Biosorption of lead by a soil isolate Aspergillus neoalliaceus

This study focused on Pb (II) elimination from aqueous solutions using fungal soil isolate which is identified as Aspergillus neoalliaceus. The sorption of lead with fungal mass studies was performed as a function of pH, biomass dose, contact time, and initial dye concentration. The solution pH value strongly affected the sorption of lead fungal mass. To examine the effect of hydrogen ions on biosorption in solutions containing lead, solutions with different pH values were used and pH 5 was found to be the most suitable pH value for lead removal. Lead biosorption remained very low below pH 4 because of the competing effect of hydrogen ions in the solution. It has been observed that the removal of lead ions based on biosorption with Aspergillus neoalliaceus is better explained by Langmuir isotherm and pseudo-second-order kinetic models compared to other used models. The biosorption of Pb (II) was determined as a spontaneous and endothermic process.


Introduction
Along with the increase in industrialization, many environmental problems have emerged. Heavy metals play a major role in this environmental pollution burden. Heavy metal contamination of the environment including soil and water, which have a crucial role in the continuity of organism life, frequently emerges from human, agricultural and industrial activities. One of the most threatening pollutants as heavy metal is lead due to its accumulation in the environment (Masindi and Muedi 2018). Pb (II) becomes harmful to the central nervous system over the concentration of 0.05 mg/L. Besides, lead pollution can cause anemia, encephalopathy, hepatitis, and kidney diseases in humans (Javanbakht et al. 2011). The developments in technological demands and industrial activities are the causes of Pb (II) released to the environment. Battery manufacturing, printing, and metalbased processes are the main origins that play important role in the contamination of lead (Tao et al. 2020).
Many studies have focused on the removal of heavy metal ions using various techniques. It has been shown that heavy metal removal is performed with various physicochemical techniques, examples of these techniques are membrane filtration, chemical precipitation, evaporation, and adsorption. (Pohl 2020). However, these methods have several drawbacks associated with high reagent requirements, toxic waste generation, and unpredictable metal removal. (Farhan and Khadom 2015). In this context, research focused on biosorbent such as new environmentally friendly, economical, and effective metal adsorbents has been carried out by the application of microbial biomass (Amini and Younesi 2009).
Activated carbon is a good adsorbent for pollutant removal processes but commercialized carbon is expensive material, in additional alternative adsorbents are always investigated (Kapoor et al. 1999). Biosorption could be defined as the process of removing heavy metals using biological material (Michalak et al. 2013). Various fungal biomass has been used to remove dyes and metals from aqueous solutions (Arıca and Bayramoğlu 2007). Previous studies showed that the biomass of Aspergillus niger and A. alliaceus are good adsorbents for the removal of environmental pollutants (Kapoor et al. 1999;Khelifi et al. 2015) Apart from fungi, different biosorbents were used for heavy metal removal. Zea Mays stalk sponge, peanut shell, Pinus sylvestris cone, and Citrus lemon peel biomass can be given as examples (Ucun et al. 2003 Taşar et al. 2014;Šehović et al. 2022). This removal platform has been reported as an easy and environmentally friendly technique providing low-cost and effective removal capability (Pham et al. 2021). Bio-active materials of bacteria, yeast, algae, and fungi have been extensively used for eliminating of metals from aqueous solutions (Mwandira et al. 2020). Fungi have gained much more attention compared to bacterial strains and have strong potential due to their high removal potential against a large number of heavy metal ions (Dhankhar and Hooda 2011;Iskandar et al. 2011). The functional groups on the cell wall of fungi such as -COOH, -SH, -OH, -NH 2 , -PO 4 H 2 enable the interaction between the surface of the cell and target heavy metal (Dhankhar and Hooda 2011;Albert et al. 2020).
In this study, the biosorption mechanisms of lead onto A. neoalliaceus surface are reported. In addition, the effects of medium pH, biomass dose, initial lead concentration,and temperature on the biosorption yield are investigated.

Isolation and identification of the fungus
Five grams of soil sample was suspended in 250 ml Erlenmeyer Flasks with malt extract broth. Afterward, the suspended soil sample in the growth medium was incubated at 25 °C for 5 days. At the end of the incubation period, 0.1 ml was taken from the suspension and inoculated on sterile potato dextrose agar media. Fungal species were identified by ITS regions/18S rRNA sequencing. Genomic DNA was isolated with the EurX GeneMATRIX isolation kit (Poland). Spectrophotometric measurement was performed to check the purity of the DNA obtained after extraction (Thermo Scientific Nanodrop 2000). Samples were Sanger Sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and ABI 3730XL instrument (Applied Biosystems, Foster City, CA).

Characterization and zeta potential
The surface morphology of I heat-inactivated Aspergillus neoalliaceus was monitored with GAIA3 + Oxford XMax 150 EDS scanning electron microscope. Fungal samples were characterized spectroscopically by 8000 Series (Shimadzu, Japan) FTIR spectrophotometer (Thermo Scientific).
Fourier transform infrared spectrums of lead-loaded and unloaded fungal masses were recorded between 4000 and 400 cm −1 wavenumber regions. The net surface charge of heatinactivated fungal biomass was analyzed by measuring zeta potentials at known pH values within 3.0 to 7.0.

Production of heat-inactivated biomass
Identified fungal species inoculated 1000 ml flasks with 500 ml malt extract broth and incubated for 5 days at 25 °C (agitation rate:150 rpm). Pellets of Aspergillus neoalliaceus were separated from growth media via filter paper and collected. The fungal mass washed with distilled water was autoclaved for 15 min at 120 °C to obtain heat-inactivated biomass. Heat-inactivated biomass was left to dry at 50 °C for 2 days and ground to obtain a powder. powdered biomass was sieved through a 0.15 mm screen.

Pb 2+ biosorption studies
Unless other was stated, all experiments were carried out in a 15 ml centrifuge tube containing 10 ml Pb 2+ solution with 10 mg fungal biomass at 25 °C and stirred at 150 rpm with a rotary shaker (Mikrotest, Turkey). Lead solutions were adjusted with 0.1 N NaOH and 0.1 N HCl solutions. The pH of the Pb (II) and 10 mg biomass-containing medium was adjusted from 2 to 7 to investigate the effect of medium pH on biosorption. To examine the effects of the amount of biomass, 5 mg to 50 mg dry heat-inactivated fungal biomass was added to Pb (II) solutions. The change of biosorption efficiency with a lead concentration in the medium was also studied by changing the metal concentration from 5 to 50 mg/L. To determine the effect of contact time on fugal sorption of Pb (II), samples were taken within the predetermined time intervals. Biomassfree solutions were prepared as a control for each run.

Pb (II) analysis
To determine lead biosorption capacity, Pb (II) solutions with biomass were filtered via syringe filters (0.45 μm). Initial and final metal concentrations in the solutions were determined by the Perkin Elmer Analyst 800 atomic absorption spectrometer in the flame module. The averages of three measurements were used in the calculations.
The Pb (II) sorption efficiency (Qe) and removal rate (%) of the fungal mass at equilibrium were calculated using the equations given below: Page 3 of 8 547 where Qe is the amount of Pb 2+ adsorbed by heat-inactivated dried fungal biomass at equilibrium (mg/g), C i is the initial concentration of lead ions (mg/L), C e is the equilibrium concentration of lead ions (mg/L), m is the amount of biomass in the lead solution (g), and V(L) is the volume of adsorption medium (L).

Isotherm studies
In isotherm studies, apart from the initial lead concentration, other variables such as pH, temperature, biomass dose, and contact time were kept constant. Ten milligrams of biomasses are put into each 10 ml Pb 2+ solution which has different lead concentrations were used in the studies. Following the biosorption period, lead concentrations of solutions were measured. Since Langmuir and Freundlich's equations are the more common models used to describe the adsorption mechanism, these two isotherm models were used.
The Langmuir and Freundlich isotherm model is expressed by the equations given below (Huang et al. 2016): where Ce (mg/L) is the equilibrium concentration, Q e (mg/g) is the adsorption capacity at equilibrium, K L (L/g) is the adsorption strength, Q max (mg/g) is the maximum adsorption capacity, K F is the adsorption coefficient, and 1/n is the adsorption index.

Kinetics
Kinetic models pseudo-first-order and pseudo-second-order kinetic models were tested for the biosorption of Pb 2+ on biomass. Lagergren's equation, used for pseudo-first-order model (Lagergren 1898): The equation for pseudo-second-order kinetic model (Ho and McKay 1999):

Identification of fungus
The fungal species, isolated from soil was identified as Aspergillus neoalliaceus (GenBank Number: MH279421.1), based on the ITS regions/18S rRNA sequence. Aspergillus is a cosmopolitan genus of fungi that can live in a wide variety of environments (Visagie and Houbraken 2020).

FTIR analyses
FTIR spectrums of lead-loaded and unloaded fungal masses are given in Fig. 1. the broadband ranging from 3600 to 3000 may cause by the overlap of -OH and -NH stretching vibrations. The absorbance peaks from 3000 to 2800 cm −1 are belonged to -CH and -CH 2 stretching vibrations. The peaks from 2363 to 2335 may represent -C ≡ C-symmetry (Imran et al. 2019). Absorbance peaks at 1742 (a) and 1743 (b) cm −1 may be attributed to C = O stretching vibrations. The adsorption band at 1643 cm −1 (a) that was shifted to 1632 cm −1 (b) after Pb 2+ biosorption may be attributed to the C = O groups of primary amides playing a role in biosorption processes (Huang et al. 2016). Amino and carboxyl groups play a major role in binding processes (Das et al.

Effect of pH on biosorption
The pH of aqueous solutions is perhaps the most important factor for adsorption and biosorption studies. The pH value of the medium influences the ionization of functional groups (amino, phosphate, and carboxyl groups) on the cell wall (Fan et al. 2008). In addition, the chemistry of the aqueous solution is closely related to the concentration of hydrogen ions in the environment. At high pH values, the solubility of heavy metals decreases significantly (Dhankhar and Hooda 2011). As depicted in Fig. 2, little or no metal uptake was observed below pH 4.0. The maximum uptake capacity of Pb 2+ biosorption was attained at pH 5.0 (19.49 mg/g). Low Pb 2+ biosorption capacities were obtained at pH values less than 4.0 due to the protonation of functional groups on the cell wall (Say et al. 2001). Since the hydrogen ion concentration is high at low pH values, these ions compete with metal ions for active metal-binding regions. (Fan et al. 2008). Hence adsorption capacity decreases with increasing H + ions. The decrease in metal uptake at pH 6-7 can be explained by the formation of hydroxylated complexes of lead ions that compete with metal ions for active sites. (Vimala and Das 2009). Lead removal capacity of Oceanobacillus profundus increased with increasing pH up to pH 5 and then decreased (Mwandira et al. 2020). The high yield of Pb 2+ biosorption capacity with Moringa oleifera was observed in the range of pH 4-6 values (Imran et al. 2019).
Another study reported that the maximum lead removal was observed at pH 5 (Bueno et al. 2008). The different study reported that maximum Pb 2+ biosorption with Penicillium cryosogenum was observed at pH 6 (Skowroński et al. 2001). The heat-inactivated Aspergillus neoalliaceus biomass (Fig. 3) gains a negative charge at pH 5 (Fig. 2). The sudden increase in the metal uptake capacity of the biosorbent above pH 3 shows the importance of the surface charge. When fungus-based sorbent negatively charged at pH 5, biosorption of lead reached maximum value. Although the biosorbent is negatively charged above pH 5, a decrease in its biosorption capacity was observed. A previous study attributed the decrease in lead adsorption over pH 5 to the hydroxylated lead compounds formed (Vimala and Das 2009).

Effect of biomass dose
The dosage effect of A. neoalliaceus mass on the removal rate and Q e were studied by varying the biomass concentrations ranging from 5 to 50 mg by keeping pH and the volume of the medium solution constant. The maximum biosorption efficiency was obtained with 1000 mg/L of A. neoalliaceus biomass dosage. The biosorption capacity of Pb 2+ ions was decreased with increasing biomass dosage in the solution. Contrary to biosorption capacity, the removal of heavy metal increases with increasing biomass dose (Fig. 4). The biosorption performance of divalent cations of Nickel, Copper, and Zinc decreased with increasing biomass amount in the solution. In addition, the removal percentage of metals increased with increasing fungal biomass (Javaid et al. 2011). The increase in the removal percentage of heavy metal with increasing biomass can be attributed to an increase in the number of metal-binding active regions. In this study, maximum Pb 2+ removal (93%) was achieved with a 1000 mg/L biomass dosage.

Effect of contact time and Kinetics
The altering lead concentration versus time is given in Fig. 5, rapid biosorption was seen within the first 10 min. Nearly 85% of the lead ions in the medium were adsorbed the first 30 min and equilibrium was reached within 60 min. The rapid adsorption of lead was observed in the early stage because of an abundance of the active binding site on biosorbent, with lead ions binding with active sites, biosorption efficiency was decreased in the later stage (Vimala and Das 2009). A previous study showed that 95% of lead ions are adsorbed within the first 60 min (Bueno et al. 2008). In another study, Pb (II) uptake with Ficus benghalensis reached equilibrium in 20 min (Surisetty et al. 2013).
The validity of each model was evaluated by R Squared value (R 2 ). The kinetics of Pb 2+ biosorption onto heattreated Aspergillus neoalliaceus biomass fits better to the pseudo-second-order kinetic model at pH 5 (Fig. 6). Zeta potential (mV) Qe (mg/g) pH Qe Zeta potential Fig. 2 Effect of pH on the Pb (II) biosorption of lead onto heat-inactivated fungal biomass (Initial concentration of lead: 20.0 mg/L, biomass amount: 10 mg, volume: 10 mL, 25 °C. All data were reported as mean ± SD, n = 3) The pseudo-second-order model, which differs from the pseudo-first-order model, predicts the behaviour throughout the entire time adsorption and is compatible with the rate-control step of the adsorption mechanism (Fan et al. 2008).The values of pseudo-first-order and pseudo-second-order model parameters were given in Table 1.
The theoretical Qe was found as 22.37 mg/g from the slope of the linear line. This value was determined as 22.47 mg/g experimentally. The theoretical Qe calculated for the pseudo-first-order was found to be 13.34, apparently quite far from the experimental Qe value. A different study reported that biosorption of lead with Pleurotus ostreatus biomass obeyed the pseudo-second-order kinetic model (Eliescu et al. 2020). In another study, it has been reported that the pseudo-second-order kinetic model better explains the lead sorption phenomenon with Penicillium simplicissimum (Fan et al. 2008).

Effect of initial lead concentration
The lead uptake efficiency of fungal mass increased with increasing heavy metal concentration in the solution and achieved saturation at 40 mg/L Pb 2+ concentration (Fig. 7). The higher metal concentrations provide an impetus for processes involving biosorbent and aqueous phase transfers (Tewari et al. 2005). In this study, the highest metal uptake was observed at 40 mg/L initial metal concentration (29.95 mg/g). A previous study reported that biosorption of lead with Moringa oleifera biomass, amount of lead ions adsorbed per unit biosorbent increased with lead ion concentration, while percent lead removal decreased (Imran et al. 2019). The reduction in percent removal can be attributed to the lack of sufficient surface area to bind the many more metal ions present in solution (Surisetty et al. 2013). In another study, authors reported that Q e (mg/g) increases with increasing amount of Pb (II) ions (Morosanu et al. 2017).

Isotherms
The Freundlich and Langmuir isotherms models are two models that are widely used to explain biosorption mechanisms. The Freundlich equation is the oldest equation used to explain the adsorption phenomenon on heterogeneous surfaces (Tran et al. 2017). The Freundlich adsorption isotherm explains the heterogeneous and multi-layered adsorption on the adsorbate surface (Hussain et al. 2021). The Langmuir adsorption isotherm explained the assumption of a fixed number of binding sites, no interaction   between adsorbate molecules, all binding sites have the same energy level and monolayered adsorption (Tran et al. 2017). According to experimental data, biosorption of Pb 2+ better fit Langmuir isotherm (R 2 : 0.995) than Freundlich isotherm (R 2 :0.898) at pH 5. Constants of isotherms were given in Table 2.
In a study with activated carbon obtained from plant material, it was reported that lead adsorption followed the Langmuir isotherm (Gerçel and Gerçel 2007). Similar results were obtained with Rhodococcus opacus (bacteria) and Lepiota hystrix (fungus) (Bueno et al. 2008;Kariuki et al. 2017). On the other hand, a recent study reported that biosorption of lead with Simplicillium chinense was obeyed Langmuir isotherm model (Jin et al. 2020).

Effect of temperature
The temperature of the medium is an important factor for biosorption studies. To investigate the effect of temperature 40 mg/L Pb (II) containing solutions with fungal biomass incubated 24 at pH 5, different temperatures (15-35 °C).The biosorption capacity of biomass, increases with increased temperature and is reached a maximum at 35 °C (33,6 mg/g). The values for the changes in standard Gibbs free (ΔG°) energies were found to be − 0.97, − 2.4,and − 3.9 kJ/mol for 288 K (15 °C), 298 K (25 °C), and 303 K(35 °C), respectively, The negative values of standard Gibbs free energies indicates that biosorption of lead is spontaneous (Fan et al. 2008). The standard enthalpy change (ΔHº) of biosorption of lead was found to 42.2 kJ/mol. The positive value of change in enthalpy indicates that the processes is endothermic. A previous study reported that biosorption of lead with the fungus Pleurotus ostreatus shows endothermic character (Eliescu et al. 2020). The increase in biosorption efficiency at increasing temperatures can be attributed to the expansion of the pore size and/or activation of the binding sites on the biosorbent surface (Fan et al. 2008). Another explanation is that higher temperature causes greater mobility in particles and reduces liquid viscosity, and Pb (II) ions affinity on adsorbent is higher at higher temperatures (Morosanu et al. 2017).

Conclusions
In this study, a fungal species which was obtained from soil and identified as Aspergillus neoalliaceus was used for the removal of lead ions, which are important to remove due to their harmful effects. Laboratory scale studies have shown that the parameter that most affects the removal efficiency of Pb (II) with fungal mass is the pH value of the solution, and the highest biosorption capacity is reached at pH 5. Studies conducted against time have shown that approximately 85% of lead uptake has been seen in 30 min. The fungal sorption of Pb (II) ions obeyed the Langmuir isotherm. Lead biosorption onto heat-treated A. neoalliaceus mass fits better to pseudo-second-order model, experimental and calculated Qe values were found to be very close to each other.
To sum up, it is thought that this biosorbent obtained from Aspergillus neoalliceus can be used in the removal of lead.

Author contributions All research (including writing) was done
Funding The authors have not disclosed any funding.

Conflicts of interest
The authors declare that they have no conflict of interest.