24 hours. These findings demonstrate the remarkable oxidative potential of fungal laccases for degrading complex aromatic dye molecules commonly found in textile wastewater streams (Šelo et al., 2021; Saldarriaga-Hernández et al., 2020).

Another fungal strain, Pycnoporus sanguineus, exhibited rapid dye degradation kinetics, removing approximately 90% of bromophenol blue within only two hours of treatment. Such rapid reaction rates highlight the catalytic efficiency of laccases derived from certain white-rot fungi. Comparable results were observed for Trametes pubescens, which achieved approximately 79.4% decolorization of Remazol Blue R in two hours (Osma et al., 2011). Although this removal efficiency is somewhat lower than that observed for other strains, it still indicates substantial enzymatic activity toward synthetic dye compounds.

Additional evidence of laccase-mediated dye degradation was observed with Trametes versicolor, which removed approximately 87.7% of methyl green dye after 24 hours of treatment. Similarly, Ganoderma lucidum achieved approximately 93.97% removal of Remazol Blue R within 32 hours (Lú-Chau et al., 2018). These results collectively indicate that fungal laccases possess broad substrate specificity and are capable of degrading multiple classes of industrial dyes.

The forest plot presented in Figure 2 visually summarizes these comparative decolorization efficiencies across fungal species. Although individual study confidence intervals vary slightly, the overall trend demonstrates consistently high removal performance across different fungal systems. The funnel plot shown in Figure 3 further suggests relatively balanced distribution of effect sizes across studies, indicating that large asymmetries—often associated with strong publication bias—are not evident in the dataset (Egger et al., 1997).

Figure 2: Forest plot of Comparative Decolorization Efficiency of Fungal Species in Dye Treatment. This plot visualizes the comparative decolorization efficiency of various fungal species. Effect sizes and confidence intervals provide insight into which fungal strains are most effective for dye treatment.

Figure 3. Funnel plot of Assessment of Publication Bias in Fungal Decolorization Studies Using Funnel Plot Analysis. This plot evaluates potential publication bias in studies reporting dye decolorization by laccases. Symmetry of the plot indicates low bias, whereas asymmetry may suggest selective reporting.

Taken together, the compiled evidence suggests that SSF-based laccase production from agro-industrial residues represents a highly promising strategy for both enzyme generation and environmental remediation. Substrate type, fungal species, and fermentation conditions collectively determine enzyme yield and functional performance. The results demonstrate that agricultural wastes such as brewer’s spent grain, cornstalk, sorghum bagasse, and fruit residues can serve as valuable feedstocks for sustainable enzyme production while simultaneously contributing to waste valorization strategies within circular bioeconomy frameworks (Šelo et al., 2021; Postemsky et al., 2017).

4. Discussion

4.1 Substrate–Strain Interactions and Environmental Applications of Laccases Produced via Solid-State Fermentation

The findings of this systematic review highlight the remarkable versatility of fungal laccases produced through solid-state fermentation systems. While the underlying biological mechanisms are relatively well established, the compiled dataset reveals how strongly substrate composition and fungal physiology influence enzyme productivity. In many ways, the results reinforce a principle frequently observed in fungal biotechnology: the natural ecological adaptations of ligninolytic fungi make them particularly well suited for fermentation processes that utilize plant-derived residues as substrates.

One of the most striking observations emerging from the dataset concerns the wide range of enzyme activity values reported across different fermentation systems. Activities ranged from modest levels around 14 U/g to extremely high levels exceeding 10,000 U/g. At first glance this variability might seem surprising, but it likely reflects differences in substrate composition, fungal strain selection, and pretreatment conditions. For example, brewer’s spent grain—one of the most productive substrates observed in the dataset—contains not only lignocellulosic components but also residual proteins, minerals, and fermentation by-products that may stimulate fungal metabolism. Such complex nutrient profiles may explain why Trametes versicolor produced dramatically higher enzyme yields on this substrate compared with cotton stalk or other agricultural residues (Šelo et al., 2021).

The importance of substrate composition in fungal fermentation has been emphasized in earlier research as well. Lignin-rich materials often act as natural inducers of laccase expression, stimulating the oxidative enzyme systems that fungi use to degrade plant cell walls. Studies examining lignocellulosic bioconversion processes have consistently shown that substrates containing aromatic lignin structures can enhance enzyme synthesis during SSF cultivation (Karp et al., 2015; Saldarriaga-Hernández et al., 2020). The results presented here appear to support this interpretation, as substrates with higher lignocellulosic complexity tended to support stronger enzyme production.

Pretreatment methods also appear to play a significant role in fermentation efficiency. The comparison between untreated cornstalk and steam-exploded cornstalk illustrates this point particularly clearly. Steam explosion disrupts the structural integrity of lignocellulosic biomass, increasing surface area and improving microbial accessibility to cellulose and lignin fractions. The higher enzyme yield observed in steam-treated substrates therefore likely reflects improved fungal colonization and nutrient availability (Šelo et al., 2021). Such observations reinforce the broader understanding that pretreatment strategies remain a critical step in optimizing biomass-based fermentation processes.

Another noteworthy outcome of the review involves the strong catalytic potential of fungal laccases for dye degradation. Synthetic dyes used in textile and chemical industries often possess complex aromatic structures that resist conventional wastewater treatment methods. However, the oxidative mechanism of laccases enables these enzymes to attack a wide range of phenolic and non-phenolic compounds. The decolorization efficiencies observed in Table 2—frequently exceeding 90%—demonstrate that fungal laccases can effectively degrade several classes of industrial dyes under relatively mild conditions.

The high removal efficiency observed for malachite green using Ganoderma species is particularly notable. Malachite green is widely recognized as a persistent environmental pollutant with potential toxicological effects in aquatic ecosystems. Achieving complete decolorization within a relatively short time frame suggests that fungal laccases could serve as valuable tools for bioremediation applications (Lú-Chau et al., 2018). Similarly strong removal rates for bromophenol blue and Violet P3P further highlight the versatility of these enzymes for treating dye-contaminated wastewater streams.

The forest plot presented in Figure 2 illustrates a consistent pattern across fungal species, with most strains demonstrating relatively high decolorization performance. Although slight variations in efficiency are evident, the general trend suggests that several white-rot fungi possess comparable catalytic capabilities. This observation aligns with earlier studies emphasizing that many ligninolytic fungi share similar oxidative enzyme systems capable of degrading complex aromatic compounds (Orlikowska et al., 2018).

Another important aspect emerging from the analysis concerns the broader implications of agro-industrial residue utilization. Agricultural wastes such as fruit peels, straw, and processing by-products are generated in enormous quantities worldwide. Historically, many of these materials have been discarded or burned, contributing to environmental pollution and greenhouse gas emissions. However, the fermentation strategies described in the reviewed studies demonstrate that these residues can instead be converted into valuable biochemical products. This transformation aligns closely with the principles of circular bioeconomy frameworks, which emphasize resource recovery and sustainable production systems (Farooque et al., 2019).

From an industrial perspective, the integration of SSF-based enzyme production with agricultural waste management could offer several advantages. Solid-state fermentation generally requires lower water consumption and energy input compared with submerged fermentation systems. Moreover, the use of inexpensive substrates reduces production costs and improves overall economic feasibility. Earlier analyses of enzyme production systems have suggested that such waste-based fermentation strategies may significantly reduce the cost of industrial enzyme manufacturing (Guerrand, 2018).

The environmental implications of this approach are equally important. Waste valorization through microbial fermentation not only reduces landfill accumulation but also contributes to the development of cleaner industrial processes. In the context of environmental biotechnology, fungal laccases represent particularly promising tools because of their ability to degrade diverse classes of organic pollutants, including dyes, phenolic compounds, and pharmaceutical residues (Patel et al., 2019; Wang & Wang, 2016).

Despite these promising outcomes, several challenges remain. One persistent issue involves the variability in reported enzyme yields across studies. Differences in fungal strains, substrate composition, and fermentation parameters make direct comparisons difficult. Standardization of experimental protocols and reporting methods would therefore improve the comparability of future research efforts. The adoption of structured methodological frameworks such as PRISMA can help ensure transparency and reproducibility in systematic reviews and meta-analyses of fermentation studies (Page et al., 2021).

Another challenge relates to scaling up SSF processes from laboratory conditions to industrial production systems. While several studies have successfully demonstrated pilot-scale fermentation systems, further research is needed to address issues such as heat accumulation, oxygen transfer, and substrate uniformity in large reactors (Postemsky et al., 2017). Advances in bioreactor design and process monitoring may help overcome these limitations in the future.

In summary, the evidence synthesized in this review suggests that fungal laccase production through solid-state fermentation represents a powerful strategy for transforming agricultural residues into valuable biotechnological products. The combination of high enzyme yields, effective pollutant degradation, and sustainable resource utilization positions SSF as an important platform within emerging circular bioeconomy models. Continued research into strain optimization, substrate pretreatment, and process scale-up will likely further expand the industrial and environmental applications of fungal laccases in the coming years.

5. Limitations

Despite providing a comprehensive synthesis of fungal laccase production through solid-state fermentation (SSF), several limitations should be acknowledged. First, considerable heterogeneity exists among the included studies regarding fungal strains, substrate composition, pretreatment methods, and fermentation parameters, which complicates direct comparison of enzyme yields across experiments. Variations in experimental design and reporting standards also limit the ability to derive fully standardized quantitative conclusions (Higgins et al., 2003; Higgins et al., 2022). Second, most studies were conducted at laboratory or pilot scale, meaning that the scalability of SSF systems for large-scale industrial enzyme production remains insufficiently validated (Postemsky et al., 2017; Šelo et al., 2021). Third, several studies reported enzyme activity without consistent normalization of units or detailed methodological descriptions, potentially affecting the precision of comparative analysis. Finally, although funnel plot analysis suggested relatively low publication bias, the limited number of studies assessing dye decolorization may still influence the robustness of the conclusions (Egger et al., 1997).

6. Conclusion

The reviewed literature demonstrates that solid-state fermentation represents a highly effective platform for sustainable fungal laccase production. Utilizing agro-industrial residues as fermentation substrates not only enhances enzyme yields but also contributes to waste valorization and resource recovery within circular bioeconomy frameworks. The ability of SSF-derived laccases to degrade complex pollutants, particularly synthetic dyes, highlights their significant potential in environmental remediation and green industrial processes. Nevertheless, challenges related to process standardization, substrate variability, and large-scale reactor design remain. Continued research focused on strain improvement, optimized pretreatment methods, and scalable fermentation technologies will be essential for translating laboratory findings into economically viable industrial applications.

References