Dairy wastewater valorization enhances white rot fungi performance in recycled paper effluent treatment

Abstract

Wastewaters produced by the recycled paper and cardboard sector (RPCE) pose significant environmental risks due to high levels of chemical oxygen demand (COD) and a lack of essential nutrients required for effective biological degradation. In response, this investigation introduces a novel and cost-effective valorization approach: employing nutrient-laden dairy wastewater (DWW) as an eco-friendly source of nitrogen and phosphorus to enhance the bioremediation performance of a fungal consortium comprising Bjerkandera adusta and Phanerochaete chrysosporium. A primary aim of the study was to optimize the RPCE concentration to achieve a balance between contaminant reduction and industrial feasibility. Results showed that although a 25% RPCE concentration achieved the peak decolorization rate, a 50% concentration emerged as the optimal operational condition for industrial scale-up. This strategy enables the processing of larger effluent volumes while maintaining high treatment quality. Under these optimized conditions, the system attained removal rates of 93.7% for COD and 35.5% for color. Moreover, the toxicity of the treated effluent was substantially reduced, evidenced by a 55.4% drop in the phytotoxicity index (PI). The proven effectiveness of this fungal co-treatment method in non-sterile settings underscores its robustness and suitability for straightforward, cost-effective implementation in large-scale industrial systems, thus advancing circular economy principles.

Introduction

Worldwide paper usage has seen a remarkable increase, rising from about 400 million tons in 2016 to 600 million tons in 2020, and forecasts suggest it could climb to 878 million tons by 2050^1,2. On a global scale, the pulp and paper sector ranks as the third biggest user of water in industrial settings. Manufacturing paper and cardboard typically relies on sources like wood, fresh pulp, or recycled cellulose fibers (RCFs). Yet, the makeup and standard of the resulting industrial discharge differ greatly depending on the particular manufacturing steps and materials involved. In recent times, there has been a noticeable uptick in the use of RCFs, drawn from items such as office documents, outdated newspapers, and assorted paper scraps³.

Facilities that recycle paper often rely on closed-circuit water recycling setups, yet they encounter a broad mix of contaminants in their operations. These include starch, volatile fatty acids (VFAs)—arising from microbial activity during the management and stockpiling of recycled materials—various minerals (for instance, calcium carbonate from additives or coatings, silicates from ink removal, and aluminum sulfate), along with intricate sticky substances⁴. Such impurities can greatly harm the final product’s quality, leading to lower effectiveness of additives, weakened visual and structural features, unpleasant smells, and practical problems like breaks in the web or rips in the paper. The buildup patterns of these elements tend to differ across plants, mainly influenced by the unique makeup of the water used in processes⁵.

Even with the clear ecological advantages of working with recycled paper, its varied input materials bring a wide array of pollutants into the operational wastewater. These arise from tainting during storage and reuse, plus the addition of diverse enhancers in preparation⁶. Substances added to the effluent include hydrogen peroxide, calcium compounds, and adsorbable organic halides (AOX)⁵. In cases where effluent is recirculated, the pollution patterns shift from one site to another. Importantly, concentrations of five-day biochemical oxygen demand (BOD₅), color, total suspended solids (TSS), chemicals, and fillers usually appear higher in discharges from operations using fully recycled paper versus those mixing recycled with fresh pulp⁵.

White-rot fungi, which are thread-like organisms that inhabit wood and infiltrate it through their filaments, stand out for their ability to break down lignin⁷. These organisms release enzymes that degrade lignin, turning it into a soft, pale substance that eventually gets fully oxidized to CO₂ and H₂O. The oxidizing power of these lignin-degrading enzymes has drawn much interest, making white-rot fungi powerful tools for dismantling stubborn organic materials, such as Persistent organic pollutants (POPs), colorants, agrochemicals, and comparable items⁸. Their system of ligninolytic enzymes, including lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac), shows strong promise in reducing color in discharges from sectors like olive oil milling, spirit production, fabric dyeing, and paper making^9,10. The broad-spectrum action of these enzymes allows for the breakdown of numerous dangerous and long-lasting contaminants, encompassing polycyclic aromatic hydrocarbons (PAHs), pesticides, fuels, alkanes, polychlorinated biphenyls (PCBs), explosives, and artificial dyes¹¹.

Bjerkandera adusta, a type of white-rot fungus, is known for its skill in breaking down aromatic substances, greatly affecting factors like color and COD in cleanup efforts. This fungus displays impressive flexibility, flourishing in diverse surroundings and successfully lowering color and harmfulness in effluents^12,13. Likewise, Phanerochaete chrysosporium, another sturdy white-rot fungus, is recognized for its prowess in dismantling organic contaminants and excels at removing color from discharges^13,14.

White-rot fungi can decompose every part of wood, including lignin, but they need sufficient supplies of nitrogen and phosphorus for best development and lignin breakdown^15,16. DWW, marked by a heavy organic burden, especially raised COD and BOD5 values, creates major issues for nearby water systems¹⁷. The dairy sector stands as a key producer of large amounts of wastewater with intense organic content¹⁶. This discharge holds considerable nutrient levels, positioning it as a notable cause of water contamination in many areas¹⁸. The elevated organic content offers a prime feeding ground for microbes, such as bacteria, algae, and fungi. In particular, DWW shows high amounts of COD, BOD₅, phosphorus, and nitrogen¹⁶.

The main drawbacks of standard biological handling for RPCE stem from its poor biodegradability ratio (owing to abundant lignin) and its typical shortage of key nutrients, especially nitrogen and phosphorus. Tackling these gaps usually calls for adding pricey external nutrients or using energy-heavy physical–chemical techniques. The fresh aspect of this work is dual: (i) the combined application of a targeted white-rot fungal group (B. adusta and P. chrysosporium) and (ii) the showcase of a circular economy framework by repurposing DWW as an independent and affordable nutrient provider to improve the bioremediation method. Thus, this investigation seeks to refine the DWW level for utmost removal of pollutants and toxicity from RPCE, offering a practical, enduring option for use in industry.

Materials and methods

Wastewater sampling

Samples of RPCE were gathered as secondary discharge from the exit point of the aeration basin at a manufacturing site for recycled paper and cardboard located in the industrial area of Yazd Province, Iran. The exact identity of the plant remains undisclosed owing to nondisclosure commitments with the collaborating industry. DWW specimens were obtained as untreated effluent from the balancing reservoir of a nearby dairy operation focused on producing milk, cheese, yogurt, ice cream, and an assortment of other dairy items. Importantly, to faithfully replicate authentic industrial scenarios and preserve the indigenous microbial population composition, the untreated and secondary effluents were used directly without prior processing, specifically omitting sterilization or pH adjustment before fungal inoculation. Baseline assessment details for the gathered effluents appear as average figures from three distinct sampling occasions spread across a single month to account for incoming flow fluctuations (Table 1). Collection techniques followed rigorously the guidelines outlined in the Standard Methods for the Examination of Water and Wastewater¹⁹. Right after gathering, measurements for pH, electrical conductivity (EC), and temperature took place at the location. Following that, the specimens were moved to the lab for additional evaluation. Moreover, chosen specimens (RPCE, DWW, and RPCE combined with 10% DWW) underwent analysis for their levels of carbon, nitrogen, phosphorus, and potassium. Organic carbon and nitrogen were determined through titration methods, whereas phosphorus was assessed using spectrophotometric techniques (Unico 2100).

Table 1 Mean and standard deviation of physicochemical parameters of wastewaters.

Fungal consortium cultivation and preparation

The selection of the fungal consortium involving Bjerkandera adusta and Phanerochaete chrysosporium stemmed from documented evidence in the literature highlighting their complementary breakdown processes: P. chrysosporium excels at dismantling large-molecular-weight substances through its LiP and MnP functions, whereas B. adusta stands out for its substantial Lac output and durability in industrial effluents^13,20.

A pair of fungal isolates, Bjerkandera adusta (IBRC-M 30423) and Phanerochaete chrysosporium (IBRC-M 30077), were acquired from Iran’s National Center for Genetic and Biological Resources. The preparation of the inoculum followed a dual-step procedure aimed at producing a uniform and vigorous fungal slurry. Initially, a solitary agar plug (8 mm in diameter) was uniformly excised from the edge of a newly developed 7-day-old culture on potato dextrose agar (PDA) medium. This segment was subsequently placed into 100 mL of potato dextrose broth (PDB) and cultivated with agitation (28 °C, 140 rpm) for another 7 days to promote rapid proliferation and peak biomass vitality. The ensuing blended liquid culture served as the ultimate inoculum. Opting for the uniformity of the starting plug dimensions and a set cultivation period in PDB, instead of measuring mycelial dry weight (MDW), was intended to assess the real-world applicability and economical aspects of the remediation approach within a non-sterile industrial setting. The prepared liquid inoculum was added straight into the effluent blend in every container²¹.

• (1) The experiments with inoculation featured a fungal alliance of Phanerochaete chrysosporium and Bjerkandera adusta, carried out across two separate stages:

• (2) Introduction of the fungal alliance into RPCE lacking any supplementary carbon or nutrient inputs.

• (3) Introduction of the fungal alliance into RPCE enhanced with DWW serving as an extra provider of carbon and nutrients²².

Additionally, two reference setups were incorporated: one made up solely of RPCE, and the other consisting of RPCE blended with DWW.

Pollutant measurement post-treatment

All pollutant parameters were determined in strict accordance with the Standard Methods for the Examination of Water and Wastewater, 23rd Edition (APHA/AWWA/WEF, 2017)¹⁹. Color measurement was determined using the American Dye Manufacturers Institute (ADMI) method. To ensure the accurate quantification of dissolved color bodies and minimize interference from suspended solids, all samples were pre-treated by centrifugation followed by filtration through a 0.45 µm membrane filter. The resulting absorbance was measured spectrophotometrically at a single, characteristic wavelength of 630 nm. COD was measured using the Closed Reflux, Colorimetric Method (5220 D); five-day BOD₅ using the 5-Day BOD Test (5210 B); total nitrogen (TN) using the Persulfate Digestion Method (4500-N); and total phosphorus (TP) using the Ascorbic Acid Method after persulfate digestion (4500-P E). Lignin concentration was calculated directly from absorbance readings at 430 nm according to equation²³:

$${\text{Lignin }}\left( {\text{mg/l}} \right) = {\text{Absorbance}}/0.000247$$

(1)

To evaluate the efficiency of heavy metals removal under optimal conditions, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was employed to quantify heavy metals concentrations in the samples.

To investigate the metabolites resulting from degradation, two samples were analyzed under optimal conditions: a control sample (process influent) and the metabolic compounds present in the effluent after biological treatment. Sample extraction was performed according to the method established by Haq (2017)²⁴, followed by analysis using Gas Chromatography-Mass Spectrometry (GC–MS).

During the biological treatment of wastewater using the fungal consortium, the phytotoxicity of the treated effluent was evaluated through a seed germination and root elongation bioassay^25,26. Seeds of Phaseolus mungo were surface-sterilized by immersion in 0.1% (w/v) HgCl₂ solution for 2 min followed by repeated rinsing with sterile distilled water. Four dilutions (25%, 50%, 75%, and 100% v/v) of both untreated (raw influent) and biologically treated effluent were tested, with tap water used as the negative control. After 72 h of incubation in the dark at 25 ± 2 °C, root length was measured and the relative seed germination (RSG), relative root growth (RRG), and PI were calculated according to Eqs. (2)–(4) ²⁷.

$${\text{RSG }}\left( \% \right){ = }\left( {\frac{{{\text{GSS}}}}{{{\text{GSC}}}}} \right) \times 100$$

(2)

$${\text{RRG }}\left( \% \right){ = }\left( {\frac{{{\text{RLS}}}}{{{\text{RLC}}}}} \right) \times 100$$

(3)

$${\text{PI}} \left( \% \right) = \left[ {1 - \left( {\frac{{{\text{RLS}}}}{{{\text{RLC}}}}} \right)} \right] \times 100$$

(4)

where GSS and GSC denote the number of germinated seeds in the treated sample and the control, respectively; and RLS and RLC represent the mean root length in the treated sample and the control, respectively.

For example, a mean root length of 3 cm in the sample and 10 cm in the control yields a PI of 70%. All removal efficiencies reported in the Results section represent net values obtained after subtraction of the contribution observed in the abiotic control.

To accurately quantify the net pollutant removal achieved by the fungal consortium, the experimental design included two critical abiotic control setups. The controls were used to account for pollutant removal due to factors other than fungal activity (such as volatilization or spontaneous chemical degradation). The two control groups were: (i) RPCE only (without DWW or fungus), and (ii) the optimal mixture of RPCE and DWW (without fungus).

Data analysis

Data were analyzed using Excel and SPSS version 26. Descriptive statistics, including Mean and Standard Deviation (SD), were used to summarize the results. The effects of varying RPCE concentrations and treatment time on pollutant removal kinetics were statistically analyzed using a Two-way Analysis of Variance, specifically the Repeated Measures ANOVA approach. This method was essential to account for the repeated measurements taken on the same experimental units over time. Assumptions of normality and homogeneity of variance were checked. Post-hoc multiple comparison tests, specifically the Bonferroni correction, were used to determine specific significant differences between treatment groups and control conditions. The level of significance for all tests was set at P < 0.05.

Results and discussion

Wastewater characteristics

Statistical Analysis of Removal Kinetics. The Two-way Repeated Measures ANOVA revealed statistically significant main effects for both factors, RPCE concentration and treatment time, and their interaction on the removal of all target pollutants (p < 0.05). Specifically, the main effect of RPCE concentration on pollutant removal was highly significant (p < 0.001). Similarly, the main effect of treatment time was also highly significant (p < 0.001), demonstrating the time-dependent nature of the biodegradation process. Crucially, a significant interaction effect between DWW concentration and time was found (p < 0.001), indicating that the highest removal efficiency was achieved by the synergistic effect of the optimized DWW ratio and prolonged treatment time. Subsequent analysis of Bonferroni post-hoc comparisons confirmed that the 10% (v/v) DWW condition achieved significantly higher removal rates compared to all other conditions after 6 days (p < 0.05).

Practical Feasibility and Cost Implications. Unlike many laboratory-scale bioremediation studies that rely on sterilized or pH-adjusted media, this research intentionally used non-sterile effluent at its native pH. The successful performance of the B. adusta and P. chrysosporium consortium under these non-ideal conditions is a key finding, demonstrating its robustness and competitiveness against the indigenous microbial community. The ability to bypass costly and energy-intensive pre-treatment steps (sterilization and chemical dosing) significantly reduces the operational expenditure, thereby positioning this co-treatment approach as a highly feasible and economical option for direct industrial integration and scale-up.The physicochemical characteristics of the effluent from the aeration unit of the recycled paper and cardboard treatment plant, raw DWW, and a mixture of these wastewater (with a ratio of 90% RPCE to 10% DWW) were determined. These characteristics are presented in (Table 1).

The characterization data (Table 1) reveals that the RPCE exhibited a high COD of 835.38 ± 216.60 mg/L. Due to the presence of complex, recalcitrant compounds like lignin and cellulose derivatives typical of paper mill discharge, this high COD value confirms the low biodegradability of the secondary effluent. Furthermore, the RPCE was severely deficient in essential macronutrients, with TN measuring only 4.4 mg/L and TP at a mere 0.265 mg/L. Based on the typical requirements for effective biological treatment, this nutrient scarcity underscores the necessity of external nutrient supplementation for effective fungal bioremediation.

The analysis of mixed wastewater physicochemical properties indicated that the addition of DWW led to an increase in mineral nutrient concentrations. Specifically, nitrogen levels increased from 4.4 to 13.76 mg/L, phosphorus from 0.265 to 4.03 mg/L, carbon from 66.15 to 89.53 mg/L, and potassium from 46.5 to 47.6 mg/L.

COD removal efficiency

This section evaluates the effectiveness of the white-rot fungal species Phanerochaete chrysosporium and Bjerkandera adusta in the biodegradation of effluent from the aeration tank of a recycled paper and cardboard treatment facility. The experimental design enabled the measurement of COD under various conditions.

The study assessed COD removal efficiency across different RPCE concentrations (25%, 50%, and 100%) over three contact periods (1, 3, and 6 days) and with varying fungal populations (4 and 8 agar plugs), both with and without the addition of DWW.

Figure 1 illustrates consistent trends in the removal of COD from effluent. Specifically, the highest average COD removal efficiency, 98.23% ± 1.06, was recorded at a 25% effluent concentration on day 6 in samples containing a fungal population of 8 agar plugs supplemented with DWW. In contrast, the lowest COD removal efficiency, 43.75% ± 10.12, was observed in samples with a fungal population of 4 agar plugs without DWW supplementation. These findings highlight the significant influence of fungal population size and substrate availability on COD removal efficiency. Dissolved oxygen (DO) levels in the effluent were periodically measured, with a minimum value of 0.8 mg/L and a maximum value of 2.87 mg/L recorded.

Fig. 1

COD removal efficiency from RPCE at various concentrations (a) 25%, (b) 50%, and (c) 100%, under different conditions, with and without the addition of a mineral source (DWW) and fungal populations of 4 and 8 agar plugs.

The COD removal efficiency in the control sample on day 6 was 12.51% ± 0.82. This trend was consistent across all wastewater concentrations, exhibiting a uniform pattern. Notably, the highest COD removal efficiency was achieved under conditions with 8 agar plugs and DWW supplementation after a 6-day contact period. It appears that COD removal efficiency decreases as effluent concentration increases.

In a study by Li et al. (2020) on effluents from ethanol and chemical industries, results indicated a COD removal efficiency of 63% over a two-day period and a color removal efficiency of 72% over three days²⁸. Diaz et al. (2021) investigated olive oil industry wastewater and observed a rapid reduction in initial sugar concentrations in samples containing glucose and inoculated fungi. Specifically, all added glucose was completely consumed within the first 24 h, whereas in the control sample, the sugar concentration decreased from 3663 to 1772 mg/L over the same period and then stabilized. This suggests that the endogenous microbiota was insufficient to degrade all added glucose²⁹. Although fungal enzyme activities were not quantified in this study, the results indicated that glucose addition facilitated the synthesis of fungal enzymes, which may account for the reduction in COD and degradation of recalcitrant compounds^30,31. Additionally, analysis of various experimental results suggests that nitrogen concentration, as a mineral nutrient, significantly impacts the COD removal efficiency of wastewater²⁸.

A notable study on biodegradation using white-rot fungi, similar to those employed in this research, was conducted by Jie Li et al. (2020). This study utilized these fungi for municipal wastewater treatment, achieving a COD removal rate of approximately 75%. It also demonstrated that COD removal efficiency significantly increased with higher fungal population growth³².

Color removal efficiency

The experimental design employed in this study enabled the evaluation of color removal from effluent under various conditions. Figure 2 illustrates the color removal efficiency at different effluent concentrations (25, 50, and 100%) over three contact periods (1, 3, and 6 days) and with varying fungal populations (4 and 8 agar plugs), both with and without the addition of DWW.

Fig. 2

Color removal efficiency from RPCE at various concentrations (a) 25%, (b) 50%, and (c) 100%, under different conditions, with and without the addition of a mineral source (DWW) and fungal populations of 4 and 8 agar plugs.

The data demonstrate that the highest average color removal efficiency, 38.7% ± 11.30, was achieved at a 25% effluent concentration on day 6 under conditions incorporating a carbon source, mineral nutrients, and a fungal population of 8 agar plugs. Conversely, the lowest average color removal efficiency, 14.11% ± 6.26, was observed in conditions without DWW supplementation and with a fungal population of only 4 agar plugs. This trend was consistently observed across all concentrations and was identified as the optimal condition for each respective concentration. Color removal in control samples was minimal, underscoring the direct influence of carbon sources, mineral nutrients, and fungal population size on the color removal process by the selected fungal species.

Furthermore, the average color removal efficiency on day 6 increased compared to day 1, indicating the significant effect of contact time on the color removal process, with removal rates improving as contact time extended.

Color removal in the control samples remained very low, with a maximum of 6.56 ± 1.37% throughout the experimental period. This negligible removal confirms that the substantial color reduction observed in the fungal-inoculated co-treatment samples was predominantly attributable to biotic degradation by the fungal consortium.

Pakshirajan et al. (2012) reported a color removal efficiency of up to 64% after treating textile wastewater with Phanerochaete chrysosporium³¹. Similarly, Ntougias et al. documented a color removal efficiency of 60–65% in olive oil industry wastewater using the fungal species Basidiomycetes Pleurotus spp.³³. Comparable reductions in color were also observed in olive oil mill wastewater treated with the fungal species Trametes versicolor in other studies³⁰.

In a study by Li et al. (2020) focusing on effluents from ethanol and chemical industries, results indicated a color removal rate of 72% over a three-day period. The study noted that color removal progressively increased over time, likely due to the depolymerization of lignin compounds and the formation of new chromophoric substances during the fungal bioremediation process. Additionally, it was observed that color and lignin removal rates from wastewater decreased after two days. This reduction may be partly attributed to biosorption and degradation of pigments such as lignin, which aligns with the findings of the present study²⁸.

In a separate investigation by Bulai et al. (2017), the treatment of textile wastewater using ligninolytic fungal species was examined, achieving near-complete color removal. Under optimal conditions, the highest reaction rates were observed within the first two days. Consequently, this approach could be effectively implemented in wastewater treatment facilities requiring the processing of significant effluent volumes within a limited timeframe. The system’s efficiency suggests that adding a small amount of glucose can facilitate color removal in half the designated time, and even minimal nutrients present in the wastewater can enhance the effectiveness and speed of fungal treatment³⁴.

The effect of varying RPCE concentrations was evaluated, ranging from 25 to 100%. Analysis of the removal kinetics revealed that the maximum color removal efficiency was achieved at the lowest concentration of 25% RPCE, resulting in 38.7 ± 11.3 color removal. However, the optimal operating condition for the bioremediation system was ultimately determined to be 50% RPCE. This selection was based on a balanced approach considering the highest overall pollutant removal rates (COD and color) and the crucial factor of industrial feasibility. Utilizing a 50% RPCE concentration maximizes the volume of treated wastewater while still achieving substantial purification, thereby offering the most practical and cost-effective solution for full-scale industrial application.

BOD₅, lignin, and heavy metal removal efficiency under optimal conditions

Following fungal inoculation and the determination of optimal conditions, the removal efficiencies of key pollutants, specifically BOD₅, lignin, and heavy metals, were evaluated after a six-day incubation period. The results indicated removal rates of 95.6% ± 2.13 for BOD₅ and 48% ± 9.25 for lignin. These findings align with a study by Li et al. (2020), which focused on effluents from ethanol and chemical industries and reported a lignin removal rate of 60% over a two-day period²⁸.

Additionally, these results are consistent with a study by Tyagi et al. (2014), which investigated paper industry wastewater using a fungal-bacterial consortium over nine days, achieving removal efficiencies of 87.2% for BOD₅ and 97% for lignin³⁵. Similarly, a study by Bardi et al. (2017), which treated landfill leachate and two synthetic effluents containing tannic acid (a polyphenol) and humic acid using Bjerkandera adusta immobilized on polyurethane with glucose supplementation, reported BOD₅ removal efficiencies of 100% in leachate, and 89 and 75% in effluents containing tannic acid and humic acid, respectively³⁶.

In a study by Diaz et al. (2021) on olive oil industry wastewater, a reduction in BOD₅ concentration was observed during fungal treatment, ultimately reaching BOD₅ levels below 100 mg/L. Enzymes produced by Phanerochaete chrysosporium demonstrated greater efficiency in degrading biodegradable organic matter compared to recalcitrant compounds, contributing to reductions in both COD and BOD₅ levels²⁹.

Figure 3 illustrates the removal efficiencies of various heavy metals under optimal conditions.

Fig. 3

The removal efficiency of heavy metals by the fungal consortium under optimal conditions) The concentration of RPCE was 50% and fungal population was 8 agar plugs and added carbon source and minerals (DWW).

The highest removal efficiencies for heavy metals were recorded as follows: aluminum decreased from 0.4 mg/L to less than 0.01 mg/L, representing a 97.5% reduction; titanium decreased from 0.2 to less than 0.01 mg/L, indicating a 95% reduction; tin decreased from 5.5 to 1 mg/L, corresponding to an 83.6% reduction; and zinc decreased from 3.9 to 1 mg/L, equivalent to a 74.3% reduction.

These findings are consistent with a study by K.R. Sharma (2020), which examined the removal of heavy metals, including cadmium, lead, and nickel, from contaminated water using the white-rot fungus Phlebia brevispora. That study reported removal efficiencies of 91.6% for cadmium, 97.5% for lead, and 72.7% for nickel. Furthermore, cadmium was identified as the most toxic metal, significantly inhibiting fungal growth³⁷.

The presence of metal ions, including lead, cadmium, and nickel, exhibited an inhibitory effect on the growth of the ligninolytic fungus Phlebia brevispora at varying concentrations, leading to morphological changes in fungal hyphae. Nevertheless, these fungi demonstrated effective removal capabilities for specific metal ions³⁷. Consequently, it can be inferred that white-rot fungi hold significant potential for the bioremediation of environments contaminated with heavy metals.

Metabolites produced during fungal bioremediation

During the treatment of RPCE with the fungal consortium under optimal conditions, metabolites resulting from fungal degradation were identified. The results of GC–MS analysis (using the NIST05a.L database) for untreated and treated effluent samples under optimal conditions are presented in (Fig. 4). The GC–MS analysis of untreated effluent revealed the presence of various organic compounds. Following fungal treatment, the majority of compounds identified in the untreated effluent were no longer detectable, and new peaks emerged.

Fig. 4

GC–MS chromatogram of extracted compounds: (A) Untreated effluent sample, (B) Effluent treated sample by the fungal consortium.

Several aromatic carboxylic acid compounds, including benzenepropanoic acid, trimethyl benzoic acid, 2-[(trimethylsilyl) 2-phenyl-2-trimethylsiloxy-prop], and 2,2-bis[(4-trimethylsiloxy)phenyl], were identified as low-molecular-weight derivatives of phenolic lignin units present in the effluent generated by the recycled paper and cardboard industry³⁸. In the treated effluent sample, a significant reduction in peak intensity was observed, and most organic compounds present in the influent wastewater were not detected in the treated sample. This observation may indicate the fungal consortium’s ability to utilize these compounds as sources of carbon, nitrogen, and energy, as well as to degrade pollutants³⁹. Additionally, similar compounds such as hexadecanoic acid, octadecanoic acid, and benzeneacetonitrile have been reported in pulp and paper industry wastewater⁴⁰. A new compound, 1,2-benzenedicarboxylic acid, was identified in the treated wastewater, likely originating from metabolites produced during the biodegradation of lignin by the fungal consortium^24,41.

In this study, the concentrations of compounds such as acetic acid and trimethylsilyl ester increased, indicating the formation of organic acids during the fungal consortium treatment process. This observation aligns with a decrease in the sample pH from 7.2 to 6.6, suggesting the production of acidic compounds during treatment. Certain compounds, including hexadecanoic acid and trimethylsilyl, remained unchanged during the treatment process. Previous studies have also reported the presence of various acidic compounds resulting from lignin degradation^24,38. Furthermore, a study by Villaplana et al. (2015) investigated the treatment of sludge in liquid and solid phases of wastewater containing pentabromodiphenyl ether using Trametes versicolor. The findings demonstrated that this fungus achieved removal efficiencies of 87, 85, and 67% for penta-, octa-, and deca-BDE in the liquid phase, respectively, and 86% in the solid phase⁴².

In a study by Li et al. (2020) on ethanol industry wastewater, GC–MS analysis of treated wastewater with the Z-6 fungus revealed the presence of new peaks compared to untreated wastewater. The findings indicated a reduction in the relative amounts of S units (syringyl; 3,5-dimethoxy-4-hydroxyphenyl) and G units (guaiacyl; 3-methoxy-4-hydroxyphenyl), while the relative amount of H units (para-hydroxyphenyl) increased after treatment with the Z-6 fungus. Specifically, para-hydroxybenzoic acid was identified as the most abundant aromatic compound extracted from the treated wastewater, suggesting the occurrence of demethylation and oxidation processes during treatment, likely involving enzymes such as fungal Lac²⁸.

The analysis of GC–MS data was complemented by the observation of a pH drop from the initial 7.2 to a final minimum of 6.6 in the optimal conditions. This drop is attributed to the fungal secondary metabolism and the production of organic acids. These acids are critical in the white-rot mechanism as they serve as chelating agents for Mn(II), thereby promoting the activity of MnP and enhancing the overall ligninolytic system’s efficacy against recalcitrant pollutants⁴³. This mechanism confirms that the acidic environment facilitates the MnP-mediated degradation pathway, which is essential for the effective removal of complex compounds in the RPCE. Complex biochemical reactions may occur during effluent treatment with the fungal consortium. These reactions may involve the cleavage of intermolecular bonds, demethylation, hydroxylation, and side-chain modifications, ultimately leading to the degradation or transformation of lignin compounds. This process results in the effective removal of color, COD, and lignin from the wastewater.

Phytotoxicity assessment of wastewater under optimal conditions

To evaluate the phytotoxicity of wastewater under optimal conditions, the acute toxicity of untreated and treated samples was assessed using a seed germination assay with Phaseolus mungo. The effects of untreated and treated effluent at concentrations of 25, 50, 75, and 100% on germination rates were systematically investigated.

As shown in Fig. 5, untreated effluent at concentrations above 25%, particularly at 100%, significantly inhibited seed growth. In contrast, after biological treatment with the fungal consortium, seeds exhibited substantial growth even at a 100% concentration.

Fig. 5

Evaluation of the toxicity of the untreated effluent sample (comprising RPCE mixed with 10% DWW) and the treated effluent under optimal conditions (72 h incubation period).

Parameters related to RRG, RSG, and the PI are presented in (Table 2). The findings indicate that in untreated wastewater at a 100% concentration, the relative root growth was 39.28%. In contrast, treated wastewater at the same concentration exhibited a relative root growth of 67.85%. Furthermore, the PI confirmed a reduction in wastewater toxicity following fungal inoculation.

Table 2 Indices for the assessment of phytotoxicity in untreated and treated effluent samples.

Based on the obtained results, it was observed that a 25% concentration of untreated effluent sample significantly inhibited seed germination. Conversely, seeds demonstrated growth even at a 100% concentration in treated effluent samples (Fig. 5). Biological inoculation with the fungal consortium at a 100% concentration resulted in a 55.4% reduction in toxicity (Table 2).

Several studies have evaluated the phytotoxicity of wastewater from the pulp and paper industry. A notable study by Raj et al. (2014) investigated the efficacy of the bacterium Paenibacillus sp. in removing color, BOD₅, COD, phenols, and lignin from pulp and paper wastewater. In that study, wastewater toxicity was assessed using a seed germination assay with Vigna radiata L., and the findings confirmed a reduction in wastewater toxicity after bacterial treatment³⁹. Similarly, a study by Kumar et al. (2021) utilized the bacterium Bacillus cereus for lignin degradation in the kraft process. The toxicity assessment of wastewater through a seed germination assay with Phaseolus mungo L. demonstrated a 70% reduction in toxicity in treated samples, which aligns with the results of the present study²⁵.

While the 25% RPCE concentration exhibited the highest efficiency for color removal, the selection of the 50% RPCE concentration as the optimal condition underscores the applied nature of this study. The choice reflects a necessary compromise between achieving maximal color reduction and maximizing the volume of wastewater treated per cycle. This operational decision is crucial for industrial sustainability, as implementing a 50% concentration provides an economically superior method for scaling up the process compared to the extensive concentration required for 25% RPCE. The resulting COD and color removal efficiencies at the 50% concentration, combined with the significant reduction in phytotoxicity, validate this choice as the most viable and cost-effective strategy for industrial implementation.

Study limitations

While the results strongly suggest highly efficient biodegradation via the ligninolytic enzyme system (LES), a key limitation of the current study is the absence of direct enzyme activity assays for Lac, LiP, and MnP. Therefore, the proposed degradation mechanism remains inferred based on the correlation between pollutant removal and the established literature for B. adusta and P. chrysosporium. Future work will focus on quantifying these enzyme activities under DWW-enhanced conditions to fully validate the LES contribution and further optimize the process.

Conclusion

The results of this study indicate that the application of a fungal consortium in the bio-inoculation process, particularly utilizing DWW as a carbon and nutrient source for fungal growth, significantly enhances the degradation of key pollutants in wastewater from the recycled paper and cardboard industry. Consequently, these fungi can be integrated as a vital component of biological treatment processes in wastewater treatment facilities for such industries.

Given the substantial volume of wastewater produced by these industries and the critical need for effective treatment and recycling, bio-inoculation with a fungal consortium appears to be a cost-effective and highly efficient biological approach for removing critical pollutants. This strategy provides a viable solution for improving the biodegradability of organic compounds, including color, COD, BOD₅, lignin, and heavy metals, thereby reducing wastewater toxicity and facilitating its recycling within this sector.

Moreover, the use of raw DWW as an effective component to enhance pollutant removal efficiency merits further investigation. Therefore, inoculating significant quantities of Bjerkandera adusta and Phanerochaete chrysosporium, combined with specific volumes of raw DWW, holds promise for improving the performance of aerobic biological treatment systems in these industries and supporting water recycling efforts.