1 Introduction

Environmental threats posed by industrial processes and improper discharge of heavy metal–containing wastewater are growing more prevalent [1]. Leather industry has grown to lead the economic development of the nation. The Indian leather industry is currently ranked sixth in worldwide rankings, and it includes one of the country’s top ten foreign currency exchange suppliers [2]. There appear to have been about 3000 tanning factories, with roughly 80% of these facilities being located in regional as well as small-scale industries [3]. A significant portion of the tanning industry is concentrated near waterways. Tannery sectors remain overwhelmingly the most hazardous owing to the wide range of chemicals (organic chemicals: tannins, phenolics, etc. inorganic: Cr, Cu, Pd, Cd, Fe, and Zn) during the processing of skins obtained from animals [4]. These chemical-enriched effluents are released into the ecosystem from the tanning facility [5].

According to the Blacksmith Institute, Vellore’s waterways are among the most contaminated in the nation, having become heavily contaminated with heavy metals including Cr, Cd, Cu, Pb, and Zn [6]. These tanning techniques make use of enzymes and chemicals (H2SO4, NH4Cl−, Na2S, and Ca(OH)2). Thus, tannery wastewater from these methods exhibits a bright color and elevated levels of dissolved as well as suspended contaminants, Cr, biological oxygen demand (BOD), and chemical oxygen demand (COD). Cr(VI) toxicity represents a major source of healthcare issues (nausea, vomiting, irritation of the skin, lungs, eyes, allergic reactions, liver and kidney toxicity) associated with tannery wastewater [7, 8]. Moreover, such colorful effluents impede light diffusion, while high COD levels lead to reduced dissolved oxygen (DO) levels throughout the aquatic ecosystem. Watering vegetation with this wastewater contaminates the soil and increases the risk of hazardous accumulation in the nutritional chain [8].

Therefore, this effluent must be cleaned before it can be properly disposed of. There are several physical (reverse osmosis, ultrafiltration, electrocoagulation, and ozonation) and chemical (ammonium salts, flocculation, and coagulation) techniques for treating tannery wastewater, all of which have advantages and disadvantages [8, 9]. For instance, these techniques have produced positive results for total suspended solids (TSS), COD removal, Cr, and turbidity, albeit potentially at the expense of increased sludge production. Similarly, the application of an electrochemical-based oxidation technique to unprocessed tannery effluent proved costly because of the excessive energy requirements that were too high and the negative effects of the effluent upon the electrodes [10]. The main disadvantage of using a membrane is the significant clogging that arises from contaminants capturing, absorbing, and coating the membrane [11]. Microbes and microbial biomasses can be exciting options for industrial wastewater remediation (e.g., bioremediation). The filamentous fungal biomass is able to remediate contaminants such as increased tannery effluent [12].

The advantages of this fungal biomass–based remediation significantly increase the removal of hazardous organic pollutants and heavy metals from effluent [13]. Enzymes such as organophosphorus hydrolases, aminohydrolases, lipase, nitrilases, hydrolases, esterases, and cutinases are produced throughout the fungal life cycle. These enzymes are also frequently detected at specific pollution levels [14]. Moreover, fungal viable or dead biomass has been recognized as a remarkably effective biosorbent substance [15] to remove harmful heavy metals from various industrial effluent and wastewater [16]. Nowadays, nearly all bioremediation studies used to treat wastewater from tanneries use a selective culture that is usually pollutant-specific and can resolve up to two pollutants, primarily Cr [17]. Effluents from tanning operations are typically complex, containing a variety of metals and organic pollutants. Furthermore, because native fungi from contaminated areas have adapted to heavy metal stress conditions, using them in metal remediation may be beneficial [18].

Using individual cells to remove various toxic heavy metals has drawbacks, including decreased cell density, decreased survival, a narrower view of bioremediation, and more challenges with effluent extraction from fungal biomass [19]. Currently, immobilization techniques are being studied to get around these drawbacks. There had been only a few studies that we were able to identify that used both viable and dead native fungus biomasses for tannery wastewater bioremediation, which could explain the study’s uniqueness [20, 21]. To effectively remediate tannery wastewater with fungal biomass, an appropriate carrier material that allows metal-tolerant fungus mycelium to develop while maintaining significant wastewater remediation activities for an extended period is required [6]. Selective metal tolerant Aspergillus sp. biomass-based approaches to treat the leather industry effluent collected from State Industries Promotion Corporation of Tamilnadu Ltd (SIPCOT) region of Erode district of Tamil Nadu is novel report as per the author knowledge.

Fungus immobilization in/on an appropriate carrier seems to represent an effective method for accomplishing this objective. The main objectives of this investigation are as follows:

  • Find sustainable and reproducible approaches to manage the leather industry effluent.

  • Successfulness of approach on tannery effluent management should be analyzed especially with the toxicity factor.

In this investigation, metal-tolerant native fungus live biomass was immobilized on an appropriate carrier to remove the heavy metals from tannery effluent. Furthermore, the phyto- and cytotoxicity of treated effluent were determined by in vitro approach.

2 Materials and methods

2.1 Tannery effluent sampling and characterization

Tannery effluent was acquired from State Industries Promotion Corporation of Tamilnadu Ltd (SIPCOT) region of Erode district of Tamil Nadu, exactly from latitude: 11.37778 and longitude: 77.71324. These areas have been designated as a chronically contaminated zone by the Pollution Control Board. This has now one of the major hub’s for the production of tanned leather in the country. The collected sample (100 L) (American Public Health Association (APHA), 2005) was shifted to laboratory for further studies. The physical and chemical properties like pH, total dissolved solids (TDS), total solids (TS), BOD, COD, and TSS and heavy metals such as Cr, Cu, Pd, Cd, Fe, and Zn were analyzed by adopting the protocol described in APHA, 2005 [22].

2.2 Mass cultivation of pre-identified metal tolerant fungal species

Aspergillus niger and Aspergillus flavus, two fungi known to be metal tolerant (pre-identified and found from metal polluted sites [23] and reported as possess metals tolerant), were subjected in this investigation. The myceliums of these test fungal cultures were cultured separately on the Potato Dextrose Broth (PDB) with typical temperature (at 28 °C) for a week, and this process was repeated until acquired the sufficient biomass for the further experiment. Meanwhile, the antagonism (grow parallel in same medium) nature among these test isolates was investigated by typical petri plate-based assay (Supplementary Fig. 1). Briefly, these test fungal cultures were inoculated on the same PDA containing petri plates and kept for typical incubation process at 28 °C for about a week. Then, the test fungal growth patterns/nature was observed.

2.3 Immobilization of fungal biomass on suitable carrier

After obtaining the mass fungal biomass of each test fungus, each one was individually immobilized using Chau et al.’s methodology, with any necessary modifications [6]. The pomegranate peel and banana peel were used as carrier material for these test fungal cultures. Briefly, well-sliced 10 g of pomegranate peel and 10 g of banana peel were individually sterilized with 1 L of PDB in conical flask. Following sterilization, 10 mL of (each) A. niger and A. flavus spore suspensions were separately inoculated in PDB medium containing pomegranate and banana peels to promote fungal growth over the surface of carrier material as for immobilization. Then, these conical flasks were kept for incubation for 7 days at typical fungal growth temperature at 150 rpm using shaking incubator (40 °C). Later, the fungal biomass with test carrier material was harvested and subjected to dry gradually under RT and then weighed the weight of fungal biomass immobilized on carrier material.

2.4 Heavy metal removal/biosorption potential analysis on leather industry effluent

The heavy metals adsorbing/absorbing efficiency of test fungal biomass immobilized on test carrier materials was investigated through following experimental setups [6] with 1 L of leather industry effluent: Set-I: control (leather industry effluent alone), Set-II: 10 g of A. niger biomass, Set-III: 10 g of A. flavus biomass, Set-IV: 10 g of A. flavus and A. niger biomasses with pomegranate peel, Set-V: 10 g of A. flavus and A. niger biomasses with banana peel. These experimental setups were accomplished in a stirred tank model bioreactor that was additionally supplemented with 1% and 0.01% of glucose as well as NH4NO3, respectively. These experimental setups were maintained for 48 h at typical temperature with magnetic stirrer. Later, the heavy metal contents and other physicochemical properties of treated water were examined with typical protocol [22].

2.5 Treatment efficiency determination

Once the experiment was over, it was discovered that the treatment had significantly changed the metal and other physicochemical properties of treated leather industry effluents were subjected (including control) to phyto- and cytotoxicity study on Vigna radiata and Artemia franciscana larvae, respectively, under in vitro condition with various proportions (25:75, 50:50, 75:25, and 100:00% of treated water:tap water, respectively).

2.5.1 Phytotoxicity analysis

The method reported in Chau et al. was adopted with minor alteration to determine the phytotoxicity of treated water with needed alterations [6]. Briefly, 20 nos of V. radiata (0.1% of HgCl2 sterilized) seeds were placed in clean filter paper containing petri plates (triplicates). This filter paper containing plates with V. radiata seeds was moistened by approximately 6 mL of abovementioned proportions, and separate clean tap water was applied as control. These plates were then kept for incubation using BOD incubator at typical seedling condition for about 2 weeks. The projection of V. radiata seedlings from each test proportions was counted and determined the germination rate.

2.5.2 Cytotoxicity analysis

The Chau et al. described methodology [6] with slight alteration as per the requirement was followed to determine the cytotoxic effects of water collected from each experimental setup with various proportions of treated water (25:75, 50:50, 75:25, and 100:00% of treated water:tap water, respectively) on A. franciscana larvae. Briefly, about 20 nos of A. franciscana larvae was incorporated in plastic container loaded with 20 mL of various proportions of water sample from each experimental setup and kept for a day at RT condition in triplicates. After a day of exposure, the larva viability was counted under microscopic observation and calculated the cytotoxic activity % (mortality %). The ocean water and 100 µg mL−1 of KMnO4 were applied as positive and negative controls, respectively.

3 Results and discussion

3.1 Leather industry effluent quality determination

Table 1 states the physicochemical traits of effluent collected from leather industry; the results clearly state that almost of the properties were beyond the permissible limit described by the pollution control board [24]. The pH was found to be 5.5 ± 0.11; the TDS, TS, DO, BOD, COD, and TSS were found to be 1469.5 ± 14, 3058.4 ± 30, 5.6 ± 0.5, 258 ± 18, 587 ± 22, and 1481.6 ± 34 mg L−1, respectively. The heavy metals such as Cr, Cu, Pd, Cd, Fe, and Zn were found as 15.81 ± 1.1, 6.74 ± 0.49, 4.2 ± 0.21, 3.9 ± 0.17, 7.9 ± 0.38, and 8.6 ± 0.29 mg L−1, respectively. These values were beyond the permissible limits of Indian standard limits. Elevated levels of harmful chemicals in untreated wastewater can lead to significant contamination of groundwater [25]. The effluents from the leather industry represent a significant ecological hazard with a significant potential for pollution of the environment. The wastewater produced by tanning and other processes contains Ca2+, S2+, and NH3−, making it naturally somewhat acidic [26]. H2SO4, Cr, Cl−, NaHCO3, and SO42− were all found in scraping as well as heavy metal–enriched leather industry effluents. The process of tanning is probably responsible for these harmful substances. Many health problems have been documented in people who engage in the leather product sectors, notably skin as well as respiratory conditions brought on by this hazardous combination of chemicals [27, 28]. Both surface and groundwater are contaminated by the effluent, which can lead to respiratory disorders, kidney disease, and skin conditions like boils, itching, and diminished feeling, among other things [29, 30].

Table 1 Physicochemical characteristics of leather industry effluent
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3.2 Antagonistic and mass cultivation of A. niger and A. flavus

Using PDB, A. niger and A. flavus were cultivated in large quantities, producing notable biomass yields of up to 12.3 g and 14.1 g, respectively. The results obtained from the typical PDA-based plate assay revealed that the antagonistic properties of A. niger and A. flavus. The assay’s results indicate that A. niger and A. flavus grow parallel to one another in a PDA plate without exhibiting any antagonistic characteristics. This clearly indicates that A. niger and A. flavus can coexist and grow altogether on same growing medium. This may be the result of the fact that both fungal species were discovered in metal-polluted environments and have the ability to tolerate metals [23]. Metal-tolerant fungi may contain a large number of enzymes such as amylases [15, 31], laccases, catalases, lipases, xylanases, proteases, and peroxidases, which contribute to the reduction, breakdown, or detoxification of the hazardous substances in the aquatic ecosystem [32, 33]. According to this, a fungal consortium is more capable than a single individual of demonstrating an efficient bioremediation technique for metals as well as dye decomposition [33]. Furthermore, even when exposed to high concentrations of toxic substances, the combination of multiple fungal species degrades, detoxifies, and removes the heavy metals and other pollutants efficiently [34].

3.3 Test fungal biomass immobilization

For successful treatment of leather industry wastewater using test fungal biomass immobilized upon firm organic substances such as pomegranate peel and banana peel can able to grow while retaining significant pollutant removal from effluent for an extended period of time. Immobilization of fungal biomass on a suitable supporting substance is a promising method for achieving this goal. Organic support substances, including pomegranate peel and banana peel, exhibited significant immobilization, as well as fungal biomass, and are able to utilize the material that supports it as an alternative form of energy/nutrition. The degree of elongation between the carrier material and mycelia allowed for better test fungal growth along with improved O2 as well as nutritive transfer effectiveness. In addition, using a carrier might offer relatively hydrodynamically steady-state circumstances, reducing hydrodynamic shear forces on such test fungi mycelia. Immobilization of A. niger and A. flavus on pomegranate peel and banana peel contained PDB media revealed that both of these species had been successfully immobilized on such solid pomegranate peel and banana peel. The active immobilization was identified by measuring pomegranate peel and banana peel after a day of colonization and then comparing it with the original weight. Remarkably, the test A. niger and A. flavus immobilized pomegranate peel and banana peel up to 82% as well as 86%, correspondingly. A research team similarly immobilized Candida antarctica on coconut fiber as well as demonstrated significant heavy metal adsorption/removal from polluted water and industry effluent [35]. Immobilization improves staying alive, coherence, and retention in A. niger and A. flavus. Immobilization has begun to grow effectively in treating the industry effluent since it provides better results compared to free microbial agents and sometimes single enzyme activity [36, 37]. Immobilized fungal cultures are now preferred over traditional microbial approaches due to inherent metabolic dependability, increased productivity, sustainability, and remarkably inexpensiveness [38].

3.4 Heavy metals and other pollutant removal

The heavy metal and other pollutant removal competence of test fungal biomass and their immobilized form on pomegranate peel and banana peel solid substrate were determined by various experimental setups (Set-I to Set-V). Upon the completion of experimental condition, the physicochemical properties were analyzed and found that Set-V showed remarkable, optimistic alterations in the treated water bioreactor-based study revealed that the fungal biomass immobilized pomegranate peel and banana peel demonstrated remarkable heavy metals and other pollutants removal potential and it followed by Set-IV, Set-II, and Set-III. The most important properties such as pH and DO level were optimistically altered up to 2.3, 1.9, 1.1, 1.4, and 0.1 (Fig. 1) and 7.9, 6.0, 1.8, 2.3, and 0.3 mg L−1 (Fig. 2) in Set-V, Set-IV, Set-III, Set-II, and Set-I treated water, respectively. The increased DO and pH level in the treated water was considered optimistic sign of effective treatment. Other properties such as TDS, TS, BOD, COD, and TSS content of treated water sample from Set-V and Set-IV were considerably reduced up to 394 and 343 mg L−1, 417 and 356 mg L−1, 122 and 106 mg L−1, 165 and 129 mg L−1, and 403 and 356 mg L−1, respectively (Fig. 3a), than other experimental setups such as Set-III, Set-II, and Set-I. Similarly, the experimental setups Set-V and Set-IV showed considerable heavy metals removal from treated leather industry wastewater sample. These experimental setups removed the metals such as Cr, Cu, Pd, Cd, Fe, and Zn up to 6.81 and 4.81, 3.64 and 3.44, 1.8 and 1.3, 2.4 and 1.8, 3.1 and 2.7, and 3.9 and 3.2 mg L−1, respectively (Fig. 3b). These reduced values were significantly higher than the other experimental setups. On the other hand, this can be revealed as A. niger and A. flavus biomass immobilized over the surface of pomegranate peel and banana peel solid support materials effectively reduced/removed considerable quantity of metals and other pollutants from the leather industry effluent (Table 2). Acclimatization of test fungi biomass in wastewater resulted in a significant reduction in pollutants in a short period of treatment [39]. The majority of COD reduction can be credited to fungal biomass–based mineralization and the consumption of highly oxidizable components [6]. In an acidic environment, biological accumulation was discovered to serve as the main mechanism responsible for removing pollutants through immobilized A. niger and A. flavus biomass over the surface of solid support material. Chitin from fungal cell walls and chitosan both possess functional groups including amino, acetanilide, and hydroxyl groups [40]. The process in acidity condition repaired all of these functional molecules, while the contaminants’ negative sulfonyl radicals became trapped to achieve equilibrium in the context of a water-based setting [41]. Hence, at acidic conditions, the biosorbent substances have a greater proclivity for absorbing additional anionic pollutants [15]. Lowering pH values favored the attraction of oxyanionic metal ions to positive charge functional elements found on A. niger and A. flavus biomass cell membranes [42]. Metal absorption occurs through a variety of mechanisms, including cell wall binding, internal accumulation, external volatilization, and precipitation [15]. Metal sorption mechanism can be categorized based on its reliance upon the fungi cell’s metabolic processes which is referred to as metabolism reliant or in accordance to the site where the metal sorbed from water is discovered which is referred to as metabolism independent including external precipitation or accumulation, other mechanisms like volatilization, detoxification, and chelation occurred [6, 43]. Biosorption via inactivated fungal biomass constitutes a passive adsorption mechanism exclusively through cell surface adsorption/binding, whereas viable fungal biomass absorption is a dynamic process that involves inside as well as outside metabolic processes including detoxification, bioaccumulation, chelation, and volatilization occurs [44]. Furthermore, a reduction in Cl−, Na+, and NO3− has been attributed to fungi that use these elements for proliferation [45]. Similarly, Ca2+ is an important intracellular signaling messenger for the development of cells as well as maintaining cytosolic accessible Ca2+ around 0.1 M through a variety of transportation processes located on plasma and vacuolar membranes which affect influx/efflux and vacuolar compartmentalization [46]. Dealings with Ca2+-binding protein may influence cytoplasmic-free Ca2+. The metal-tolerant fungal biomass immobilized on the corncob and coir progressively adsorbed the harmful substances from the leather industry effluents via the adsorption mechanisms [6, 43].

Fig. 1
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Optimistic alteration in pH of treated water sample under different experimental setups

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Fig. 2
figure 2

Considerable raise in the DO level in treated water sample under different experimental setups

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Fig. 3
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Quantity of various physicochemical parameters including heavy metals removed from test water sample. a Parameters such as TDS, TS, BOD, COD, and TSS. b Heavy metals

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Table 2 Physicochemical characteristics of pre- and post-treated water sample
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3.5 Treatment efficiency determination by toxicity analysis

3.5.1 Phytotoxicity analysis

Table 3 and Fig. 4 showed the percentage of germination and phytotoxicity, respectively, on V. radiata seedling exposed to water derived from each experimental setups (Set-I–Set-V). Interestingly, the Set-V and Set-IV treated water at all dilution ratio (25:75%, 50:50%, 75:25%, and 100:00%) showed 100% germination and 0% toxicity on V. radiata seedling. The treated water from Set-III and Set-II showed reduced germination as 70 and 60% at 25:75% and 40 and 10% at 100:00%, respectively. The phytotoxicity was not found in Set-V and Set-IV water-exposed V. radiata seedling [47]. Remaining experimental setup treated water samples showed certain percentage of toxicity on growth of V. radiata seedling (Fig. 4). Ultimately, fungal biomass immobilized pomegranate peel as well as banana peel exhibited significantly lower toxicity in leather industry effluent (treated) than non-immobilized A. flavus and A. niger biomass. Unprocessed (untreated) wastewater had no V. radiata seed sprouting, indicating that the wastewater that was untreated remained extremely harmful thereby completely inhibiting V. radiata germination. A research team stated that the Penicillium commune and Cladosporium perangustum have a greater capacity for tanning facility wastewater treatment when immobilized on organic solid support substances than non-immobilized fungal biomass, reducing leather wastewater toxic effects and supporting the establishment of Triticum aestivum on remediated effluent [48]. Additionally, they discovered that wastewater that was untreated was harmful to seed sprouting. The detrimental impact of unprocessed leather industry effluent on crop seedlings could be attributed to its increased heavy metal content, which also promotes osmotic pressure under anaerobic environments [49]. Because under raised osmotic pressure as well as anaerobic circumstances, various physiological and biochemical mechanisms of sprouting and growth, such as solute movement, rate of respiration, as well as seed germination, as well as developmental parameters, occur [44].

Table 3 V. radiata germination % under the exposure of treated effluents under various experimental setups
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Fig. 4
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Phytotoxicity (seed germination inhibition) effect on V. radiata exposed to treated water samples from various experimental setups

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3.5.2 Cytotoxicity analysis

Table 4 and Fig. 5 exhibited the viability % of A. franciscana larvae and cytotoxicity % which was subjected to water sample acquired from each experimental setup. Set-V as well as Set-IV treated water exhibited 100% larvae viability and 0% cytotoxicity on A. franciscana larvae growth regardless of all dilution ratios (25:75%, 50:50%, 75:25%, and 100:00%). Set-III as well as Set-II water that were treated exhibited lower larvae viability of 80 and 75% at 25:75% as well as 40 and 35% at 100:00%, correspondingly. The cytotoxicity of A. franciscana larvae growth subjected to treated water in Sets V as well as IV was not determined. The remaining experimental setup treated water samples demonstrated a particular level of cytotoxicity in A. franciscana larvae growth (Fig. 5). A research investigation discovered that fungal biomass–based biosorption substantially cleanses the hazardous chemicals present in leather industry effluent during biological remediation/biosorption/adsorption, as evidenced by reduced toxicity on crops and aquatic fauna [50]. Hazardous materials, like pollutants that are both inorganic and organic, are able to accumulate and diffuse throughout the food, having catastrophic effects on organisms [51]. As a result, it is critical to assess the negative effects of treated leather industry effluent as well as cereal cultivars’ compatibility with agricultural irrigation [52]. The transition from dormancy to reproduction represents a critical stage in the developmental process of flora and fauna, since it governs trends in population as well as effectiveness, leading to an important ecological as well as industrial aspect [53]. Similarly, the microbes along with microbial products effectively removed the heavy metals and other pollutants from industrial effluents like leather industry effluents, leading to less cytotoxicity on A. franciscana larvae than untreated effluent [54]. A research investigation looked at sprouting seeds, growth, and biological characteristics to assess germination and the early growth stages that govern plant adaptations towards treated and untreated effluents [55]. Untreated harmful elements enriched effluents significantly reduced sprouting seeds and accelerated the progression of V. radiata as well as caused A. franciscana larvae fatality. Adsorbents containing active fungal biomass can remove a significant amount of harmful metals and other pollutants from effluent and thus reduce phytotoxicity by increasing chlorophyll and biomolecule content, as well as certain vital protein concentrations, when exposed to treated as well as untreated water samples [56, 57].

Table 4 Viability of A. franciscana larvae exposed to treated water sample from experimental setup
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Fig. 5
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Cytotoxicity effect of treated water sample from various experimental set up on A. franciscana larvae

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4 Conclusion

Leather wastewater comprises harmful elements that can kill microbes and organisms found in water, fauna, flora, and human beings, as well as induce genotoxicity, genetic modification, and cancer-causing potential. As a result, the sustainable remediation approach, methods, or agents required for effluent management must be discovered. The results show that the filamentous A. flavus and A. niger produced a significant amount of biomass and, when immobilized on organic solid support materials including pomegranate peel and banana peel, have a high ability to remove heavy metals and other pollutants from leather industry effluent (adsorption/absorption). Surprisingly, this fungal biomass immobilized on pomegranate peel and banana peel, as well as their high pollutant sorption potential from leather industry effluents, had no harmful impact on V. radiata and A. franciscana larvae. This also endorses seed growing at unique proportions, suggesting this substance decreases toxic effects (phyto- and cytotoxicity) while promoting germination of crops and larvae growth. The findings conclusively show that the filamentous A. flavus and A. niger have significant remediation capability when immobilized. To determine the viability of this strategy on leather industry effluent, a real-world evaluation is required, and this will be the investigation’s future focus.