Enhanced Biosorption of Triarylmethane Dyes by Immobilized Trametes versicolor and Pleurotus ostreatus: Optimization, Kinetics, and Reusability

Abstract

The discharge of synthetic dyes from industries poses severe environmental challenges, necessitating eco-friendly remediation strategies. This study investigated the biosorption of triarylmethane dyes Crystal Violet (CV), and Brilliant Green (BG) using self-immobilized and sponge-immobilized biosorbents of Trametes versicolor (strain CB8, CB8/S2) and Pleurotus ostreatus (strain BWPH, BWPH/S2). Tests were conducted with live and autoclaved biomass under varying conditions of dye concentration (100–400 mg/L), temperature (15–55 °C), and pH (2–10). Sponge-immobilized live biomass (CB8/S2 and BWPH/S2) showed superior performance, removing up to 90.3% and 81.7% of BG and 43.9% and 39.3% of CV, respectively, within 6 h, demonstrating 3–5 times higher efficiency than self-immobilized biomass for both dyes. Maximum sorption of 379.4 mg/g of BG and 48.9 mg/g of CV was achieved by CB8/S2 at 400 mg/L. Principal Component Analysis biplot confirmed immobilization efficacy, where Dim1 (85.9–91.8% variance) dominated dye concentration and contact time. The optimized conditions for BG removal by CB8/S2 was 20.85–32.17 °C and pH 3.4–6, and for CV, at pH 6.5–7.5 and 30 °C. The percentage of dye sorption data fitted well with the quadratic model (p < 0.05). Fourier transform infrared spectroscopy (FT-IR) analysis indicated that hydrogen bonding and electrostatic interactions facilitated dye binding onto fungal mycelium. Notably, sponge-immobilized biosorbents were reusable without additional treatment. The findings support fungal biomass immobilization as a viable strategy to augment the bioremediation potential in treating dye-laden wastewater.

1. Introduction

There has always been a significant issue with the coloring of wastewater from the manufacturing of dyes and pigments as well as from industries that use these colorants, including the leather, textile, fiber, carpet, paper, printing, automotive, plastic, ceramic, glass, food coloring, cosmetics, pharmaceutical, and other industries [1,2,3]. Approximately 70 billion tons of dye-containing wastewater are generated annually, and 80% of the synthetic dyes produced are used in the textile industry alone. Most dye winds up in effluents, with about 10–15% actually binding to textiles [4]. As a result, around 100,000 tons of dye are discharged into the environment annually. More than 280,000 tons of dye enter surface waters annually. Wastewater containing dyes is a serious environmental issue that hinders sustainable development since synthetic dyes worsen water contamination [5]. Given the mounting challenges of water scarcity, dye removal requires quick, efficient treatment.

According to Amran et al. [6], cationic dyes are typically more poisonous and dangerous than anionic dyes. Crystal Violet (CV) and Brillian Green (BG), two triarylmethane dyes, are classified as cationic synthetic dyes. These cationic dyes are the most adaptable and are used extensively in a wide range of commercial applications, including paints, paper-printing inks, and textile dying [7,8,9]. As a biological stain for recognizing the bloody fingerprints, Crystal Violet, a member of the triarylmethane group, is frequently employed in animal and veterinary medicine [10]. The coloration is a visible issue that indicates the water is unfit for drinking, domestic use, and irrigation, but more importantly, it conceals potential carcinogenicity, reproductive toxicity, neurotoxicity, and chronic toxicity within itself, and can cause skin and eye irritation, respiratory and kidney failures, and other serious health problems when absorbed in harmful amounts through the skin [11,12].

Adsorption is a cost-effective technique for purifying high-concentration dyes without generating byproducts, facilitating recovery and reuse, which has contributed to its increasing popularity. The adsorbents demonstrate significant affinity for dyes by physisorption and/or chemisorption through electrostatic interactions, hydrophobic interactions, hydrogen bonding, pore filling, and π–π interactions [13]. Carbon-based materials, including activated carbon, charcoal, carbon nanotubes, carbon nanofibers, and graphene, exhibit an inherently superior adsorption capacity for many dyes compared to traditional absorbents like zeolite and clay [14]. Nevertheless, substantial energy and chemicals are required for their regeneration which questions their sustainability. From a techno-economic perspective, cost-effective, eco-friendly, and reusable adsorbents for dye removal must be developed.

The treatment of wastewaters by fungi has been emphasized as an eco-friendly and perhaps successful method, as it is effective against different pollutants such as dyes, heavy metals, and drugs [15]. Numerous microorganisms, including bacteria, mold, yeast, and algae, have been demonstrated to be capable of sorption, accumulation, and dye degradation [16,17,18]. Microorganisms use their degradative enzymes, such as laccase, lignin peroxidase, and manganese peroxidase, to break down dyes [19,20]. Although biodegradation-based removal techniques are quite effective, enzymatic remediation has some severe drawbacks, including the dependence on metabolism, the limited degradability of many dye compounds, and the production of very hazardous byproducts [19,21].

Biosorption is described as a process of metabolically independent sorption that can be carried out utilizing either dead or live biomass, in contrast to biodegradation [22,23]. The two categories of physisorption and chemisorption are the most common divisions of the sorption process. Adsorbent and adsorbate are connected by powerful forces during chemisorption, and typically, these connections are irreversible [24]. However, there exist modest, reversible forces between the adsorbent and the adsorbate in physisorption. Because of its adaptability, straightforward design, convenience of use, absence of creation of poisonous chemicals, and lack of sensitivity to harmful compounds, the biosorption process is more appropriate than other approaches. Numerous studies on various adsorbents for dye adsorption have already been conducted [24,25,26]. However, the physicochemical adsorbents method faces several challenges, such as costly regeneration and disposal issues. Hence, the ease of accessibility and low cost of macro fungal biomass make it an attractive biosorbent. It can be produced using basic fermentation procedures on inexpensive growth media or obtained as a by-product of several industrial operations [27]. After undergoing a minimal amount of treatment, industrial organic and/or inorganic wastes and byproducts are employed as biosorbent. Increased sorption capacity has also been demonstrated to benefit from the immobilization of fungal cells on natural or synthetic supports. According to Pandey and Singh [28], batch sorption operates quicker than fixed-bed mode sorption for the cationic dyes Brilliant Green and Crystal Violet. Therefore, batch sorption is considered in this work. However, the current research has numerous limitations with fungal biosorbents, as they are efficient only at extremely low dye concentrations, whereas wastewater may have higher levels, such as 400 mg/L. Typically, fungal biomass encounters the challenge of reduced adsorption capacity per gram of biosorbent utilized at elevated dye concentrations due to restricted pore size and structural integrity. The primary focus should be the reusability of fungal sorbent without any additional treatment to achieve sustainability.

To overcome the challenges of mycoremediation of dye-containing wastewater, the use of self-immobilized or support-immobilized fungal biomass should be the focus of alternative and innovative wastewater treatment procedures, as these materials can improve their commercial utility as dye biosorbents by overcoming issues related to their physical features [29,30]. The separation of the biomass’s dispersed solid-phase effluents from their liquid-phase counterparts may be challenging due to the low density and mechanical strength of the material, which in turn places restrictions on the creation of efficient process designs. The fragmentation of the hyphal biomass and cell mass sedimentation that results in flow limits in the continuous-flow contact vessels is another issue. In a recent process for the immobilization of fungal hyphae, the structural fiber network of papaya wood and polyurethane sponge was successfully applied to solve these issues. In our previous study, we explored the biodegradation potential of sponge-immobilized Trametes versicolor (strain CB8) against five different synthetic dye [31]. Because of its large surface area, depressions, and cavities, the immobilization matrix of the fibrous network of the polyurethane sponge is perfect for immobilizing microbial cells. While immobilized fungal biomass from loofa sponges has been used to remove heavy metals and chlorinated compounds with success [32,33], there has not been much information on polyurethane sponge usage as a dye biosorbent.

As a rationale, this study investigates the application of self-immobilized and sponge-immobilized fungal biomass of Trametes versicolor (strain CB8) and Pleurotus ostreatus (strain BWPH) as a simple, cost-effective, and eco-friendly biosorption technique for the removal of triarylmethane dyes from aqueous solutions across various dye concentrations, pH levels, and temperatures. Principal component analysis has been conducted to ascertain the significance of immobilization, contact duration, and dye concentration. The developed fungal biosorbents have been analyzed using FT-IR and Scanning Electron Microscopy (SEM) for the biosorption removal procedure. The potential of immobilized fungus has been evaluated for reusability to confirm the sustainability of developed fungal biosorbents for treating dye-contaminated wastewater.

2. Materials and Methods

2.1. Dye Solution Preparation

Two triarylmethane group dyes, Brilliant Green (CAS:633-03-4, VWR Chemicals, LLC, Solon, OH, USA) and Crystal Violet (CAS:548-62-9, Fluka, Switzerland), were utilized to prepare concentrated stock solutions (5000 mg/L) in deionized water. These solutions were then autoclaved at 121 °C for 15 min and stored in the dark at 4 °C. Stock solution was diluted with deionized water to prepare dye solutions at the required concentration.

2.2. Fungal Strain Collection and Culture Condition

The pure culture of White Rot Fungi (WRF), Pleurotus ostreatus (strain BWPH), and Trametes versicolor (strain CB8) were collected from the depository of Fungal Strain Collection of Environmental Biotechnology Department, Silesian University of Technology, Gliwice, Poland. The isolation and identification of the fungal species were previously described by Jureczko et al. [34]. Both strains were maintained in liquid organic medium containing glucose–5 g/L, peptone–1 g/L, MgSO₄·7H₂O–1 g/L, and KH₂PO₄–1 g/L (pH-5.7).

2.3. Preparation of Fungal Biosorbents Variants

Commercial polyurethane sponge (S2) (purchased from Chempak DOMERS, 32-800 Brzesko, Poland) was used as the immobilization matrix. The sponge was cut into cubes of approximately 1 cm³ and used without further chemical pretreatment. Polyurethane sponges were chosen because of their high porosity, mechanical stability, and suitability for microbial immobilization. For both fungal strains, two types of fungal live biomass (LB) were generated, namely self-immobilized fungal biomass pellets (CB8 and BWPH) and fungal biomass immobilized on sponge (CB8/S2 and BWPH/S2), as described in Upadhyay et al. [31]. Prior to the experiment, the culture flasks were pre-cultivated for 7 days at room temperature (20 ± 2 °C) on a rotary shaker at 150 rpm. Then, the culture medium was removed, the pellets were washed twice with deionized water, and, in the case of the CB8 strain, which forms large pellets, they were homogenized using a bag mixer. To create the dead biomass, the seven-day-old biomass was autoclaved at 121 °C for 15 min. Autoclaved biomass (AB) was denoted as CB8-A, CB8/S2-A, BWPH-A, and BWPH/S2-A, based on the kind of fungal strain and type of immobilization. As a control sample, only dye and dye with sponge support were used, and they underwent an identical incubation process without inoculation with fungi.

2.4. Characterization of Fungal Biosorbents

All types of biomasses were incubated for 24 h in both dye solutions with a concentration of 250 mg/L, collected using filter, and dried at 37 °C until the constant weight was attained. Then, the dried biomass was converted into powder form using mortar and pestle. Likewise, the control CB8 and BWPH strain were also prepared without incubation with dye. The adsorbent characterization was performed by FT-IR Attenuated Total Reflectance (ATR) spectroscopy using a FTIR spectrometer (Perkin Elmer Spectrum Two with UATR diamond accessory, Perkin Elmer, Shelton, CT 06484, USA) over a range of 650–3700 cm⁻¹. A Phenom ProX scanning electron microscopy with EDS analyzer (SEM) (Phenom ProX, Thermo Fisher Scientific, Waltham, MA 02451, USA) operating at 10–15 kV in BSE mode was used for the morphological characterization of biosorbents.

2.5. Biosorption Assay

Each variant of biosorbents was tested to eliminate the triarylmethane dyes BG and CV. In each vial, 0.5 g of wet live biomass or autoclaved dead biomass of free-fungal biomass pellets (CB8 and BWPH), or one piece (uniform cubes of approximately 10 mm³) of immobilized sponge fungal pellets (CB8/S2 and BWPH/S2), was added to 10 mL of dye solution at 200 mg/L concentration. The dye solutions were at their natural pH, which was 4.36 for BG and 4.35 for CV. The incubation was carried out for 24 h at 22 °C. The absorbance of the cell-free supernatant was measured using a Hitachi U-1900 UV-VIS spectrophotometer (Hitachi High-Tech Analysis Corporationi, Tokyo, Japan) at 0, 1, 2, 4, 6, and 24 h, and the dye removal by sorption method was evaluated by indirectly measuring the pollutant concentration in the solution. The concentrations of dyes in the samples were determined by measuring the absorbance of BG dye at 623 nm and CV at 584 nm. Standard curves were prepared in the range of 1–20 mg/L. Blanks containing no dyes were used for each series of experiments. All the assays were conducted in at least three experimental replicates. This experiment allowed for a test of the effect of contact time and type of biomass. In parallel, the dry matter content was measured after drying for 7 days at 35 °C to constant weight. These values were used to determine the removal by gram of dry matter of the mycelium. After accounting for the loss seen in the abiotic sample (control), the amount of dye adsorbed was determined. The following equation was used to compute the overall percentage of dye decolorization by sorption process:

$$\mathrm{D}\mathrm{y}\mathrm{e}\mathrm{S}\mathrm{o}\mathrm{r}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}[\mathrm{\%}]={\displaystyle \frac{{\mathrm{C}}_0-{\mathrm{C}}_{\mathrm{t}}}{{\mathrm{C}}_0}}\times 100,$$

(1)

where C₀ is the initial concentration of dye in a control sample with support or only medium (mg/L) and C_t is the current concentration of dye in test samples with immobilized or free-fungal biomass (mg/L).

The maximum amount of dye sorption at measurement points was determined using the following equation:

$${\mathrm{q}}_{\mathrm{t}}=\left({\mathrm{C}}_{\mathrm{i}}-{\mathrm{C}}_{\mathrm{t}}\right){\displaystyle \frac{\mathrm{V}}{\mathrm{M}}}$$

(2)

where C_i is the initial dye concentration, C_t is the concentration at time t (mg/L), V is the solution volume (L), and M is the mass of fungi biomass used (g).

Welch’s t-test for two independent samples, assuming unequal variances, was conducted using Microsoft Excel (Microsoft Corp., Redmond, WA, USA) to assess differences in dye removal efficiency between the immobilization methods. The analysis contrasted sponge-immobilized biomass with self-immobilized biomass across each fungal species and biosorbent variant, encompassing both living and dead biomass. A one-tailed test was conducted based on a prior hypothesis that sponge-immobilized biomass would demonstrate superior dye removal efficiency compared to self-immobilized biomass. Statistical significance was evaluated at α = 0.05.

2.6. Optimization of Biosorption at Different Physicochemical Parameters

2.6.1. Effect of Initial Dye Concentration and Contact Time

The effect of initial dye concentration was checked by containing appropriate concentration of both dyes—100, 200, 300, 400 mg/L—by preparing dilution of stock solution (1000 mg/L) using deionized water. For determining the effective contact time for dye removal, the absorbance of the cell-free supernatant was measured at following time points: 0, 1, 2, 4, 6, and 24 h. Principal component analysis (PCA) was performed to evaluate the combined effect of initial dye concentration and contact time on dye sorption.

2.6.2. Effect of pH and Temperature

For biosorption optimization, we applied a statistical method to find optimal values of temperature and pH, two important parameters with high influence on the tested process. We used a central composite design (CCD), followed by response surface methodology (RSM), to obtain a mathematical relationship for independent parameters’ influence, along with their interactive effects on the response, which was approximated by a polynomial quadratic formula, as shown in Equation (3):

$$\mathrm{E}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{t}=\mathrm{a}\cdot \mathrm{p}\mathrm{H}+\mathrm{b}\cdot \mathrm{T}+\mathrm{c}\cdot \mathrm{p}{\mathrm{H}}^2+\mathrm{d}{\cdot \mathrm{T}}^2+\mathrm{e}\cdot \mathrm{p}\mathrm{H}\cdot \mathrm{T}+\mathrm{f},$$

(3)

where T is temperature and a, b, c, d, e, and f are coefficients.

Since we have a two-factor analyses (k = 2), our CCD consisted of 9 experimental set-ups, specifically one center point and eight so-called “star points”, among which we differentiated factorial points (k2) and axial points (2⋅k). The former points, along with the center point, are required for the first-order regression coefficients, while the latter allow us to estimate the second-order model. The distance from the center to all “star points” is equal and amounts to α = √2, which results in a spherical, rotatable design. The graphical representation of CCD is shown in Figure S1. In order to improve the precision of the experiment, the center point is repeated 4 times; therefore, the entire experiment consisted of 12 experimental sets [35,36,37]. In our study, the temperature range was established between 15 °C and 55 °C, while pH was tested from 2 to 10. The pH values were adjusted with 0.1 M HCl and/or 0.1 M NaOH. Due to the protonation or deprotonation of the functional groups at a wide range of pH, the shift in absorption maxima of each dye were corrected by control dye samples at individual pH and temperature. The CCD experimental plan with coded and natural values has been displayed in

Table 1. Central Composite Design experimental plan with coded values and natural values of temperature (°C) and pH; α ≈ 1.41.

Experiment Number	Coded Values	Natural Values
		Temp. (°C)	pH
1	0/0	35	6
2	−α/0	15	6
3	−1/−1	20.85	3.2
4	0/0	35	6
5	1/1	49.15	8.8
6	0/0	35	6
7	0/α	35	10
8	0/0	35	6
9	0/−α	35	2
10	1/−1	49.15	3.2
11	α/0	55	6
12	−1/1	20.85	8.8

. In the results’ evaluation, we used the Design Expert (Stat-Ease 360) software to calculate the coefficients of the second-order polynomial equation based on regression analysis. The obtained mathematical model was tested using analysis of variance—ANOVA.

2.7. Evaluation of Reusability of Fungal Biosorbent

In order to make this procedure more sustainable, the effectiveness of the fungal pellets were tested for reusability. Both dyes were incubated with CB8/S2 and BWPH/S2 for 6 h, and dye removal was calculated. In order to desorb the dye, one set was then incubated with 5 mL of a 70% methanol solution overnight (~8 h), while the second set was incubated without conducting any treatment. The following day, the dye was reintroduced to test its ability to once more be absorbed by the medium.

3. Results and Discussion

3.1. Effect of Immobilization

The primary experiments were set up to check the biosorption efficiency of CB8 and BWPH strains in self-immobilized and sponge-immobilized forms. As indicated in

Table 2. Biosorption efficiency of fungal biomass for two dyes: Brilliant Green and Crystal Violet (conditions: C₀ = 200 mg/L, biomass dose = 0.5 g wet/10 mL, Biomass type= Live/Autoclaved-Dead, T = 22.5 °C, t = 6 h).

Biosorbent	Biomass Type	Brilliant Green Sorption (%)	Crystal Violet Sorption (%)
Trametes versicolor (CB8)	Live	14.2	15.6
Immobilized Trametes versicolor (CB8/S2)	Live	90.3 ****	43.9 **
Immobilized Trametes versicolor (CB8/S2-A)	Autoclaved dead	48.4 ***	22.8 *
Pleurotus ostreatus (BWPH)	Live	23.9	12.1
Immobilized Pleurotus ostreatus (BWPH/S2)	Live	81.7 *	39.3 *
Immobilized Pleurotus ostreatus (BWPH/S2-A)	Autoclaved dead	30.3 -	18.9 -
Sponge (S2)	No fungal biomass	49.9 ***	6.5 -

(Note: **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, ^- p ≥ 0.05 for one tail t-test compared with self-immobilized biomass).

, when the initial BG and CV dye concentrations were 200 mg/L, the highest decolorization was achieved by live immobilized fungal biomass as compared to non-immobilized fungal biomass. The sorption capacity for both tested dyes was enhanced by at least threefold through the use of CB8/S2 and BWPH/S2. The BG dye was removed by 90.3% using CB8/S2 in just 6 h, showing great removal efficiency and speed. In this case, sponge cubes were covered with live fungal biomass, which promoted interaction between dye molecules and the fungal hyphae’s functional groups. The compound’s bioaccumulation in the cytoplasm and cell wall is the primary mechanism by which alive biomass removes dyes [38]. Sponge use may result in increased surface area and, thus, enhanced dye sorption by living fungal biomass. While testing autoclaved-dead biomass of CB8/S2, it removed 48.4% of BG and 22.8% of CV dye. This rate is greater than that of self-immobilized biomass, but, at the same time, less effective than that of live biomass. Similarly, dead immobilized P. ostreatus biosorbent was not as effective as live biomass.

Both dyes belong to the triarylmethane group, but the difference in functional group and its interaction with fungal mycelium may result in difference in sorption capacity. The BG dye contains an organic hydrogen sulfate salt, whereas the CV dye contains an organic chloride salt. Similar results were achieved by Iqbal M. and Saeed A. [29], where immobilization of Phanerochaete chrysosporium fungal biomass on loofa sponge helped enhance Remazol Brilliant Blue R dye sorption by 18.60% compared to non-immobilized fungal biomass. In a recent study, loofah sponge has been successfully fabricated with Ca-alginate to achieve 180 mg/g methylene dye sorption [39], whereas the zinc nanoparticle–loofah sponge composite was not highly effective in enhancing the sorption of trypan blue [40]. Therefore, it is necessary to explore alternative sponge fabrications to enhance dye sorption capacity. Our research is valuable, as it presents an environmentally friendly, cost-effective sponge alteration that simplifies waste disposal.

3.2. Effect of Dye Concentration and Contact Time for Living Biomass

Biosorption experiments for both dyes, BG and CV, were carried out by selecting a concentration range between 100 mg/L and 400 mg/L, an adsorbent dosage of 0.5 g of wet T. versicolor CB8 or P. ostreatus BWPH biomass, and one piece of sponge-immobilized biomass. As seen in Figure 1a, the T. versicolor CB8 strain biomass was able to remove 46.9% BG dye in 6 h at 100 mg/L initial dye concentration. As the dye concentration increased to 200 mg/L, the dye removal capacity was halved. At higher dye concentration, fungal biomass could not sorb BG dye in considerable amount. The experimental results show that from 0 to 2 h, the unstable interaction was found between dye and CB8 fungal biomass. Primarily bioaccumulation, physisorption, and chemisorption are responsible for the removal of dye from the aqueous medium [21]. On a positive note, this problem was not faced by sponge-immobilized T. versicolor biomass, as it started taking dye as soon as it encountered dye-containing medium. It is observed that the amount of BG adsorbed by sponge-immobilized T. versicolor CB8/S2 biomass in just one hour was more than the amount absorbed by CB8 by end of 24 h (Figure 1a). Even at the highest BG dye concentration, 400 mg/L, CB8/S2 was able to sorb nearly 90% of dye from the medium. While evaluating the maximum amount of dye sorbed by CB8/S2 per gram of biosorbent used, it stands out with 379.4 mg/g for Brilliant Green dye just in 6 h (Figure 3a). Table 3 makes it evident that our biosorbent has a very high capacity compared to the results of other researchers who have attempted other biological or physicochemical sorbents for the removal of BG dye. Compared to other fungal sorbents created for the sorption of various dye classes, our study is notable for its high efficiency and quick removal rate. The Remazol Brilliant Blue R dye was sorbed by loofa sponge-immobilized Phanerochaete chrysosporium where 101.06 mg/g was achieved [29]. Nouri et al. [41] achieved a maximum Remazol Black dye biosorption capacity of 58.48 mg/g by dried Sarocladium sp. biomass. Chew and Ting [42] used free-cells and alginate-immobilized forms in biosorption studies to investigate the dye-removal capacity of Trichoderma asperellum on four triarylmethane dyes. When compared to free cells, T. asperellum showed the ability to remove these dyes more effectively when alginate-immobilized forms were employed. While free cells absorbed 12.97, 12.54, 14.34, and 11.44 mg/g of CV, methyl violet, cotton blue, and malachite green, immobilized forms adsorbed 60.64, 50.29, 49.91, and 16.61 mg/g, respectively. The increase in the sorption of dye with dye concentration is attributed to the retardation of resistance toward dye uptake, which increases the diffusion of the dye [43]. This is very much desirable for treatment of dye-containing wastewater on large scale, which demands high removal capacity in the shortest duration of time.

Figure 1b represents the sorption of BG dye by P. ostreatus BWPH fungi at varying dye concentrations (100–400 mg/L). In the first few hours (0–5 h), BG dye sorption increases significantly, especially at lower dye concentrations (100–200 mg/L). This trend is consistent with studies on fungal biosorption, which suggest that the initial phase is controlled by surface adsorption due to abundant binding sites on the fungal biomass, wherein electrostatic interactions between dye molecules and fungal cell wall constituents such as proteins, glucans, and chitin cause initial rapid adsorption [44]. Due to the saturation of active binding sites and the slower diffusion of dye molecules into deeper layers of fungal biomass, the sorption rate decreases after the initial fast phase. Figure 1b indicates that higher dye concentrations (300–400 mg/L) result in lower overall dye sorption percentages which was opposite in case of T. versicolor fungi. The curves indicate that BWPH/S2 conditions at lower dye concentration (100, 200 mg/L) generally show higher dye uptake than BWPH alone and show 246.7 mg/g and 302.5 mg/g amount of dye sorption at their respective initial dye concentrations (Figure 3a). This may be attributed to the enhanced surface area or increased functional group availability in the BWPH/S2 condition, likely by improving the functional properties of the fungal biomass.

Similarly, as seen in Figure 2a,b, there was a steep increase in dye sorption within the first 5 h, especially at lower dye concentrations (100–200 mg/L). T. versicolor CB8 and P. ostreatus BWPH self-immobilized biomass were more successful in the removal of Crystal Violet dye as compared to Brilliant Green. The removal difference can be attributed to the difference in molecular weight of both dyes. More than 60% of CV dye was removed by CB8/S2 and BWPH/S2 at lower dye concentrations. However, per gram of biosorbent, the maximum amount of CV was quite good even at higher dye concentration (Figure 3b). Other physicochemical and biological techniques for removing CV dye are compared in Table 4. Ceriporia lacerata (CLB)-powdered mycelial biomass gave a CV biosorption capacity of 239.25 mg/g at a comparatively lower initial dye concentration of 100 mg/L [12], whereas the fungal sorbents CB8/S2 and BWPH/S2 were even for CV dye sorption at higher concentrations. Moturi and Singara Charya achieved similar decolorization of Crystal Violet and Malachite Green dye by WRF, but it took approximately 15 days for decolorization using the biodegradation method [45]; in our research, the biosorption mechanism was able to remove dyes in shorter duration. The biosorption process did not reach complete equilibrium after 24 h for lower dye concentrations. Longer contact durations may be necessary due to the slow occupancy of particularly active sites at a low driving force (concentration gradient). Although this does not lessen the biosorbent’s usefulness, it does imply that, in practical applications, longer treatment times or continuous-flow systems may be required to optimize dye removal effectiveness, especially for diluted wastewater streams. Therefore, it could be applicable in the primary stages of wastewater treatment, where most of the dye can be sorbed by the immobilized biomass. The immobilized biomass provides an added advantage for easy biomass separation after wastewater treatment.

Table 3. Comparison of physicochemical and biological sorbents’ optimized condition and its sorption capacity for the removal of Brilliant Green dye.

Sorbent (Physicochemical or Biological)	Optimized Condition: Initial Dye Concentration (mg/L), Time (min), Sorbent Amount (g), pH, Temperature (°C)	Biosorption Capacity (mg/g), Decolorization (%)	Adsorption Kinetics	Reference
Poly(acrylic acid) hydrogel composite (PAA-K hydrogel) with kaolin clay conventional method	30, 250, 1, 7, 35	6.25, 50%	Pseudo-second-order model, Freundlich and Langmuir Model	[46]
Poly(acrylic acid) hydrogel composite (PAA-K hydrogel) with kaolin clay ultrasound method	30, 375, 1, 7, 35	12.5, 84%	Pseudo-second-order model, Freundlich and Langmuir isotherm
Activated carbon(AC) derived from guava tree wood	25, 20, 0.8, 7, -	90, 99%	Pseudo-second-order model, Freundlich isotherm	[47]
Trichoderma asperellum- free cells	100, 350, 0.25 5, 30 ± 2	12.97, -	-	[42]
Trichoderma asperellum alginate-immobilized forms	100, 350, 0.25, 5, 30 ± 2	60.64, -	-
Salix alba leaves (SAL)	50, 210 0.15, 6, 25	15.89, 95.2%	Pseudo-second-order model, Langmuir isotherm	[48]
Sponge-immobilized Trametes versicolor (CB8/S2)	400, 360, 0.5, 4.3, 22.5	379.4, 88%	-	Our study
Sponge-immobilized Pleurotus ostreatus (BWPH/S2)	200, 360, 0.5, 4.3, 22.5	302.5, 80%	-	Our study

Table 3. Comparison of physicochemical and biological sorbents’ optimized condition and its sorption capacity for the removal of Brilliant Green dye.

Sorbent (Physicochemical or Biological)	Optimized Condition: Initial Dye Concentration (mg/L), Time (min), Sorbent Amount (g), pH, Temperature (°C)	Biosorption Capacity (mg/g), Decolorization (%)	Adsorption Kinetics	Reference
Poly(acrylic acid) hydrogel composite (PAA-K hydrogel) with kaolin clay conventional method	30, 250, 1, 7, 35	6.25, 50%	Pseudo-second-order model, Freundlich and Langmuir Model	[46]
Poly(acrylic acid) hydrogel composite (PAA-K hydrogel) with kaolin clay ultrasound method	30, 375, 1, 7, 35	12.5, 84%	Pseudo-second-order model, Freundlich and Langmuir isotherm
Activated carbon(AC) derived from guava tree wood	25, 20, 0.8, 7, -	90, 99%	Pseudo-second-order model, Freundlich isotherm	[47]
Trichoderma asperellum- free cells	100, 350, 0.25 5, 30 ± 2	12.97, -	-	[42]
Trichoderma asperellum alginate-immobilized forms	100, 350, 0.25, 5, 30 ± 2	60.64, -	-
Salix alba leaves (SAL)	50, 210 0.15, 6, 25	15.89, 95.2%	Pseudo-second-order model, Langmuir isotherm	[48]
Sponge-immobilized Trametes versicolor (CB8/S2)	400, 360, 0.5, 4.3, 22.5	379.4, 88%	-	Our study
Sponge-immobilized Pleurotus ostreatus (BWPH/S2)	200, 360, 0.5, 4.3, 22.5	302.5, 80%	-	Our study

Table 4. Comparison of physicochemical and biological sorbents’ optimized condition and its sorption capacity for removal of Crystal Violet dye.

Sorbent (Physicochemical or Biological)	Optimized Condition: Initial Dye Concentration (mg/L), Time (min), Sorbent Amount (g), pH, Temperature (°C)	Biosorption Capacity (mg/g), Decolorization (%)	Adsorption Kinetics	Reference
Coriolopsis sp. (1c3) filamentous fungi-free- mycelium forms	100, 2880, 1, 5, -	-, 58.3%	-	[49]
Coriolopsis sp. (1c3) filamentous fungi-filamentous biofilm	100, 2880, 1 g biomass on muslin cloth, 5, -	-, 85.1%	-
Biochar derived from palm kernel shell (BC-PKS)	50–500, 1440, 0.5, - 25	24.45, -	Pseudo-second-order model, Langmuir isotherm	[50]
Ceriporia lacerata (CLB)- powdered mycelial biomass	100, 780, 0.01, - 20	239.25, -	Pseudo-second-order model, Koble–Corrigan model	[12]
Adiantum capillus-veneris plant leaves	30, 90, 0.06, 3, -	9.05, 90.36%	Pseudo-second-order model, Freundlich isotherm	[51]
Sponge-immobilized Trametes versicolor (CB8/S2)	300, 360, 0.5, 4.3, 22.5	54, 40%	-	Our study
Sponge-immobilized Pleurotus ostreatus (BWPH/S2)	400, 360, 0.5, 4.3, 22.5	31.6, 21%	-	Our study

Table 4. Comparison of physicochemical and biological sorbents’ optimized condition and its sorption capacity for removal of Crystal Violet dye.

Sorbent (Physicochemical or Biological)	Optimized Condition: Initial Dye Concentration (mg/L), Time (min), Sorbent Amount (g), pH, Temperature (°C)	Biosorption Capacity (mg/g), Decolorization (%)	Adsorption Kinetics	Reference
Coriolopsis sp. (1c3) filamentous fungi-free- mycelium forms	100, 2880, 1, 5, -	-, 58.3%	-	[49]
Coriolopsis sp. (1c3) filamentous fungi-filamentous biofilm	100, 2880, 1 g biomass on muslin cloth, 5, -	-, 85.1%	-
Biochar derived from palm kernel shell (BC-PKS)	50–500, 1440, 0.5, - 25	24.45, -	Pseudo-second-order model, Langmuir isotherm	[50]
Ceriporia lacerata (CLB)- powdered mycelial biomass	100, 780, 0.01, - 20	239.25, -	Pseudo-second-order model, Koble–Corrigan model	[12]
Adiantum capillus-veneris plant leaves	30, 90, 0.06, 3, -	9.05, 90.36%	Pseudo-second-order model, Freundlich isotherm	[51]
Sponge-immobilized Trametes versicolor (CB8/S2)	300, 360, 0.5, 4.3, 22.5	54, 40%	-	Our study
Sponge-immobilized Pleurotus ostreatus (BWPH/S2)	400, 360, 0.5, 4.3, 22.5	31.6, 21%	-	Our study

3.3. Principal Component Analysis

Furthermore, this research was validated by applying principal component analysis by using R 4.5.0 version. The PCA biplot (Figure 4 and Figure 5) visualizes the relationships between contact time (T1, T2, T4, T6, and T24), biomass types, and immobilization (CB8, CB8/S2, BWPH, BWPH/S2), and their dye removal percentage under varying initial dye concentrations (100–400 mg/L).

In Figure 4a, Principal Component 1 (Dim1) accounts for 85.9% of total variance, representing the dataset’s major tendencies. Contact time and initial dye concentration are highly related to Dim1, as seen by their vector orientations and magnitudes. The pronounced correlation between contact time and initial dye concentration vectors with Dim1 indicates that this axis predominantly represents the synergistic influence of dye availability and exposure duration on fungal biosorption capacity. Biologically, elevated initial dye concentrations enhance the driving force for mass transfer, while extended contact durations provide a more thorough occupation of binding sites, both of which are essential factors influencing biosorption efficiency in fungal systems. A similar pattern was seen in Figure 4b. Longer vectors show that these variables have a considerable influence on sample separation, along with Dim1. Higher dye concentrations and longer contact times are likely to explain the observed variation in sorption efficiency. The CB8 (free-fungal) and CB8/S2 (immobilized) clusters are clearly separated by Dim1. CB8/S2 samples (e.g., score: 76.7) are located further down the positive axis of Dim1 than CB8 (e.g., score: 72.4), demonstrating improved Brilliant Green dye sorption effectiveness under sponge-immobilized experimental conditions. Similarly, the PCA biplot analysis (Figure 4b) demonstrates the effectiveness of immobilizing P. ostreatus (BWPH/S2) for BG dye removal. Dim1 (84.7% variance) demonstrates the significant impact of contact time and initial dye concentration on sorption efficiency, with BWPH/S2 outperforming BWPH under high-stress conditions. In both fungal species, the immobilization matrix (S2) appears to offset the limits provided by high dye concentrations, as CB8/S2 and BWPH/S2 clusters are closer to locations associated with good sorption despite varying dye levels. Prolonged contact time correlates positively with sorption efficiency in both strains, whereas CB8/S2 achieves better efficiency at shorter durations, implying faster saturation kinetics. CB8/S2 and BWPH/S2 demonstrate reduced sensitivity to elevated dye concentrations, likely due to improved stability and active site accessibility conferred by immobilization. Comparative analysis of P. ostreatus (BWPH/S2) with T. versicolor (CB8/S2) reveals consistent benefits of immobilization across fungal species, though species–specific metabolic traits may modulate matrix interactions. This improvement is attributed to the protective effects of the S2 matrix, which likely stabilizes the fungal biomass and enhances reusability.

The PCA biplots (Figure 5a,b) illustrate the relationship between contact time, initial CV dye concentration, and sorption efficiency for two fungal species: T. versicolor (CB8 and CB8/S2) and P. ostreatus (BWPH and BWPH/S2). Both biplots are dominated by Dim1, which explains 91% of T. versicolor (CB8) and 91.8% of P. ostreatus (BWPH) of the total variance, respectively, indicating that the primary trends in the data are strongly driven by experimental conditions (contact time and dye concentration). Dim2 accounts for only 6.6–6.7% of the variance, suggesting minor secondary influences of self-immobilized biomass on dye removal percentage, as it was separated clearly from sponge-immobilized biomass. Contact time and initial CV dye concentration are the primary drivers of sample separation, along with Dim1, as evidenced by their strong alignment with this axis. Immobilized strains (CB8/S2 and BWPH/S2) are distinctly separated from their free counterparts (CB8 and BWPH), along with Dim1, occupying positions associated with higher sorption efficiency. This separation suggests that immobilization enhances dye uptake, likely due to improved structural stability, increased active site availability, and resistance to dye toxicity. P. ostreatus (BWPH/S2) shows a marginally higher Dim1 variance, suggesting slightly more predictable sorption behavior than T. versicolor (CB8/S2). As a result, immobilization significantly enhances CV dye sorption for both fungal species by possibly providing protection against dye toxicity and improved mechanical stability [52]. Immobilization is highly recommended for scaling up fungal bioremediation of CV dye. Both CB8/S2 and BWPH/S2 are promising candidates, with slight variations in performance, which may guide species selection based on specific industrial needs. These findings advocate for immobilization as a scalable strategy to enhance fungal bioremediation in dye-contaminated effluents [53,54].

3.4. Effect of Autoclaved-Dead Biomass

The thermally inactivated T. versicolor (CB8/S2-A) immobilized biomass was incubated with BG or CV dye solution at different dye concentrations. As compared to live biomass, the autoclaved biomass did not show desorption in the starting period. It showed gradual increase in the absorption of dye with increasing contact time. For both dyes, the highest dye removal was observed with the lowest dye concentration. Autoclaved CB8/S2-A was able to remove 73% of BG (Figure 6a) and 63.1% of CV (Figure 6b). When self-immobilized CB8-A and BWPH-A biomass was subjected to autoclaving, it encountered tremendous reduction in fungal biomass. When it was utilized for dye removal, there no notable amount (less than 8%) of sorption of dye was observed. Therefore, it was discontinued for further experiments, and the sponge-immobilized was chosen instead. Due to autoclaving, the functional groups present on the surface of fungal biomass may lead to alteration, which is responsible for changes in biosorption capacity. It is may also result in changes in the porosity of sponge, which may result in different dye-sorption behaviors. In contrasts to the obtained result, the highest Congo Red eliminations (95.37–100%) against live pellets and pellets that have been acid and alkali pretreatment are demonstrated by the autoclaved pellets of T. versicolor [38,55].

3.5. Effect of Temperature and pH

Temperature and pH are two crucial physicochemical factors that can affect WRF’s ability to absorb dyes. The interaction between dye molecules and the functional groups on the mycelial surface may be influenced by the pH level. Similarly, temperature affects the fluidity of membranes, kinetics of enzymes, and the rate at which fungi absorb dye molecules. Here, we used a central composite design to create an experiment to evaluate the combined effects of temperature and pH on dye sorption.

The interactive effect of pH and temperature on the biosorption efficiency of Brilliant Green dye by immobilized T. versicolor is illustrated in Figure 7 S(a), C(a) (contour plot). The response surface reveals a significant dependency of dye removal percentage on both variables. The experimental results fitted well with the quadratic model (p < 0.05), and ANOVA showed the lack of fit as non-significant. The predicted R² of 0.9484 is in reasonable agreement with the adjusted R² of 0.9825; i.e., the difference is less than 0.2. T. versicolor’s biosorption ability, which was much higher at lower and moderate temperatures (20.85–32.17 °C) and at mildly acidic to neutral pH values (pH 3.4–6), peaking at roughly 50%. This implies that moderately acidic and mesophilic environments are ideal for CB8/S2 enzymatic and surface activity. Specifically, the lower left quadrant of the contour plot (low pH, low temperature) showed the maximum dye removal, suggesting ideal sorption efficacy under these conditions. On the other hand, biosorption effectiveness significantly decreased at temperatures over 37 °C, and the pH moved towards alkaline conditions (>7.3). The contour plot’s rightmost area, which corresponds to high temperatures (up to 49.15 °C) and high pH levels (up to 8.6), revealed little dye elimination (<10%), most likely because of the impairment of fungal cell surface and enzymes involved in dye binding and the breakdown being denatured or inhibited. The notion that both too low and too high extremes in pH and temperature can impair biosorption effectiveness is supported by the transition zone represented by the central region of the Figure 7 S(a), C(a) (pH ~6, temperature ~32–37 °C), where considerable dye removal (30–40%) is seen. According to these results, it is crucial to maintain moderate temperatures (25–35 °C) and somewhat acidic conditions (pH 4.0–6.0) in order to optimize the biosorption of Brilliant Green dye employing T. versicolor. When BWPH/S2 was employed for BG dye sorption, it followed the 2FI model and showed that the maximum dye sorption (~50%) was observed at low pH levels (3.4–4.7) and lower temperatures (20.85–26.5 °C), indicating that acidic conditions and lower thermal energy favor dye uptake by P. ostreatus (Figure 7 S(b), C(b)). These conditions likely promote enhanced surface binding or electrostatic attraction between the fungal biomass and the cationic Brilliant Green molecules, as fungal cell walls bear more negative charges under acidic pH levels. Similarly to the T. versicolor strain, increased temperature and pH led to progressive decline in dye removal, with sorption efficiency falling below 30% above pH 6 and temperature 32 °C. The lowest biosorption performance (<10%) was recorded at the extreme top-right quadrant of the plot-high pH (8.6) and high temperature (49.15 °C), suggesting that thermal and chemical conditions are unfavorable for dye interaction and fungal metabolism.

The CV sorption by immobilized T. versicolor and P. ostreatus is depicted in Figure 7c,d. Similarly to BG dye, a variety of pH and temperature ranges were examined to determine the ideal conditions for fungal dye sorption. In the instance of CV dye removal by CB8/S2, the highest dye sorption (~40%) occurs at pH 6.5–7.5 and temperature ~30 °C, according to the plot’s surface shape evaluation. At both high temperatures and low pH levels, sorption dramatically drops, creating a downhill slope on the 3D surface. Negative or near-zero values suggest low activity or desorption effects in extreme conditions. When assessing the fit summary for CB8/S2, the quadratic model suited the data well (p < 0.05) and the ANOVA test revealed a non-significant lack of fit (p > 0.05). Predicted R² and Adjusted R² differed by more than 0.2, which is a limitation of the model’s predictive capability, even though the model helped determine the optimized condition for dye removal. Meanwhile, the 2FI model was significantly preferred by immobilized P. ostreatus (p value = 0.0003) over the linear model (p value = 0.1296). An ANOVA test of the derived model revealed a non-significant lack of fit (p > 0.05). Furthermore, the difference between the Adjusted R² of 0.8451 and the Predicted R² of 0.7042 was less than 0.2, indicating a satisfactory agreement.

As a cationic dye, CV attaches itself to negatively charged compounds on the fungal cell wall, such as phosphate, carboxyl, or hydroxyl. Functional group protonation decreases electrostatic interactions at low pH levels (<5), which lowers dye uptake. Deprotonated sites improve binding effectiveness at higher pH values (~7). Membrane fluidity is a key factor in determining dye sorption, since biosorption comprises both chemisorption and physisorption. A moderate temperature improves dye uptake and degradation by increasing membrane fluidity and enzyme kinetics. Higher temperatures (>40 °C) have the potential to reduce biosorption by denaturing enzymes, damaging membranes, and inhibiting metabolism. The phenomenon can be explained by a gradual decrease in sorption as the temperature rises above about 35 °C. Our findings are consistent with those of Lin et al. [12] and P. Grassi et al. [56]. For the uptake of CV from aqueous solution, the former has employed powdered mycelial biomass of the basidiomycetous fungus Ceriporia lacerata. Over a pH range of 2–10, it was observed that the amount of adsorbed CV per unit biomass weight by C. lacerata increased as the dye solution’s pH rose, noticeably up to pH 6. There was no discernible increase in dye sorption between pH 6 and pH 10. The dye sorption also rose with pH, according to Grassi et al., reaching about 87% of dye removal at pH 6. The clearance percentage remained at 87% between pH 6 and 10.

3.6. Mycelial Matrix and Dyes Sorption Characterization

3.6.1. FT-IR Spectroscopy

The absorption mechanism of triarylmethane dyes involved several possible non-covalent interactions between dye molecules and fungal mycelium peptide and polysaccharide species, e.g., dipole–dipole, dipole-induced dipole, charge–charge, charge–dipole, charge–π electrons, and aromatic rings’ π–π stacking [57]. The functional groups involved in the dye biosorption process by the fungi Trametes versicolor (CB8) and Pleurotus ostreatus (BWPH) are shown by the FT-IR spectra in Figure 8a,b. A broad peak was seen in both graphs, around 3200–3500 cm⁻¹, indicating the potential involvement of the hydroxyl (-OH) and amino (-NH) groups. In dye-sorbed fungal samples (CB8-BG, CB8-CV, BWPH-BG, and BWPH-CV), a slight shift and decrease in intensity were observed in this area, suggesting that the fungal mycelium and dye molecules were interacting via hydrogen bonds and electrostatic interaction [58] with the hydroxyl and amino groups of fungal mycelium. Furthermore, C-H stretching was seen to occur between 2800 and 3000 cm⁻¹, which accounts for the weak van der Waals interaction [59]. Compared to P. ostreatus (BWPH), a more noticeable shift between 1600 and 1700 cm⁻¹ was seen in T. versicolor (CB8), showing C=O stretching and possibly ion exchange interaction with dye molecules [60]. The participation of C-N or C-O functional groups is indicated by a prominent peak close to 1100 cm⁻¹, which implies complexation with dye molecules [61]. The negatively charged carboxyl and hydroxyl groups on fungal biomass are important for dye binding since both BG and CV are triarylmethane cationic dyes [44]. More prominent changes are visible in the spectra of CV-absorbed fungal samples (BWPH-CV and CB8-CV), where the aromatic ring stretching at 1332 cm⁻¹ and out-of-plane C-H vibration of the aromatic ring at 700 cm⁻¹ of the absorbed dye are easily noticed. In the case of samples after BG absorption, no direct evidence of dye absorption originating from its molecules was observed in the BWPH-BG and CB8-BG spectra, which has been already reported in several absorption studies of green dyes [62,63,64]. The aforementioned shifts in peak position and changes in intensity are considered evidence of the interaction between fungal mycelium and absorbed dyes. Strong peak changes and the appearance of additional peaks following dye sorption indicate a characteristic interaction between dye and fungal biomass. Immobilization of fungal biomasses (CB8/S2 and BWPH/S2) onto sponge (S2) matrices have been confirmed by increased intensity and the broadening of the O-H/N-H band (~3300 cm⁻¹), indicating the addition of fungal hydroxyl and amino groups (Figure S2). New peaks appeared near 1650 cm⁻¹ and 1540 cm⁻¹ in immobilized fungal biomass, corresponding to Amide I (C=O) and Amide II (N-H) bands of fungal proteins. Enhanced intensity was observed between 1200 and 1000 cm⁻¹, associated with the C-O stretching of fungal polysaccharides (glucans, chitin). These additional functional groups could be responsible for the enhanced biosorption capacity of immobilized fungal biomass.

3.6.2. Scanning Electron Microscopy

The scanning electron microscopy (SEM) image (Figure 9a) of the polyurethane sponge (S2) displays a highly porous and interconnected three-dimensional architecture, which is optimal for microbial immobilization [39]. The sponge’s fibrous structure offers an extensive surface area for fungal adhesion and proliferation, while its mechanical integrity guarantees resilience during bioprocessing. The open pores enable effective nutrition transport and oxygen transfer, essential for sustaining fungal metabolic activity. The SEM picture (Figure 9b,c) of CB8/S2 and BWPH/S2 illustrates effective fungal colonization. The hyphae are intricately intertwined within the sponge matrix, creating a fibrous surface [33]. The sponge’s architecture seems to facilitate uniform hyphal distribution, reducing diffusion constraints and improving dye sorption efficacy, as evident by the results.

The SEM image of CB8 (Figure 10a) displays a compact, fibrous network of hyphae with homogeneous surface shape, characteristic of filamentous fungal development. The mycelial structure remains intact, exhibiting no apparent abnormalities, which suggests strong pre-sorption conditions. Meanwhile, BWPH hyphae (Figure 10b) have a loosely organized but broad network, characterized by visible pores and channels indicative of a high surface area for possible dye interaction. Following BG and CV dye sorption (Figure 10c–f), CB8 and BWPH hyphae exhibit a marked decrease in pore size, as it supports dye penetration. CB8 exhibits a better dye-binding capability, which is counterbalanced by potential structural stress, whereas BWPH provides a harmonious equilibrium between efficiency and stability. Immobilization on supports (e.g., sponge S2) may alleviate morphological alterations and improve reusability.

3.7. Reusability of Immobilized Mycelial Pellets

The removal of BG and CV dyes, respectively, using immobilized T. versicolor (CB8) and P. ostreatus (BWPH) fungal mycelial pellets, is demonstrated in Figure 11a,b. In the first cycle sorption studies, mycelial pellets were exposed to dye solution for six hours in a standard manner. Both fungi absorbed dye with an extremely high efficiency (almost 40–80%) in the first cycle. CB8/S2 demonstrated a strong sorption capacity for both colors in the second round after the pellets were soaked in water overnight and reused. In the second cycle, the BWPH/S2 variant’s capacity for BG dye sorption was cut in half, suggesting a decrease in binding sites or fungal biomass saturation. Nevertheless, it had a very high capacity for CV sorption. Following the methanol treatment of fungal pellets, both fungal strains’ dye sorption efficiency was significantly decreased (almost 10–15%), suggesting that the functional groups involved in biosorption may have been changed or harmed by the methanol exposure. The second cycle’s decline in sorption is consistent with earlier research, which found decreased biosorption effectiveness after biomass reuse, mostly as a result of site saturation or structural alterations in the fungal surface [65]. Since methanol can function as a denaturing agent, the sharp decline in sorption following methanol pre-treatment raises the possibility of changes in the composition of the fungal cell wall, possibly as a result of the loss of lipids and proteins [55]. Fungal pellets soaked in water demonstrated more reusability than those treated with methanol, which made them more appropriate for repeated dye biosorption applications. The structure of the biomass was likely altered by methanol exposure, which decreased its capacity to hold dye molecules in later cycles.

The developed fungal-based biosorbent demonstrates significant promise for large-scale application. White rot fungi can be cultivated on low-cost support, reducing production expenses and minimizing waste generation. The preparation process is simple and does not require hazardous chemicals, thereby lowering operational complexity and environmental impact. Additionally, the biosorbent is reusable without additional treatment, supporting sustainable wastewater treatment practices. These characteristics suggest strong potential for cost-effective and environmentally friendly scale-up in industrial dye remediation.

4. Conclusions

This study demonstrates the high efficacy of sponge-immobilized fungal biomasses, T. versicolor (CB8/S2) and P. ostreatus (BWPH/S2), in the rapid and efficient biosorption of triarylmethane dyes such as Brilliant Green and Crystal Violet. Among the tested configurations, immobilized live biomass outperformed self-immobilized and dead forms, achieving over 90% dye removal within six hours. The principal factors influencing sorption were dye concentration and contact time, as confirmed by PCA, with optimal performance observed at moderately acidic pH levels and temperatures around 30 °C. Most importantly, the reusability of sponge pellets highlights a viable, affordable, biodegradable, and ecologically benign substitute for traditional physicochemical techniques. It is suitable for use in both centralized facilities and decentralized treatment units. Overall, the findings demonstrate that fungal immobilization is a reliable, environmentally friendly, and scalable method of reducing the negative effects of dye-contaminated industrial effluents on the environment, promoting cleaner water supplies and developing sustainable wastewater treatment techniques.