Biosorption of Pb(II) and Cd(II) by Endophytic Fungi Isolated from Eichhornia crassipes: Screening, Identification, and Adsorption Performance

Lead (Pb) and cadmium (Cd) are among the most hazardous heavy metals released into the environment as a result of anthropogenic activities, including industrial processes, mining, agricultural runoff, and domestic waste discharge. Cadmium is primarily associated with battery, pigment, and plastic production, whereas lead is commonly derived from smelting activities, mining, and the degradation of lead-based materials [1]. These metals tend to accumulate in industrial wastewater and subsequently contaminate freshwater systems [2, 3]. Although natural processes such as geological weathering contribute to their presence, human activities remain the dominant source of elevated concentrations [4]. The persistence and non-biodegradable nature of Pb and Cd result in severe ecological impacts, including physiological disturbances in aquatic organisms, biodiversity loss, and significant health risks to humans through bioaccumulation and biomagnification in the food chain [1, 5].

To address these challenges, various remediation technologies have been developed, including chemical precipitation, ion exchange, membrane filtration, and adsorption. However, these conventional methods often suffer from limitations such as high operational costs, sludge generation, and reduced efficiency at low metal concentrations. As a result, bioremediation approaches, particularly biosorption, have gained increasing attention as environmentally friendly and cost-effective alternatives for heavy metal removal. Biosorption utilizes biological materials to bind and remove metal ions through physicochemical interactions, offering advantages such as low cost, high efficiency, and minimal secondary pollution [6, 7].

Among biological agents, fungi have been widely reported as effective biosorbents due to their unique cell wall composition. Fungal cell walls contain biopolymers such as chitin, chitosan, and melanin, which provide abundant functional groups, including carboxyl, hydroxyl, amine, and phosphate, that facilitate metal binding [6, 7]. The biosorption process involves both passive (non-metabolic) mechanisms, such as ion exchange and complexation, and active (metabolic) processes, including intracellular accumulation mediated by compounds such as metallothioneins and phytochelatins [8, 9]. Several genera, including Aspergillus, Penicillium, and Rhizopus, have demonstrated high biosorption capacities for various heavy metals [10, 11].

Despite these advances, studies focusing on endophytic fungi that live symbiotically within plant tissues remain limited, particularly those isolated from aquatic plants. Endophytic fungi may possess enhanced tolerance to environmental stress and metal exposure due to their close association with host plants growing in contaminated habitats. However, research exploring endophytic fungi from a single aquatic plant source, such as Eichhornia crassipes, for the simultaneous removal of Pb and Cd under controlled conditions is still scarce. This gap highlights the need for a systematic investigation into their biosorption potential and comparative performance.

Therefore, this study aims to isolate and identify endophytic fungi from Eichhornia crassipes and to evaluate their biosorption performance for Pb(II) and Cd(II) removal using a single-metal system. The study further compares the adsorption efficiency of Aspergillus sp., Penicillium sp., and Rhizopus sp. at different metal concentrations. The findings are expected to provide scientific insights into the development of efficient, economical, and sustainable bioremediation technologies, while also promoting the utilization of locally available aquatic plants for mitigating heavy metal pollution in tropical environments.

3.1 Isolation and identification of endophytic fungi

The isolation of endophytic fungi from the leaves of Eichhornia crassipes (water hyacinth) was successfully performed using tissue culture techniques on Potato Dextrose Agar (PDA) medium. The macroscopic morphological observations showed colonies with distinct colors, textures, and growth rates for each isolate, as presented in Table 1.

The EC1 isolate displayed irregular colonies with umbonate (domed) elevation, smooth and fluffy surfaces, and a black-and-white coloration. Microscopic examination revealed round conidia with thick walls and septate hyphae, characteristic of Aspergillus sp. These results are consistent with the findings of Mawarni et al. [16], who reported that Aspergillus sp. colonies typically appear black, with spherical conidia and septate, branched hyphae. Similarly, another study described the macroscopic characteristics of Aspergillus colonies on PDA as light to dark green, sometimes turning black with a cream-colored base and a powdery texture [17].

The EC2 isolate, obtained from the roots of water hyacinth, exhibited irregular and flat colonies with a smooth surface, white coloration, and yellow pigmentation. Microscopic observation showed thin-walled, round conidia with septate hyphae, identifying the isolate as Penicillium sp. Penicillium colonies commonly appear in yellowish-green to bluish-green shades, often releasing yellow or hyaline exudates. The conidiophores form vesicles, and the conidia are typically round to elliptical in shape, with fast-growing, flat, wool-like textures [18].

Table 1. Macroscopic and microscopic observation results of the isolates

The EC3 isolate exhibited rhizoid-shaped colonies with convex elevation, smooth surface, and grayish-white color. Microscopic examination revealed non-septate hyphae and thin-walled, spherical sporangiospores, identifying the isolate as Rhizopus sp. This species is characterized by coenocytic hyphae (multinucleate and aseptate), grayish-white colonies, and the presence of stolons and dark rhizoids [19, 20]. The sporangium is spherical, supported by a smooth sporangiophore opposite the rhizoid, and terminates in a rounded spore head [20].

Microscopic observations reinforced these identifications. Aspergillus species showed branched conidiophores ending in vesicles with radially arranged conidia. Penicillium species displayed brush-shaped conidiophores with phialides bearing conidia. Rhizopus species were characterized by broad aseptate hyphae and spherical sporangia located at the tips of sporangiophores with visible rhizoids [21-23]. These results are consistent with morphological approaches commonly used in fungal taxonomy [24, 25].

3.2 Biosorption efficiency for lead (Pb)

The variation in heavy metal concentration significantly affected the adsorption capacity of the fungi. This phenomenon occurs because the number of active sites on the fungal cell wall is limited, determining the amount of metal ions that can be adsorbed at each concentration level. At lower concentrations, the active binding sites remain abundant, allowing metal ions to interact easily with functional groups such as carboxyl (-COOH), amine (-NH₂), and hydroxyl (-OH). As a result, adsorption efficiency is relatively high since most of the metal ions are captured by the fungal biomass.

Table 2. Absorption of heavy metal lead (Pb) by endophytic fungi

The biosorption results of heavy metal lead (Pb) are summarized in Table 2. An apparent inconsistency was observed in the biosorption results of Aspergillus sp., where the residual Pb concentration at an initial concentration of 20 mg/L (0.10 mg/L) was lower than that at 10 mg/L (0.12 mg/L). This trend deviates from the typical expectation that higher initial concentrations result in higher residual metal levels. One possible explanation is that the biosorption process reached an optimal interaction condition at intermediate concentration (20 mg/L), where the availability of Pb ions and the accessibility of active binding sites were balanced, leading to more efficient adsorption. At lower concentrations, the driving force for mass transfer may be insufficient to fully utilize available binding sites, whereas at higher concentrations (30 mg/L), partial saturation of adsorption sites may begin to occur, reducing overall efficiency.

In addition, it is possible that supplementary removal mechanisms, such as precipitation or surface complexation, contributed to the reduction of Pb concentration in solution. These physicochemical processes may occur alongside biosorption, particularly under controlled pH conditions, and can enhance the apparent removal efficiency. However, due to the absence of detailed mechanistic analysis in this study, these explanations remain speculative and should be verified in future studies through more comprehensive experimental design and analytical techniques.

Figure 1. Comparison of lead (Pb) by Aspergillus sp., Penicillium sp., and Rhizopus sp.

Based on Figure 1, the highest biosorption efficiency of lead (Pb) was observed in Aspergillus sp. at an initial concentration of 20 ppm, reaching 99.52%, whereas the lowest adsorption was recorded in Rhizopus sp. at 30 ppm (90.12%). This non-linear trend supports the observation that optimal biosorption may occur at intermediate concentrations, where the interaction between Pb ions and available binding sites is maximized. The superior performance of Aspergillus sp. is consistent with previous studies reporting that species such as Aspergillus piperis exhibit high biosorption capacity for heavy metals, including lead. This capability is generally attributed to the composition of the fungal cell wall, which contains functional groups such as carboxyl and phosphate that act as active binding sites for metal ions. However, considering the exploratory nature of this study and the absence of replication, these findings should be interpreted as preliminary, and further validation is required to confirm the observed trends and adsorption performance [26].

The differences in biosorption efficiency are associated with variations in the structural and chemical properties of each fungal species. Aspergillus niger possesses cell walls rich in carboxyl and hydroxyl groups, enhancing its ability to adsorb metal ions [27]. Penicillium sp. has complex conidiophores and large colony surfaces that facilitate metal binding. Although Rhizopus sp. shows relatively lower efficiency compared to Aspergillus, it remains an effective and easily cultivable biosorbent, making it suitable for large-scale bioremediation applications [28].

3.3 Biosorption efficiency for cadmium (Cd)

As shown in Figure 2, the highest cadmium (Cd) biosorption was recorded for Rhizopus sp. at 20 ppm, achieving 98.83%, while the lowest was found in Penicillium sp. at 10 ppm with 95.03%. Biomass from Rhizopus oligosporium was reported to effectively reduce cadmium concentrations by 97.27% within 24 hours [29]. This efficiency is mainly attributed to the presence of functional groups such as amine, hydroxyl, and carboxyl, which play crucial roles in metal ion binding. Meanwhile, Penicillium sp. also shows biosorption potential; P. oxalicum was reported to adsorb Cd with a 71% reduction after 96 hours [30]. These findings suggest that Penicillium species tend to exhibit lower adsorption capacity compared to Rhizopus sp., possibly due to differences in cell wall structure and the presence of active functional groups involved in biosorption [31]. As shown in Table 3, the biosorption efficiency of cadmium (Cd) varied among the tested fungi.

Figure 2. Comparison of cadmium (Cd) by Aspergillus sp., Penicillium sp., and Rhizopus sp.

Table 3. Absorption of heavy metal cadmium (Cd) by endophytic fungi

Hydroxyl groups have been identified as the main contributors to enhancing biosorption capacity, as also observed in microbes such as Bacillus megaterium and Rhodotorula sp. [32]. Rhizopus sp. can remove significant amounts of Cd from contaminated media, as reported by Douglas et al. [33].

3.4 Comparative efficiency and bioremediation potential

Based on the biosorption efficiency results obtained through AAS, Aspergillus sp. exhibited the highest removal efficiency for Pb, whereas Rhizopus showed relatively better performance in Cd removal. This divergence suggests that biosorption in this study is not governed by a uniform mechanism but rather by species-specific surface properties that determine selective metal affinity. Such selectivity indicates that fungal biosorption should be interpreted as a competitive interaction between metal ions and heterogeneous binding sites, rather than a generalized adsorption process.

A critical limitation of this study is the absence of spectroscopic characterization (e.g., Fourier Transform Infra Red (FTIR)), which restricts direct identification of the functional groups involved. Therefore, any mechanistic interpretation must be treated as inferential rather than confirmatory. Nevertheless, previous studies have consistently demonstrated that fungal biomass contains multiple reactive sites capable of binding heavy metals, supporting the plausibility of the observed biosorption trends [34, 35]. Within this constraint, the present study contributes empirical evidence of biosorption performance but does not claim molecular-level validation.

Importantly, the use of dried fungal biomass strongly indicates that the dominant mechanism in this study is passive biosorption rather than metabolically driven active transport. This distinction is crucial, as passive biosorption is governed primarily by physicochemical interactions such as ion exchange and surface complexation, which are highly dependent on the structural composition of the fungal cell wall [36]. Consequently, differences in biosorption efficiency among Aspergillus, Penicillium, and Rhizopus should be interpreted as a function of surface heterogeneity rather than biological activity.

The higher Pb uptake by Aspergillus sp. can be critically linked to its potentially higher density or accessibility of binding sites with stronger affinity toward Pb²⁺ ions. Pb, with its larger ionic radius and higher polarizability, tends to form more stable complexes with available binding groups compared to Cd. In contrast, the relatively higher Cd removal by Rhizopus suggests that its cell wall structure may provide binding environments more favorable for Cd²⁺ interaction. This indicates that biosorption efficiency is not solely dependent on the number of binding sites, but also on their chemical compatibility with specific metal ions. Similar species-dependent selectivity has been reported in fungal biosorption studies, emphasizing the role of physicochemical compatibility in adsorption processes [34, 37].

Furthermore, these differences can be critically associated with variations in fungal cell wall composition, particularly the relative proportions of chitin, chitosan, glucans, and other structural polymers. These components create a heterogeneous matrix of binding sites with varying affinities and reactivities. In some fungi, additional compounds such as melanin may further enhance metal binding by introducing extra coordination sites. Therefore, the observed differences between Aspergillus and Rhizopus are likely rooted in intrinsic structural variability rather than experimental conditions alone [38].

However, without direct compositional or spectroscopic evidence, this explanation remains hypothetical and highlights a key research gap. Future studies integrating FTIR, Scanning Electron Microscope-Energy Dispersive X-ray Spectroscopy (SEM-EDX), or surface chemistry analyses are necessary to validate the proposed mechanisms and to quantify the contribution of specific functional groups. This is particularly important for advancing fungal biosorption from empirical observation toward predictive and design-oriented applications.

Despite these limitations, the present findings remain significant in demonstrating that all three fungal isolates possess practical biosorption potential. Moreover, the relatively faster growth and adaptability of Rhizopus suggest an operational advantage in time-sensitive remediation systems, even if its maximum adsorption capacity is lower than that of Aspergillus. This trade-off between adsorption efficiency and growth kinetics should be considered in designing large-scale bioremediation strategies.

In a broader context, fungal biosorption represents a robust and adaptable approach to heavy metal remediation, supported by extensive prior research on fungal tolerance and metal-binding capacity [29, 37]. However, to transition from laboratory-scale feasibility to real-world application, future work must address current limitations by integrating mechanistic validation, process optimization, and scalability assessment.