The solution pH affects the speciation of metal ions, and also influences the surface properties of the sorbents ^[38][41], affecting the dissociation of binding sites and their surface charge, the activity of functional groups (mainly carboxylate, phosphate, and amino groups) in the biomass ^[54], as well as the competition of metallic ions with hydronium ions for the adsorption sites ^[38].

The effect of pH in biosorption processes depends on the strain and the metal ion to be removed. Binupriya et al. ^[39] evaluated the biosorption of metal ions in aqueous solutions with pellets of Aspergillus japonicus; in the case of Ni(II), the removal increased as the pH increased from 2 to 10, reaching a maximum removal of 100% at pH 9. In the case of Fe(II), the removal improved as the pH increased from 2 to 6; at pH 6, the removal was 100%, and when the pH was extended above this value and until pH 10, the removal remained higher than 90%. This trend has also been reported for Zn removal by Phanerochaete chrysosporium pellets ^[45], as the adsorption capacities increased as the pH values rose from 2 to 7. The maximum removal efficiencies (48.6 ± 4.5%) were observed at pH 7.0, which was attributed to the dissociation of protons from the carboxylic acid group, one of the major components of the fungal cell wall. As the pH of the aqueous solution increased, the dissociation of protons also augmented, as well as the presence of negatively-charged groups in fungal surface and therefore the number of binding sites available for complexation of metal cations ^[45]. Concerning the removal of U(VI) with Funalia trogii pellets, the optimum pH was 5, which was attributed to the presence of ligands such as amino, carboxyl, and phosphate groups on the surface of the pellets at this pH value ^[55]. The authors indicated that the decrease in the amount of metal absorbed at pH higher than 5 was due to the formation of uranyl carbonate complexes ^[55]. Concerning the biosorption of Cu, it has been reported that when pH value is higher than 5, the adsorption on fungal pellets decreases due to the precipitation of copper hydroxide ^[34][40]. As most metals complex and precipitate at alkaline pH values, biosorption is usually evaluated in a 2–7 pH range to attribute the metal removal only to the biosorption process.

On the other hand, at very acid pH values, low metal adsorption capacities have been reported, which is mainly attributed to the hydronium ions competing with the metal ions for the binding sites. For instance, the removal of Cu, Ni, and Zn is negligible at pH 3, due to the effects of the competition between the cations and the hydronium ions for the biosorption sites in Aspergillus niger pellets ^[38]. In the case of Cu, a low biosorption capacity was observed at pH values below 3.0 when alkali-pretreated and viable pellets of Rhizopus oryzae were tested ^[56]. In column experiments, Gabriel et al. ^[34] reported a low Cu biosorption capacity with pellets of Pleurotus ostreatus at pH 3 (1.92 mg·g⁻¹) compared to the value obtained at pH 4 (7.92 mg·g⁻¹). Many authors agree that the pH optimum for Cu biosorption is at 4–5 pH range ^[36][38][56]. Mishra and Malik ^[36] obtained the best metal uptake yields (79.8% and 77.2%) at pH 4 and 5, respectively, with Aspergillus lentulus pellets. For Rhizopus oryzae pellets, the maximum Cu adsorption capacities (52.91–61.73 mg·g⁻¹) were reported at pH 4 ^[40]. In another study, the maximum Cu removal with alive pellets of Phanerochaete chrysosporium (50%) and with Funalia trogii pellets (61%) was obtained at pH 5 ^[40].

In the case of Cr(VI) biosorption, the removal by Aspergillus japonicus pellets decreased from 100 to 40% as the pH value increased from 2 to 10 Biosorption of meta ^[39]. Filipović-Kovačević et al. ^[38] reported that the biosorption of Cr(VI) on Aspergillus niger pellets followed this same trend, since the maximum capacity of adsorption was obtained at pH 2 and then it decreased as the pH increased from 2 to 7. This was explained by the speciation of Cr(VI), which predominates as HCrO₄⁻ at concentrations less than 500 mg·L⁻¹ and low pH values, although another species as Cr₂O₇²⁻, Cr₃O₁₀²⁻, and Cr₄O₁₃²⁻ coexist in acid media ^[57]. As all these Cr(VI) species are negatively charged, a decrease in pH leads to a higher protonation of the fungal surface, creating a stronger attraction between the adsorbate and the biosorbent. Therefore, as the pH increases, the surface charge of the fungal pellets becomes negative, leading to weak bondings with the negatively-charged Cr species. Besides, in alkaline environments other negative ions such as OH^- are likely to compete with the anion predominant at higher pH values (CrO₄²⁻) for the biosorption on the fungal biomass ^[57][58].

Finally, several works report a negligible effect of pH in the biosorption process. It is the case of As(III), for which almost complete removal was obtained at several concentrations of both Phanerochaete chrysosporium pellets and adsorbate, while a negligible effect of pH values comprised between 5 and 9 was observed ^[44]. In preliminary experiments with Pb(II) and Aspergillus lentulus pellets, Mishra and Malik ^[36] concluded that pH did not significantly alter the metal uptake.

Another important parameter in the biosorption of metals with fungal pellets is the initial metal concentration in the solution. On the one hand, if the metal biosorption and bioaccumulation of the metal are being evaluated in live pellets, its initial concentration can affect the growth of the pellets ^[36]. On the other hand, the initial concentration of metal ions in the solution plays a key role as a driving force to overcome the mass transfer resistance between the aqueous phase and the biosorbent ^[59].

Mishra and Malik ^[36] evaluated the growth of Aspergillus lentulus pellets in presence of Cu(II), Cr(III), Ni(II), and Pb(II). A lower biomass production was observed, compared to a control, when metals were added. The biomass growth reduced 19% in presence of 70 mg Ni²⁺·L⁻¹; this reduction was of 76% when the metal concentration increased at 140 mg·L⁻¹. In the same study, after five days of growth, the addition of 80 mg Cu²⁺·L⁻¹ reduced 16% the pellets’ biomass, while this reduction was of 77% at 800 mg Cu²⁺·L⁻¹.

The metal uptake capacity of Aspergillus lentulus was also evaluated as a function of the initial concentration of metal ions (Cu(II), Cr(III), Ni(II), and Pb(II)) in the medium ^[36]. The removal of Cu(II) and Ni(II) ions was enhanced by increasing the initial metal ion concentration. The maximum specific metal uptake was determined as 124.5 mg·g⁻¹ (at 800 mg·L⁻¹) for Cu(II), as 11 mg·g⁻¹ (at 140 mg·L⁻¹) for Ni(II), 331.5 mg·g⁻¹ for Cr(III), and 1120.6 mg·g⁻¹ for Pb(II) (at 4000 mg·L⁻¹ for both metal ions) ^[36]. A high initial concentration provides an increased driving force to overcome all mass transfer resistance of metal ions between the aqueous and solid phase, resulting in a higher probability of collision between metal ions and biosorbents ^[36][59].

The biosorption capacity of Pb(II) (1120.6 mg·g⁻¹) obtained with live Aspergillus lentulus pellets ^[36] is 70-fold higher than the values obtained with live pellets of Phanerochaete chrysosporium, namely 16 mg·g⁻¹ [68], and around 120-fold higher than the capacity (9.0 mg·g⁻¹) measured by Yetis et al. ^[48]. By using alkali-pretreated pellets of the same strain, low Pb(II) biosorption capacities were reported (15.2–23.7 mg·g⁻¹ and 20.1–48.2 mg·g⁻¹, respectively) ^[47][48]. This differences between adsorption capacities are mainly due to the initial metal concentration used (50 mg·L⁻¹) in these works ^[47][48] compared to the (4000 mg·L⁻¹) evaluated by Mishra and Malik ^[36].

An increase in the Ni concentration (from 100 to 500 mg·L⁻¹) resulted in an approximately fivefold increase in the biosorption capacity of this metal onto Rhizopus arrhizus pellets from 61.2 to 348.8 mg·g⁻¹. However, the biosorption capacity decreased when the nickel concentration exceeded 500 mg·L^{−1 [51]}.

Some general comments on the literature reviewed so far can be made. First of all, research work concerning the evaluation of fungal pellets for the biosorption of low concentrations of metal ions is scarce. The concentrations being evaluated should be as realistic as possible, and for some metals (i.e., precious metals or radionuclides) their concentrations in wastewater are hardly higher than 1–10 mg·L^{−1 [44]}. Besides, other compounds that can intervene negatively in the biosorption processes of metals should be more explored, such as the most common anions present in groundwater and industrial effluents (i.e., chlorides, nitrates, fluorides, phosphates, and sulfates) ^[38]. In the Section 2.1.6, the few articles that have reported the interference of these anions and their competition with different metal ions will be discussed.

The pellet diameter determines the surface area of the biosorbent, which is a key factor in biosorption processes. It also determines the number of metal binding functional groups readily exposed to the metal ions in solution. Fu et al. ^[40] evaluated the effect of the diameter of Rhizopus oryzae pellets on the removal of Cu(II) at pH 4 and an initial concentration of 100 mg·L⁻¹. The adsorption capacity decreased from 37.1 to 18.4 mg·g⁻¹ when the diameter of the pellet increased from 0.4 mm to 2.0 mm. This was attributed to a decrease in the surface area, which was responsible for a lower availability of exposed binding sites in biomass of larger diameter. When the diameter was higher than 1.2 mm, the autolysis of the pellets was likely to further decrease the availability of metal-binding functional groups.

Li et al. ^[46] evaluated the effect of pellets diameter (from 1.32–1.57 mm to 3.5–5.50 mm) on the Cd adsorption on Phanerochaete chrysosporium, at pH 4.5, 25 °C, and an initial metal concentration of 50 mg·L⁻¹. The maximum Cd uptake (15.2 mg·g⁻¹) was obtained when the pellet diameter was comprised between 1.58 and 2.03 mm. As for Fu et al. ^[40], the adsorption capacity decreased to 9.86 mg·g⁻¹ when the diameter of the pellet increased to 3.5–5.5 mm.

Gabriel et al. ^[34] also studied the Cu biosorption by pellets of different strains, namely Aspergillus carbonarius, Lepista nuda, Oudemansiella mucida, Phanerochaete chrysosporium, Pleurotus ostreatus, and Pycnoporus cinnabarinus, among others. Although the variety of conditions at which the adsorption experiments were carried out hinders the comparisons, it is noteworthy that some of the reviewed papers about Cu biosorption report the same environmental conditions, i.e., pH 4, 25 °C, and a stirring rate of 50 rpm. In these conditions, the highest adsorption capacities were observed for the following fungal species: O. mucida (8.8 mg·g⁻¹), L. nuda (6.3 mg·g⁻¹), P. cinnabarinus (5.1 mg·g⁻¹), and P. ostreatus (4.8 mg·g⁻¹). The differences in these values were attributed to dissimilarities in the cell wall composition and to the physical properties of the pellets. From a biotechnological point of view, the fungal pellets of O. mucida, P. chrysosporium, and P. ostreatus were considered as having the best mechanical properties among the species studied ^[34]. These pellets had a size big enough for handling (3–7 mm) but at the same time with an adequate surface area. Thus, due to their high adsorption capacity, ease of handling, and resistance to mechanical disintegration, these fungal species were deemed well-suited to environmental applications.

It has been reported that the biosorption capacities of fungal pellets can improve with the modification of their surface area, either with acid ^[51], alkaline ^[48], or thermal pretreatments ^[41]. Other strategies are based on the chemical modification of the pellet surface with ligands having oxygen donor atoms, such as amidoxime ^[55], or the immobilization of nanoparticles of some metals on the fungal pellet surface ^[42][45]. Table 2 summarizes the results of some studies that have evaluated the pretreatment effect on the biosorption of metals.

Some of the reasons for using dead or inactive pellets in metal biosorption are that maintaining the viability of the microbial cells during the process requires a continuous supply of nutrients, and that avoiding toxicity against the cells might need a certain control of the metal concentration. Therefore, the use of non-living microbial cells can eliminate these problems, while enabling the biomass regeneration and reuse for several cycles ^[60].

Bayramoglu and Arica ^[41] evaluated the removal of Hg(II), Cd(II), and Zn(II) with alive (active) and dead (heat-inactivated) pellets of Lentinus edodes. The surface areas of the alive and heat-inactivated fungal pellets were measured by the BET method and were found to be 0.89 and 1.18 m²·g⁻¹ of fungal biomass, respectively. The surface area in the native pellets was increased after thermal treatment, which enhanced the biosorption of the metals evaluated. So, the heat-inactivated pellets showed better adsorption capacities for Hg (419.1 mg·g⁻¹), Cd (299.4 mg·g⁻¹), and Zn (63.3 mg·g⁻¹) compared to the alive fungal pellets (358.1, 86.4, and 37.7 mg·g⁻¹, respectively).

The chemical modification with amidoxime appears to increase the biomass surface area and to favor the adsorption of heavy metals, as reported for Trametes trogii pellets evaluated for the biosorption of U(VI) ^[55]. Maximum biosorption capacities of modified and native pellets were found to be 447 and 238 mg g⁻¹, respectively. This was explained by the fact that the amidoxime pretreatment increased 3.3-fold the surface area of fungal pellets, as well as the total available surface amino groups (2.54 mmol·g⁻¹ of dry modified biomass against 0.36 mmol·g⁻¹ dry native biomass).

One of the most recent proposals for improving metal removal is the synergy between biosorbents and nanomaterials ^[45]. Particles in the nano-size range possess altered properties compared to their bulk materials, including larger surface areas, higher reactivities, and faster adsorption kinetics ^[42], which makes them particularly attractive as sorbents ^[61]. Recently, fungal pellets have been proposed as biomass carriers of nanoparticles to make up new biocomposites for the treatment of wastewater. Abundant functional groups on the mycelium surface provide the feasible environment for the assembly and enhance the dispersibility of nanoparticles ^[42]. Phanerochaete chrysosporium pellets provided with nanosized Se showed to be better biosorbents as they removed more Zn (88.1 ± 5.3%) compared to Se- free fungal pellets (56.2 ± 2.8%) at pH 4.5 and an initial Zn concentration of 10 mg·L⁻¹. This improvement in biosorption performance was attributed to a more negative surface charge density, and hence to a higher concentration of sorption sites ^[45].

Ding et al. ^[42] studied the immobilization of iron oxide nanoparticles in pellets of Penicillium sp., which showed the following advantages: the nano-Fe₃O₄ particles can uniformly grow on the surface of Penicillium sp. with no aggregation; Penicillium sp. can be used as the template to direct and control the structure of the nano-Fe₃O₄ from the micro-scale level; and fungal pellets are more environmentally-friendly and cost-effective than other reported templates. The sorption results for three radionuclides (Sr(II), Th(II), and U(VI)) on both the alive native pellets and the Fe₃O₄-pellets of Penicillium sp. are shown in Table 2. Yet the increase of sorption capacity for the composite fungus-Fe₃O₄ toward the radionuclides was not obvious, probably because some surface functional groups of Penicillium sp. were occupied by nano-Fe₃O₄, leading to a partial loss of the ability of binding with radionuclides.

Little attention has so far been given to the multi-component biosorption of metal ions. However, these studies are more environmentally-relevant than those carried out with single metal ions, because they reflect more closely the state of the actual aquatic media. In these multi-systems, the biosorption depends, as shown above, on the biosorbent surface features, physicochemical parameters such as the solution pH, and the initial concentrations of both the adsorbate and the adsorbent, but also on the number and characteristics of the involved ions, among other factors ^[46]. Some relevant studies dealing with multi-metal ion biosorption will be discussed below.

Lead represents a serious and well-known environmental issue, because it induces dysfunctions in the neurologic, renal, and reproductive systems, particularly in young children. It is often released with cadmium from certain chemical processes and battery manufacturing ^[46][62]. Li et al. ^[46] studied the competitive biosorption of Cd²⁺ and Pb²⁺ by pellets of P. chrysosporium in the optimal physicochemical conditions determined for each metal separately. The comparison between the competitive biosorption of Cd²⁺ and Pb²⁺ showed that the biosorption of Pb²⁺ by P. chrysosporium pellets was preferential to that of Cd²⁺. Since both electronegativity and ionic radius of Pb²⁺ were larger than Cd²⁺, there might be a stronger chemical and physical affinity for Pb²⁺ on P. chrysosporium.

Bayranoglu et al. ^[41] evaluated the biosorption of cadmium, mercury, and zinc, because these three metals are commonly discharged together in wastewater by many industrial activities; however, cadmium and mercury raise the largest human health concerns. Cadmium is classed as a human and animal carcinogen (group 1) by the IARC (International Agency for Research on Cancer) ^[63]. As to mercury, it has been related to the induction of more than 250 symptoms ^[64]. The biosorption of the Cd²⁺, Hg²⁺, and Zn²⁺ multi-system was studied with dead and alive fungal pellets of Lentinus edodes ^[41]. For both types of pellets, and for all the metal ions (at individual concentrations varying from 25 to 600 mg·L⁻¹), the highest adsorption capacity occurred at pH 6. Dead and alive pellets showed the same affinity order for the metal ions both individually and in mixture: Hg²⁺ > Cd²⁺ > Zn²⁺. In fact, the adsorption capacity of Hg²⁺ was one order of magnitude higher than the Zn²⁺ adsorption capacity in live pellets, and two orders of magnitude higher in inactivated pellets. The authors attributed this selectivity to differences in the ionic properties, such as electronegativity, ionic radius, or redox potential of these metals. Thus, larger ionic radius and more electronegative metal ions would result in greater adsorption efficiencies ^[65]. However, the overall adsorption capacity of the pellets (dead and alive) was lower in the multi-metal system than in the single-metal assays ^[41][65].

Mishra and Malik ^[36] evaluated the simultaneous removal of multiple metals from electroplating effluents using Aspergillus lentulus pellets. First, the tolerance of A. lentulus against Cr, Cu, Pb, and Ni was evaluated in synthetic solutions after 5 days of growth. The removals followed the trend Pb²⁺ (100%) > Cr³⁺ (79%) > Cu²⁺ (78%) > Ni²⁺ (42%). When the pellets were applied to the treatment of a real electroplating effluent, the metal concentrations decreased by 71%, 56%, and 100% for Cr, Cu, and Pb, respectively, within 11 days, thereby showing that in a multi-metal system the preferential uptake can be different to that observed for single metal ions.

Domestic and industrial wastewaters usually contain significant amounts of different anions, that may influence the biosorption of heavy metals. Filipović-Kovačević et al. ^[38] studied the effect of adding separately Cl⁻, NO₃⁻, SO₄²⁻ and ClO₄⁻ on the removal of several metal ions. Among the anions added, only chlorides significantly decreased the efficiency of Cu²⁺, Zn²⁺, Ni²⁺ and CrO₄²⁻ biosorption, i.e., more than 50%.

Pakshirajan et al. ^[44] evaluated the effect of interfering ions such as F^-, Fe(III), Cl⁻, and NO₃⁻, which are commonly present in groundwater, on As(III) removal by Phanerochaete chrysosporium pellets. Among the studied ions, only Fe(III) significantly enhanced As(III) sorption. This was explained by the precipitation of Fe(III) as Fe(OH)₃, which is already known to be involved in the removal of As(III) from aqueous solutions. At higher concentrations of F⁻and NO₃⁻ (1.5 and 75 mg·L⁻¹, respectively), it was noticed that whereas F⁻ enhanced the biosorption of As(III), NO₃⁻ reduced it. These different effects of fluoride and nitrate maybe attributed to differences in their reactivity towards the As(III) binding sites on the biosorbent, thereby influencing As(III) removal either positively or negatively.

4. Conclusions

Biosorption constitutes an eco-friendly technology for the removal of heavy metals, as important adsorption capacities have been reported for several of them. This is mainly due to the properties of the fungal cell wall, which possess a large variety of functional groups able to interact with heavy metals through various chemical forces. Moreover, fungal granules offer process advantages over disperse mycelia, such as an improved biomass separation from treated aqueous media.

Effort has been made to increase the biosorption efficiency by several biomass pretreatments, including thermal and acidic inactivation. The underlying hypothesis is that such pretreatments increase the number of surface binding sites. Besides, dead fungal cells are considered as an adequate solution to the toxicity or inhibition problems that alive cells could endure due to adverse operating conditions. Some pretreatments do increase the biomass adsorption sites and seem enhance the adsorption capacity towards some metals, but it is not always the case. From our perspective, dead fungal pellets, unable of biodegrading organic matter or bioaccumulating metals or nutrients, are not the best option for treating real, complex water or wastewater. 

The biosorption of complex mixtures of pollutants on fungal pellets should be more explored, as well as the interfering effect of common anions such as chlorides, nitrates, carbonates or sulfates. Earlier reviews already signaled that, in spite of the profusely-available scientific literature available on fungal biosorption, there is a lack of studies carried out with real wastewater and at pilot and large scale. It is also necessary to propose studies for the post treatment of fungal biomass after being used in the treatment of water pollutants. Further research on these topics is needed to take full advantage of fungal biotechnology on improving the quality of aquatic ecosystems.