Considering the laboratory dye usage, four structurally different synthetic dyes; one azo dye - CR, phenazine dye - Safranin, and two Triphenylmethane dyes - CV and MG were obtained from Himedia Laboratories Pvt. Ltd., Mumbai, India, and used for primary mycoremediation screenings. All the synthetic dyes were of analytical grade.

Water Agar medium (WA; 1.5% agar) was employed for isolation of fungi (Yang et al., 2016), while potato dextrose agar medium (PDA; 20% potato, 2% dextrose, 2% agar) was used for fungal colony growth, and as a solid medium for synthetic dye decolourisation experiment (Jayasinghe et al., 2008; Rani et al., 2014; Ekanayake and Manage, 2022). Potato Dextrose Broth (PDB; 20% potato, 2% dextrose) was used for the decolourisation test in a liquid medium (Jayasinghe et al., 2008; Purnomo et al., 2019).

Submerged dead woody specimens with fungal fruiting bodies were collected from lentic freshwater habitats (Mahakanadara tank; 8.38683° N, 80.38683° E, and Mihintale tank; 8.36267° N, 80.50591° E) in Anuradhapura district, Sri Lanka ( Figure 1 ). The specimens were brought to the laboratory in Ziplock plastic bags and incubated using moist chambers at room temperature for three days. Pure cultures were obtained following the single spore isolation technique described by Chomnunti et al. (2014) and Senanayake et al. (2020) on PDA media under aseptic conditions at room temperature (28 °C – 30 °C) for three to five days, and stock cultures were maintained at 4 °C. Living cultures were deposited at the Rajarata University Fungal Culture Collection (RUFCC) in Sri Lanka.

Macro and micro-morphological characters were observed using a stereomicroscope (Nikon SMZ18; Nikon Corporation Shinagawa Intercity Tower C, 2-15-3, Konan, Minato-ku, Tokyo, 108–6290 Japan) and an inverted trinocular microscope (Nikon ECLIPSE Ts2R-FL; Nikon Corporation Shinagawa Intercity Tower C, 2-15-3, Konan, Minato-ku, Tokyo, 108–6290 Japan), respectively. Colony characteristics (colour, form, elevation, margin, and texture) were examined and recorded over a period ranging from three to seven days, and images were subsequently processed using the extended version of the online photo editor Photopea (https://www.photopea.com/; accessed on 10 January 2024).

The solid media were prepared by adding 5 mL of a 0.01% (w/v) concentration of each synthetic dye separately, mixed with 1 L of PDA medium under aseptic conditions (Rani et al., 2014; Ekanayake and Manage, 2022). Tetracycline (50 mg L^-1) was added to prevent bacterial growth, and sterile media were poured onto Petri plates in volumes measuring about 10 mL each. The media plates were allowed to solidify within a laminar hood chamber under UV (Ultraviolet) exposure, ensuring the enhanced neutralization of any potentially viable bacterial or fungal spores.

Mycelium plugs (diameter ∼5 mm) obtained from the edge of pure cultures of fungal strains grown on PDA medium plates for four days at room temperature (28 °C–30 °C) were transferred to the center of a solid medium plate and inoculated at room temperature for seven to ten days in the dark to prevent polymerization of the dye. Un-inoculated plates containing each dye were maintained under the same conditions as a control. The plates were examined daily for the visual disappearance of colour compared to the control. Fungal strains that exhibited the most substantial colour disappearance during solid media screening were selected for further evaluation in liquid medium screening (Khambhaty et al., 2015). The decolourisation ability of synthetic dyes on solid media was assessed through visual observation every 24 hours until ten days after fungal inoculation. The Munsell colour notation system, including hue, chroma, and value, was used to visually detect the disappearance of synthetic dye discolouration, aiding in the identification of colour changes in synthetic dyes in solid media (Yi and Cho, 2008; Raluca, 2016). The visual disappearance of colour on both the up and down sides of the plate indicated the decolourisation ability of the fungal strains.

The decolourisation of dyes in the liquid phase was evaluated using 1 L of PDB medium containing tetracycline (50 mg L^-1) and 5 mL of a 0.01% (w/v) concentration of each synthetic dye separately. Falcon tubes containing 30 mL of liquid medium with each dye were inoculated with five agar plugs of mycelia (diameter ∼5 mm) obtained from the edge of actively growing colonies on pure culture plates containing the selected fungi. The liquid cultures were incubated at 30 °C for four weeks in a shaking incubator (180 rpm) (Jayasinghe et al., 2008; Purnomo et al., 2019). Un-inoculated falcon tubes, each containing liquid medium and dye, were employed as controls. Each set undergoes a liquid media dye decolourisation assay conducted in triplicate.

After incubation, falcon tubes were centrifuged at 4000 rpm for 10 min. to obtain biomass and supernatant, which were measured using a UV-VIS spectrometer (Thermo Scientific Multiskan GO with cuvette 100–240 V, SN 1510-049220, Ratastie 2, Fl 01620 Vantaa, Finland). Changes in absorbance were recorded at the maximum wavelength ranges of 585 nm for CV, 500 nm for CR, 520 nm for Safranin, and 625 nm for MG, respectively. The absorbance values were used to calculate the percentage of decolourisation (%DP) based on the equation (Purnomo et al., 2019; Ekanayake et al., 2022).

Experimental data from three replicates in the liquid media assay were statically analysed using a one-way ANOVA with Duncan’s t-test, applying SPSS software (version 25.0), with a significance level set at p < 0.05. Principal Component Analysis (PCA) was performed using R software (https://www.r-project.org/) to interpret the data.

To address the challenge of synthetic dye decolourisation, we designed a scalable prototype leveraging the mycoremediation potential of Lasiodiplodia pseudotheobromae for CV decolourisation. The development process focused on optimizing critical growth conditions for the fungal species, including shaking speed, temperature, pH, nutrient composition, and surface sterilisation techniques, to maximize decolourisation efficiency. The prototype was developed in two stages, with the first-stage design serving as the basis for improvements incorporated into the second-stage system.

Five fungal strains isolated from freshwater habitats in Sri Lanka were identified and confirmed as Aspergillus sp., Lasiodiplodia crassispora, L. pseudotheobromae, Neopestalotiopsis saprophytica, and Trichoderma sp. using morphological and molecular characteristics. These strains were selected for the dye decolourisation experiment based on their rapid growth rate in the PDA medium within four days.

Visual observation facilitated the comparison of synthetic dye colour variations between control and treated samples, revealing significant changes in colour after 14 and 28 days of decolourisation.

The concatenated dataset (ITS and tef1-α regions) contained 40 strains in the sequence analysis, which comprise 750 characters with gaps. Single gene analysis was carried out and compared with each species, to compare the topology of the tree and clade stability. Diplodia mutila (CMW 7060) and D. seriata (CBS 112555, ex-type) are set as the outgroup taxa. The best-scoring RAxML tree with a final likelihood value of -3104.886120 is presented. The matrix had 217 distinct alignment patterns, with 4.91% of undetermined characters or gaps. Estimated base frequencies were as follows; A = 0.215130, C = 0.285729, G = 0.257826, T = 0.241315; substitution rates AC = 0.865801, AG = 4.151570, AT = 1.525331, CG = 1.008008, CT = 5.192424, GT = 1.000000; gamma distribution shape parameter alpha = 0.159782 ( Figure 5 ). GTR+I+G model was selected as the best model based on MrModeltest and was used for the Bayesian analysis. Overall, tree topologies based on ML and BI analyses were similar and not significantly different. In the phylogenetic analysis ( Figure 5 ), our new collections RUFCC2463 and RUFCC2464 grouped with Lasiodiplodia crassispora (CBS 118741 (ex-type), CMW 13488 and CBS 110492) and L. pseudotheobromae (CBS 116459 (ex-type), CBS 116460, MFLUCC 23–0141 and MFLUCC 20-0137) with high statistical support (99% MLBP, 1.00 BYPP) and (95% MLBP, 1.00 BYPP), separately.

Based on the current in-vitro experiments, the strain numbers RUFCC2463 and RUFCC2464 have been revealed as effective in the decolourisation process. Mega blast results in the GenBank regarded these strains as belonging to Lasiodiplodia crassispora (strain numbers: RUFCC2463) and L. pseudotheobromae (strain numbers: RUFCC2464). We carried out multi-gene analyses based on ITS (ITS4/ITS5), and tef1-α (EF-725F/EF-986R) gene regions for further confirmation ( Figure 5 ). Despite numerous adjustments to the PCR time range and annealing temperatures, L. pseudotheobromae failed to amplify using tef1-α. Consequently, the phylogenetic analysis for L. pseudotheobromae was based solely on the ITS locus.

Based on liquid and solid media assays, Lasiodiplodia pseudotheobromae was identified as the most effective of the five tested fungal strains for CV decolourisation and was subsequently applied in the prototype. Apparently, it is essential to identify the species precisely before its applications; thus, here we provide the identification procedure and taxonomy (microscopy and molecular phylogenetics) of the newly isolated Lasiodiplodia pseudotheobromae.

Ascomycota Caval.-Sm

Dothideomycetes O.E. Erikss. & Winka

Botryosphaeriales C.L. Schoch, Crous & Shoemaker

Botryosphaeriaceae Theiss. & Syd.

Lasiodiplodia Ellis & Everh.

Notes: The genus Lasiodiplodia showed 94 records in Index Fungorum 2025 (https://www.indexfungorum.org/names/Names.asp; accessed on 12^th June 2025). Species belonging to the genus Lasiodiplodia are widely distributed in tropical and subtropical regions and are increasingly documented in temperate areas (Jayawardena et al., 2019; Rathnayaka et al., 2023). Since 2004, over 70 Lasiodiplodia species have been identified and characterised based on morphology and molecular data (Ko et al., 2023). The genus has been extensively studied for its pathogenic impact on numerous significant agricultural plants, with thousands of host plants recorded (Gunamalai et al., 2023; Ko et al., 2023).

Lasiodiplodia pseudotheobromae A.J.L. Phillips, A. Alves & Crous, Fungal Diversity 28: 8 (2008)

Index Fungorum Registration Identifier: 510941

Description: Saprobic on a submerged woody stem. Sexual morph: Undetermined. Asexual morph: Conidiomata 190–300 µm high × 120–230 µm diam. (x̅ = 240 × 170 µm, n = 10), pycnidial, semi-immersed, unilocular, solitary, scattered, globose or subglobose, dark brown. Conidiomata wall thick, outer layers dark brown to black, inner layers thin-walled, pale brown to hyaline, comprising 2–3 layers of dark brown cells of textura angularis. Conidiogenous cells 20–35 × 5.5–10 µm (x̅ = 25 × 7 µm, n = 10), holoblastic, hyaline, thin-walled, smooth, cylindrical. Conidia 28–50 µm × 11.5–14 μm (x̅ = 38 × 12.5 µm, n = 30), initially hyaline and aseptate when immature, becoming medianly one-septate, dark brown, thick-walled, ellipsoid to obovoid, base truncate or rounded, with longitudinal striations from apex to base.

Culture characteristics: Initially, the colonies on the plate appeared white with sparse, fluffy aerial mycelia on the surface. After one week, they developed a pale grey colour with dark pigmentation, eventually turning greyish-black. The reverse side of the plate also displayed a greyish-black colouration after a seven-day incubation period at 28–30 °C.

Material examined: SRI LANKA, North Central Province, Anuradhapura District, Mihintale, Mihintale tank (8.38683° N, 80.38683° E, 117 m), on Submerged woody stem, 10^th January 2023, Madhara K. Wimalasena, living culture (RUFCC2464).

Figure 6 represents the morphological and colony characteristics of Lasiodiplodia pseudotheobromae.

Mycoremediation to remove synthetic dyes should be extended to outdoor settings, particularly in industrial wastewater plants that use synthetic dyes. The treated wastewater must be released into the environment without causing any adverse effects from using fungal strains. Therefore, it is important to design the mycoremediation system for future implementations. An exciting prospect is the use of capsule mycoremediation in the process of removing synthetic dyes ( Figure 7 ).

This innovative system is composed of a temporary wastewater storage chamber, capsule incubators, and a water sterilisation chamber. The wastewater, which contains synthetic dyes, flows from the temporary storage tank to the capsule incubator through a filtering system that removes debris before releasing the water into the capsule chamber. The capsule incubator receives wastewater through a dropper system from the temporary storage chamber, which allows the wastewater to be released into the liquid media without causing damage to the fungal mycelia growth. The system comprises four capsule chambers, each containing a liquid medium suitable for specific fungal growth. The incubation temperature within the liquid media are automatically adjusted according to standard parameters specific to the growth requirements of each fungal strain. To ensure proper fungal mycelia growth, fungal inoculation is carried out seven days prior to operating the capsule mycoremediation. Each capsule chamber retains the wastewater for seven days with periodic shaking. After this period, the treated wastewater is automatically transferred to the second capsule chamber through the filtering system. The second capsule chamber holds the water for an additional seven days for synthetic dye remediation and then automatically advances to the third chamber. The third and fourth chambers further decolourize the wastewater using the previously described process. In the water sterilisation chamber, the treated wastewater is held for two days while undergoing sterilisation through either heat sterilisation or chlorinated water. This step directly contributes to eliminating any harmful effects that occur in the environment resulting from using fungi in the remediation process.

Based on assays conducted on both solid and liquid media, Lasiodiplodia pseudotheobromae was selected for the prototype application due to its superior ability to decolourise CV compared to the other fungal isolates tested. Crystal Violet was chosen for the prototype because it exhibited the most rapid and consistent decolourisation among the dyes evaluated.

During the development of the first-stage prototype, one of the primary challenges was providing adequate agitation for fungal growth. This was particularly important because PDB, the main medium used in the prototype, had a tendency to clot if proper shaking was not maintained. Additionally, since the PDB contained CV, thorough mixing was critical to ensure both effective fungal growth and dye exposure. As the initial design lacked a built-in mechanical shaker, the prototype was placed on an orbital shaker (Stuart SI600 Incubator, manufactured by Cole-Parmer Ltd., Beacon Rd, Stone ST15 0SA, United Kingdom), which enabled controlled shaking speed and temperature conditions to optimize fungal development and dye degradation.

Shaking speeds were tested from 180 rpm to 90 rpm, with 90 rpm found to provide the most favorable conditions for mycelial growth. Temperature trials conducted between 28 °C and 32 °C revealed that 30 °C supported the most vigorous fungal development, whereas growth was slower at 28 °C and declined rapidly at 32 °C. Under these optimised conditions, the average pH of the PDB medium was 6.5, supporting efficient dye decolourisation.

The prototype containers were initially designed to retain dye-contaminated water for seven days, with the total experimental period planned for 21 days (seven days per container across four containers). However, visual observations demonstrated that CV was completely decolourised within just four days in the first container. Consequently, the total retention period for the prototype was revised to 16 days instead of 21.

The containers and other components of the prototype were tested to ensure they could be sterilised by autoclaving. All containers and relevant parts were sterilised by autoclaving before use. The PDB medium was also sterilised prior to the addition of dye. To prepare the dye mixture, 5 mL of a 0.01% (w/v) CV solution was added to 1 L of sterilised PDB. Importantly, the CV introduced PDB mixture was not autoclaved, as this could reduce dye intensity. To minimize contamination during CV solution preparation, autoclaved distilled water was used, and the dye solution was sterilised by membrane filtration to remove microbial contaminants.

The first-stage prototype system comprised four plastic containers (each 16 cm × 10 cm × 5 cm) connected via ball valves and pneumatic pipes. These containers were mounted on a treated hardwood frame in a stepwise arrangement, allowing passive water flow between containers using gravity. This mechanism, as described by Jones (2011), reduces energy consumption and enhances the eco-friendliness and sustainability of the prototype for wastewater treatment.

Each container operated sequentially in the decolourisation process. Initially, dye-contaminated water was added to the first container, and 10 g of pre-cultured mycelial plugs (~5 mm diameter) of L. pseudotheobromae were introduced. The entire prototype was maintained on an orbital shaker at 90 rpm with 30 °C to ensure optimal fungal activity. The first container was treated for four days, after which the water flowed into the second container via the gravity-driven mechanism. Fresh mycelium plugs were added at each stage, continuing until the dye-degraded water exited through the final ball valve. This stepwise design ensures thorough treatment of CV contaminated water and effective fungal activity at each stage ( Figure 8 ). Before releasing the treated water from the fourth container into the environment, it was autoclaved to prevent any potential adverse effects of L. pseudotheobromae on the ecosystem.

The first stage prototype demonstrated the potential for fungal dye decolourisation wherein several limitations became apparent. The water storage capacity of the system was low, preventing continuous filtration and durability. The use of an orbital shaker, while effective in small-scale testing, was impractical for larger-scale applications, and the overall efficiency of the process was found to be limited.

The second-stage prototype was designed using SolidWorks 2022 software (accessed on January 5, 2025) to identify design requirements and optimize the system layout, as shown in Figure 9 .

To overcome these challenges, a second-stage prototype was developed ( Figure 10 ), incorporating several key improvements. The new design replaced the plastic containers with larger borosilicate glass bottles (3-L capacity) serving as treatment chambers, copper valves and polypropylene lines, which are commonly used in laboratory-scale fluid storage, transport and wastewater treatment setups due to their cost-effectiveness, chemical resistance and thermal resistance (Kutz, 2002). An electronically controlled shaking mechanism was introduced for a continuous agitation mechanism. This system uses NEMA stepper motors programmed to control both speed and bi-directional rotation (clockwise and counterclockwise), preventing the mycelium from blending while ensuring optimal agitation for dye degradation. The prototype was designed with an integrated water pumping system to facilitate continuous circulation. The system comprises three treatment capsule chambers connected via pumps: the first pump transfers dye-containing water from the storage tank to the initial capsule chamber, while subsequent pumps ensure regulated transfer among the first, second, and third capsules. The second-stage design further incorporates independent control of agitation speed and direction for each tank, enabling a more efficient and continuous decolourisation process.

For operational implementation, the temporary wastewater chamber was initially filled with dye-containing PDB, to which 30 g of pre-cultured mycelial plugs (~5 mm diameter) of Lasiodiplodia pseudotheobromae were added. The dye mixture was prepared by adding 15 mL of a 0.01% (w/v) CV solution, sterilised by microbial membrane filtration, to 3 L of sterilised PDB and thoroughly mixing. This medium was subsequently transferred to the second chamber, integrated into the automated system, and incubated for five days under shaking at 90 rpm and 30 °C to promote decolourisation. Following this period, the water was sequentially circulated through the remaining capsules. The complete decolourisation process spanned 15 days, with each of the three active capsules receiving five days of treatment. The third and fourth chambers were maintained under identical conditions to the second, and sequential circulation facilitated the collection of clear, decolourised water from the final chamber. Prior to environmental release, the decolourised water was sterilised using the wet-heat sterilisation method of autoclaving to mitigate any potential ecological impact of L. pseudotheobromae. According to the operational process of the second-stage prototype, it was observed that the shaking speed should be reduced from 90 rpm to 50 rpm, as this facilitates proper mycelial growth in PDB.

The second-stage prototype demonstrated significant improvements in scalability, durability, and operational efficiency, representing an advancement in the application of fungal mycoremediation for synthetic dye removal in wastewater treatment. Decolourisation was initially assessed visually against a control; however, more precise quantification using advanced analytical techniques such as HPLC-MS and total organic carbon (TOC) analysis is planned. Although beyond the scope of the current study, future research will employ UPLC MS-MS facilities at Rajarata University of Sri Lanka to elucidate detailed degradation pathways and overall treatment efficiency.

At this stage, the focus was on evaluating the operational performance of the second-stage prototype. Subsequent studies will investigate a broader range of dye concentrations (0.01–0.2% w/v Crystal Violet) and incorporate advanced analytical techniques such as HPLC-MS and TOC testing to provide comprehensive and quantitative results.

The improper disposal and uncontrolled release of synthetic dyes from industries into aquatic environments are significant concerns contributing to environmental pollution in contemporary times (Li et al., 2021; Bharathi and Ga, 2022; Ganaie et al., 2023). Compounds such as CR, CV, MG, and Safranin, commonly utilised in laboratories (Islam et al., 2019; Bharathi and Ga, 2022; Shah et al., 2023), are often released without proper treatment. Such indiscriminate discharge poses a significant threat to the environment and human well-being (Li et al., 2021; Bharathi and Ga, 2022). Conventional physicochemical treatment methods for dye removal have proven largely ineffective in mitigating the environmental impacts and addressing the challenges, necessitating the development of more efficient treatment technologies (Yang et al., 2016; Routoula and Patwardhan, 2020; Ardila-Leal et al., 2021; Shah et al., 2023).

The degradation of synthetic dyes involving fungi is mainly driven by biotransformation enzymes, which are crucial for breaking down complex dye molecules (Shi et al., 2021; Ngo and Tischler, 2022). The biosorption mechanism is recognised as an important factor in dye decolourisation by living fungi, contributing to the potential recovery of synthetic chemicals from spent dye baths (Fu and Viraraghavan, 2001; Khan et al., 2013). Mycoremediation has emerged as a prominent alternative in addressing synthetic dye pollution. Fungi, with their distinctive characteristics, play a crucial role in the remediation process, making them essential contributors to mitigating the environmental issues associated with synthetic dyes (Yang et al., 2016; Chen et al., 2019; Melati et al., 2022; Shourie and Vijayalakshmi, 2022; Thoa et al., 2023; Vaksmaa et al., 2023). The ability of fungi to degrade pollutants is attributed to their extracellular enzyme system, which is non-specific and non-stereoselective (Hofrichter, 2002; Khan et al., 2013). Fungal species capable of producing ligninolytic enzymes, including laccase, manganese peroxidase (MnP), and lignin peroxidase (LiP), have been widely used in treating resilient synthetic dyes in wastewaters (Hofrichter, 2002; Torres-Farradá et al., 2017; Dayi et al., 2018; Borham et al., 2023). These enzymes, derived from lignolytic fungi, have demonstrated significant effectiveness in decolourising dyes in textile wastewater systems (Khan et al., 2013; Daphedar et al., 2023).

Laccase, along with immobilised laccases, has been extensively investigated for its role in degrading recalcitrant compounds, including synthetic dyes (Abadulla et al., 2000; Saratale et al., 2009). Fungi can transform dyes, particularly azo dyes, into cation radicals, making them susceptible to nucleophilic attacks by water or hydrogen peroxide. This transformation leads to both symmetrical and asymmetrical cleavage of the azo bonds, generating intermediate compounds that undergo multiple redox reactions before stabilising into less toxic forms (Khan et al., 2013). Unlike direct cleavage of azo bonds, laccase-mediated decolourisation operates through a highly non-specific free radical mechanism, preventing the formation of toxic aromatic amines (Kalme et al., 2009). Furthermore, MnP isoenzymes efficiently decolourize azo dyes and phthalocyanine complexes in a Mn²⁺-independent manner. This enzymatic treatment not only disrupts chromophoric groups but also significantly alters the overall chemical structure of the dyes, facilitating their breakdown (Chagas and Durrant, 2001; Khan et al., 2013).

In this study, the main contributors to synthetic dye decolourisation among the tested fungi were Lasiodiplodia crassispora and L. pseudotheobromae, particularly in the decolourisation of CV. Of these two species, L. pseudotheobromae was selected for prototype application due to its superior performance in CV decolourisation. Considering its strong dye-decolourising ability, the genus Lasiodiplodia is recognised for producing ligninolytic enzymes, especially laccase (Esteves et al., 2014; Salvatore et al., 2020; Borham et al., 2023).

In our studies, L. pseudotheobromae demonstrated laccase enzyme production, as identified through qualitative enzymatic assays. Laccase activity was further assessed using application of 0.1 M 1-Naphthol (Research Lab Fine Chem. Industries, Mumbai, India) on five-day-old cultures of L. pseudotheobromae. After reagent application, plates were incubated for an additional 24 hours at 28–30 °C. A visible colour change at the colony edge, particularly the development of a blue–purple colouration, was considered a positive indication of laccase activity, following the methods described by Robledo-Mahón et al. (2020). The laccase-producing ability of the same L. pseudotheobromae strain used in this study was also confirmed by Wimalasena et al. (2024).

Lasiodiplodia sp. belongs to the family Botryosphaeriaceae (Jami et al., 2013; Wijayawardene et al., 2022), and is identified as an endophytic fungus living within the host plant tissues (Borham et al., 2023). Lasiodiplodia sp. has been identified as a producer of lignocellulolytic enzymes, with a focus on laccase and MnP, showcasing potential applications in mycoremediation against diverse synthetic dye contaminants (Arunprasath et al., 2019; Borham et al., 2023). Additionally, Félix et al. (2018) documented the ability of Lasiodiplodia strains to produce various extracellular enzymes, including cellulases, lipases, xylanases, and pectinases.

Considering previous studies, Borham et al. (2023) documented a remarkable 94.8% decolourisation ability of Lasiodiplodia sp. for 250 mg L^-1 of CR, while Asses et al. (2018) reported a higher decolourisation efficiency of 97% for CR (200 mg L^-1) by the same fungal species. Besides, Arunprasath et al. (2019) highlighted the mycelium of Lasiodiplodia sp. and its role in the adsorption of MG, achieving an impressive decolourisation efficiency of 96.9%. Nguyen et al. (2017) reported CV decolourisation ability of Lasiodiplodia sp. was 93.2% (10 mg L^-1). Likewise, this study also provided significant evidences of the synthetic dye decolourisation ability of Lasiodiplodia sp. in CV, CR, and MG.

Nevertheless, Aspergillus sp. has been utilised for dye removal through processes such as absorption by mycelium, degradation with ligninolytic enzymes, or a combination of both, as outlined by Liu et al. (2020). Namnuch et al. (2021) reported the capability of Aspergillus sp. to produce cellulolytic enzymes. Ekanayake and Manage (2022) highlighted that A. niger could produce laccase enzymes, directly contributing to textile dye decolourisation. Other previous studies have demonstrated the significant contribution of Aspergillus sp. in synthetic dye decolourisation. For instance, the isolated fungus A. flavus exhibited the ability to achieve a substantial reduction of synthetic dyes, including CV, MG, and safranin, ranging from 80% to 90% within a one-week period (Lalitha et al., 2011). Furthermore, Asses et al. (2018) reported a 97% decolourisation of CR (200 mg L^-1) by A. niger, and Skanda et al. (2021) found that A. arcoverdensis achieved an impressive 98.6% decolourisation of CR (100 mg L^-1).

In this study, we investigated the potential efficacy of fungi isolated from Mahakanadara and Mihintale tanks in the Anuradhapura district of Sri Lanka for the remediation of synthetic dyes. Specifically, Aspergillus sp., Lasiodiplodia crassispora, L. pseudotheobromae, Neopestalotiopsis saprophytica, and Trichoderma sp. were evaluated for their ability to decolourize CR, CV, MG, and Safranin through in vitro screening. The selected fungal strains demonstrated robust growth, reaching a colony diameter of 6.5 cm on PDA within one week. Their rapid growth rate minimised the risk of contamination by other fungi and bacteria. Furthermore, these fungi exhibited adaptability to both solid and liquid PDA media, underscoring their resilience under different culture conditions. Synthetic dyes, commonly discharged from laboratories and textile industries without adequate treatment, pose a significant environmental hazard. The in vitro assay conducted in this study revealed substantial decolourisation potential among the selected fungal strains, with Aspergillus sp., L. crassispora, and L. pseudotheobromae exhibiting the most pronounced effects. Notably, L. crassispora demonstrated the highest decolourisation efficiency, surpassing 70% for all tested dyes. These findings highlight the potential application of Lasiodiplodia sp. as a bioremediation agent for synthetic dye removal, offering an effective strategy for mitigating environmental pollution.

Beyond laboratory-scale experimentation, these findings hold significant potential for industrial applications. The prototype was developed in two progressive stages to bridge the gap between research and large-scale implementation. The initial prototype successfully revealed the feasibility of fungal-based decolourisation of synthetic dyes through a stepwise, gravity-driven system. The integration of Lasiodiplodia pseudotheobromae within sequential treatment chambers effectively facilitated dye degradation, with CV achieving complete decolourisation within four days, surpassing initial expectations. The reliance on passive water flow and controlled agitation further enhanced the system’s sustainability, offering an eco-friendly approach to wastewater treatment.

Despite its success, the first-stage prototype exhibited limitations in water storage capacity, sterilization compatibility, and scalability, necessitating design refinements for industrial applicability. In response, a second-stage prototype was engineered, incorporating larger glass treatment chambers, an electronically controlled shaking mechanism, and a water pumping system to enable continuous treatment. These modifications significantly improved the system’s efficiency, operational durability, and scalability, making it more suitable for large-scale wastewater remediation.

The transition from the initial to the advanced prototype highlights the viability of fungal mycoremediation as a sustainable and efficient strategy for synthetic dye removal. Future optimizations and pilot-scale evaluations will be critical to refining the system for seamless industrial integration, ensuring its practical applicability in large-scale wastewater treatment operations.

According to our experience, the total cost of preparing the second-stage prototype was approximately 900 USD, which comprised raw materials and consumables (USD 399), equipment and tools (USD 166), prototype development and fabrication (USD 166), labour costs (USD 116), and logistics and maintenance (USD 33). This prototype effectively decolourised 100 ppm CV using Lasiodiplodia pseudotheobromae within 15 days. The cost figure reflects only the development expenses; subsequent costs are limited to maintenance and the recurrent expense of PDB. Overall, this design is eco-friendly, cost-effective, and durable, with a projected usable lifespan exceeding one year.

Saad et al. (2022) reported that the Malaysian textile industry generates azo dye wastewater, which is difficult to treat biologically due to its chemical stability. Conventional treatment methods, such as electrocoagulation (EC) and photocatalysis, are often costly and energy-intensive. In their study, both standalone EC and integrated electrocoagulation–membrane (ECM) systems achieved a 96% dye removal efficiency. Cost analysis revealed that the ECM system (1 V, 1.0 g NaCl) incurred a total cost of 1.079 million MYR (approximately USD 256,800), whereas the standalone EC system (1 V, 1.0 g NaCl) was even more cost-effective, with a total cost of 0.325 million MYR (approximately USD 77,000).

This study highlights the potential of fungal strains in synthetic dye degradation; however, certain limitations should be recognised. Only a single dye concentration of 0.01% (w/v) was tested in both solid and liquid media. While this concentration was selected as a standard baseline commonly reported in fungal dye decolourisation studies, it may not be sufficient to fully evaluate the tolerance and degradation capacity of the fungi investigated. Future studies should therefore employ a broader concentration gradient (0.01–0.2% w/v) to provide a more comprehensive understanding and improve the reliability of results.

In addition, the observed colour removal does not necessarily indicate complete mineralisation of the dye, as the assessment was based solely on visual decolourisation relative to the control. More rigorous evaluation requires advanced analytical techniques such as HPLC-MS and total organic carbon (TOC) testing. Although beyond the scope of the present study, future research will utilise UPLC MS-MS facilities at Rajarata University of Sri Lanka to gain deeper insights into the degradation pathways and overall efficiency.

This section highlights the importance of commonly encountered fungi and their potential applications in various remediation fields. It stresses the need to go beyond fungal isolation and identification to explore their broader roles in bioremediation. The fungi examined in this study, including Aspergillus spp., Lasiodiplodia spp., Neopestalotiopsis spp., and Trichoderma spp., are frequently found in diverse environments, often as plant pathogens or laboratory contaminants.

Among these, Lasiodiplodia theobromae is a well-known plant pathogen prevalent in tropical and subtropical regions (Salvatore et al., 2020). Similarly, species within Pestalotiopsis are commonly reported as saprobic fungi (Maharachchikumbura et al., 2014; Belisário et al., 2020). Aspergillus spp., Alternaria spp., and Penicillium spp. are frequently observed as laboratory contaminants (Leifert and Woodward, 1998). Despite their ubiquity and potential for contamination, these fungi possess significant yet often overlooked roles in mycoremediation. Recognizing their bioremediation potential is crucial for maximizing their positive environmental impact.

Kang et al. (2019) reported that Neopestalotiopsis spp. are involved in the degradation of pharmaceutical compounds, while Lasiodiplodia spp. have demonstrated efficiency in heavy metal degradation (Deng et al., 2014) and textile dye degradation (Borham et al., 2023). Among these fungi, Trichoderma spp. are well-documented for their bioremediation capabilities, particularly in degrading various pollutants (Ren et al., 2022; Racić et al., 2023; Voloshchuk et al., 2024; Conte et al., 2025). Trichoderma harzianum (AUMC14897) has also been reported for efficient dye decolourisation in wastewater treatment through laccase enzyme production, effectively degrading Novatic Green XBN, Red 4BL, and Reactive T. Blue G (Salem et al., 2024). Furthermore, T. viride exhibited decolourisation efficiencies of 36%, 73%, and 87% for Synozol Red, Yellow, and Navy Blue dyes, respectively (Ali et al., 2023). Al-Hawash et al. (2018) reported that T. harzianum demonstrates significant hydrocarbon degradation potential, mainly through laccase enzyme production.

Similarly, Aspergillus spp. play a significant role in bioremediation processes (El-Bondkly and El-Gendy, 2022; Zango et al., 2023; Ghanaim et al., 2025; Ameen et al., 2024). These species serve as prime examples of fungi with substantial remediation potential. The role of Aspergillus spp. in synthetic dye decolourisation has also been well documented. For instance, A. fumigatus and A. flavus were found to degrade Sumifex Turquoise Blue and Navy Blue dyes (Ghafoor et al., 2024). Aspergillus candidus decolourised Direct Blue 71 (Rathi and Kumar, 2022), while A. niger, A. candidus, and A. iizukae were effective in decolourizing Direct Yellow (Kořínková et al., 2019). Additionally, A. fumigatus exhibited degradation potential for Rhodamine B (Vithalani and Bhatt, 2023), and A. tamarii demonstrated a 97.87% decolourisation efficiency for Remazol Black B (Ayu and Kasiamdari, 2022

The role of fungi in petroleum hydrocarbon remediation is another significant area of study. Al-Hawash et al. (2018) reported that A. fumigatus, A. ochraceus, A. niger, and T. harzianum exhibit remarkable hydrocarbon degradation potential, primarily through the production of extracellular enzymes. Aspergillus niger demonstrated a hydrocarbon degradation efficiency of 94.4%, while P. commune displayed a degradation activity ranging from 80% to 90% (Myazin et al., 2021; Mahmud et al., 2022).

Fungal involvement in heavy metal remediation has been extensively studied. Several fungi and yeast strains, including Aspergillus spp., Candida spp., Fusarium spp., Mucor spp., Penicillium spp., and Saccharomyces spp., have been reported to effectively remediate heavy metals such as Cd, Cr, Cu, Ni, Co, Pb, and Zn in contaminated soils (Archana and Jaitly, 2015; Siddiquee et al., 2015; Kumar and Bharadvaja, 2020; Kumar and Dwivedi, 2021).