Statistical Optimization of Bioleaching for Simultaneous Recovery of Cu, Sn, Pb, and Zn from Computer-Printed Circuit Boards

Electronic garbage is one of the fastest-growing waste streams. Its disposal and appropriate management are a worldwide concern. Printed circuit boards (PCBs) are critical components in contemporary electronic gadgets that contain toxic elements. Bio-processing of PCBs for metal recovery employing microbial methods has evolved as a green solution in metallurgical operations. Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans were used in this study to leach metals from powdered waste PCBs. Te RSM is used for optimizing the leaching conditions. Te optimal conditions obtained were a bacterial activation period of 28days, a pulp density of 23g/L, and a temperature of 31 ° C. A confrmatory experiment under these optimal circumstances yielded recovery rates of


Introduction
Te rapid development of electrical and electronic equipment use and its fast obsolescence due to rapid technical innovation lead to the generation of massive volumes of electronic waste (e-waste) globally [1].Rapid economic expansion and increased transboundary fows of secondary materials will necessitate 3R (reduce, reuse, and recycle) activities [2].E-waste is becoming a global concern as a result of informal recycling and reuse activities, particularly in poor nations.Due to the presence of numerous harmful substances, electronic waste management is a major international environmental problem.Despite the fact that there are various restrictions concerning what to do with electronic trash, the majority of it is discarded or transported to developing nations without authorization.
Printed circuit boards (PCBs) are the most prevalent source of high-value critical metals in e-waste, and they are typically processed using conventional pyrometallurgical and/or hydrometallurgical processes [3].Te pyrometallurgical process consumes more energy, emits unpleasant and toxic gases, and creates massive volumes of secondary waste (slag).Hydrometallurgical recycling demands the use of a variety of toxic chemicals, some of which are carcinogenic.Furthermore, the hydrometallurgical process demands additional treatment operations.As a result, considerable efort has been expended in developing ecologically acceptable biotechnology for e-waste processing.In comparison to smelting and chemical processing, the use of microbial activity in metal recycling is rapidly expanding.Because bioleaching procedures are less expensive and use less harmful biogenic lixiviants, they may be used to recycle e-waste.Other potential benefts include the ability to modify how it operates, using less energy, and metal selectivity.
Te most common source of high-value critical metals in e-waste is PCBs, which are normally treated using standard pyrometallurgical and/or hydrometallurgical procedures.Since these processes have a lot of limitations, as discussed, there has been a quest for alternative technologies that are efective, environmentally benign, and long-lasting.In terms of capital investment, labor efort, and energy consumption, bioleaching has the potential to be one of the most promising technologies.As a result, contemporary eforts have focused on improving biological e-waste processing.Bacteria and metals interact in bioleaching via reduction, oxidation, sorption, and sulfate precipitation.Metal recovery from low-grade ores has been accomplished using bacterial leaching techniques.Using less toxic and less expensive biogenic lixiviants provides additional benefts such as operational fexibility, lower energy use, and metal selectivity.
Table 1 summarizes the literature on heavy metal recovery by microorganisms.
In this study, experiments are carried out by generating perfect conditions for microorganism development.To establish the optimal conditions for recovering heavy metals such as copper, lead, tin, and zinc from dumped PCBs, the bacteria A. ferrooxidans and A. thiooxidans were used.Te efect of process factors such as time, temperature, and pulp density on leaching was investigated.Furthermore, RSM was employed to fnd the optimal conditions.

Raw PCB Collection and Sample Preparation. Te PCBs
were retrieved from an e-waste disposal site.To remove dust particles, the sample was frst cleaned with an air blower.Mechanical tools (a saw metal cutter, a sheet metal cutter, a metal lathe cutting tool, cutting pliers, and a material separation tool kit) were employed to separate other parts such as capacitors, resistors, integrated circuits, diodes, and transistors.Te crushed sample was ground into powder using a pulverizer with a disk diameter of 175 mm and a 3phase motor running at 1400 rpm on a 225-445 volt supply.Te weight fraction of the bottom products obtained (sieves from 52 B.S.S. to pan) is insufcient for the expected recovery since the decrease in size increases the rate of metal ion recovery [30][31][32].As a consequence, the crushed PCB powder is further processed in a ball mill with a 500 g ball weight, a mill diameter of 200 mm, and a 0.25-HP, 3-phase motor operating at 60-120 rpm.

2.2.
Microorganism's Cultivation.Te microbe's A. ferrooxidans and A. thiooxidans were procured from the National Chemical Laboratory in Pune, India.Te A. ferrooxidans were grown in a 9 K medium containing the following components: Ca (NO 3 ) 2 : 0.01 g/L, (NH 4 ) 2 SO 4 : 3.0 g/L, KCl: 0.1 g/L, K 2 HPO 4 : 0.5 g/L, MgSO 4 •7H 2 O: 0.5 g/L, and FeSO 4 .7H 2 O: 45 g/L. A. thiooxidans were also cultivated on a 9 K medium in the same way; the only diference is that instead of ferrous sulfate, sulfur powder at a concentration of 20 g/L is employed.Using 1 N sulfuric acid, the pH of the growth medium was changed to the desired level of 2.5.Following that, the strain inoculation fasks were shaken in an incubator for 48 hrs at 30 °C with an agitation speed of 170 rpm.Te bacterial cells were collected by fltration after growth, and the medium was centrifuged at 10,000 rpm for 20 min to eliminate any leftover bacteria.Te bacterial count was obtained as 1 × 10 9 cells/mL.Te cell pellets were collected and kept in deionized water at 4 °C for further investigation.

Bioleaching Experimentation. A. ferrooxidans and
A. thiooxidans bacteria were used in bioleaching experiments.Te PCB powder samples are added to the nutrient broth solutions, and the infuence of various leaching parameters is studied.At the beginning of the bioleaching experiment, the stock cultures of A. ferrooxidans and A. thiooxidans are injected into the PCB sample in a conical fask.Te parameters studied were PCB size fraction (0.25 mm-3 mm), temperature (20-30 °C), pulp density (5-25 g/L), and time intervals (7 days, 14 days, 21 days, and 28 days).Te metal leaching rate was analyzed using the following equation: where C o is the initial concentration of metal ions from sample PCBs.C e is the concentration of metal ions after bioleaching.Te PCB powdered samples are added into the nutrient broth solutions, and the various leaching parameters are studied.Te bioleaching is carried out under specifed parameters and constants.Te composition of metals is analyzed by the energy-dispersive X-ray spectroscopy (EDXs).Journal of Chemistry (−1), middle (0), and high (1), with regularly spaced gaps between them.Equation ( 2) was used to calculate the uncoded actual levels, Equation (3) was used to calculate the connection between the actual and coded values, and equation ( 4) was used to calculate the total number of experiments (N) in a Box-Behnken design (4).As a result, 17 trials were required for a three-variable (n � 3) and threereplicate (c p � 3) centre point.Based on the experimental data collected in equation ( 5), the second-order mathematical models were developed as follows [8,33]:

Modelling and Statistical
where X actual , X centre , and X min are the corresponding actual values, the actual value in the canter, and the minimum (low) actual value, respectively, and X Coded is the coded value.Te number of parameters (variables) and replicates in the central point is n and c p , respectively.c is the predicted response, β is the model constant, β 1 , β 2 , β 3 , and β 4 are the linear coefcients, β 12 and β 13 are the interaction coefcients, β 23 , β 11 , β 22 , and β 33 are the quadratic coefcients, and X 1 , X 2 , X 3 , and X 4 are the symbols for the independent variables.In this present study, the efect of the parameters (i.e., pulp density, time of bioleaching, and temperature) on the bioleaching was studied.Table 2 lists the diferent parameters (variables) and their associated values used in the bioleaching experiments.Te experimental data were statistically analyzed using Design Expert 13 software.Tree-dimensional response surface graphs are shown.Te ft model and optimal conditions for independent variables were estimated using the ANOVA technique.

Metal Content of PCBs.
Te collected PCBs were processed in the manner specifed in Section 2.1.Figure 1 displays the sequence of processes.Using energydispersive X-ray spectroscopy (EDXs), the fnal collected samples (size range of 4 mm-0.05mm) revealed that the principal metals contained in the sample were copper (13.15 wt%), tin (4.24%), lead (2.78%), zinc (1.16%), and other metals (2.55%) (Table 3).Te total metallic content was found to be 23.88%.Te samples evaluated in other studies had an average metal level of 27% [30].Te typical PCBs comprise 30% organic fraction, 40% inorganic fractions, and 30% metallic fraction.Te vast range of board types employed, the varying characterization methods used by the various researchers, and the change in PCB composition through time can all explain this diference.

Parameter Optimization Studies.
Bioleaching is used to remove the metallic part of PCBs.Te efect of process factors such as the particle size, time, pulp density, and temperature was investigated.

Efect of Pulp Density on the Recovery Rate.
Pulp density is a key factor in the bioleaching process.For PCBs with a diameter of 0.25 mm, samples with varying pulp densities (5, 10, 15, 20, and 25 g/L) are prepared.Other factors, such as temperature and time, are held constant at 200 °C and 21 days.After inoculating the bacteria, the mixture is placed on a magnetic stirrer, and the fasks are incubated.Te leaching efciency progressively improves with an increase in the pulp density, but it is essentially steady at pulp densities over 12 g/L.Te toxic metal removal was computed using (1), and the results are Cu 2+ of 92.80%, Sn 2+ of 95.83%, Zn 2+ of 87.61%, and Pb 2+ of 88.88%.Poor bioleaching at higher pulp densities might be owing to the toxic efects of WPCB metallic and nonmetallic components on bacteria or to oxygen mass transfer restrictions, which are a barrier to the process's practical industrial applicability.Te precipitate appears to form near the facks' bottom.With a prolonged incubation period, the precipitate is continually generated, and the heavy metal lixiviating effectiveness is somewhat lowered (Figure 2).Te microorganism's A. ferrooxidans and A. thiooxidans do not exist in the sample due to the increase in pulp density.Tis might be because the precipitate formed diminishes with increasing metal ion pulp density in the solution [34,35].

Efect of Temperature on the Recovery Rate.
Temperature is an important operational parameter in microorganism activation.Leaching tests for PCB metallic waste will be conducted up to 30 °C [36,37].Previous research has shown that A. ferrooxidans and A. thiooxidans work well at the optimal temperature of 28-35 °C [13,14].As a result, the current study was also conducted at tempera-  3).Te leaching efciency exceeds 90%.

Efect of Time on the Recovery Rate.
Te rate of leaching grows with time until a certain point is reached and then declines [4].Te time period during which the measured leaching rate is at its peak is referred to as the "efective leaching rate" [30].Te samples used in this study were grown for 7 days, 14 days, 21 days, and 28 days, respectively.Figure 4 depicts the efect of inoculation on the long-term leaching of metal ions from PCB samples, such as Zn 2+ , Sn 2+ , Cu 2+ , and Pb 2+ .Te highest leaching efciency was 96.49% for Cu 2+ , 96.31% for Sn 2+ , 97.64% for Zn 2+ , and 98.6% for Pb 2+ when the leaching experiments were extended from 14 to 21 days.Te leaching procedure was extended for 28 days, but no signifcant improvement in metal removal was observed after 21 days.As a result, the best time interval between treatments is 21 days [31,38].

Efect of Size on the Recovery Rate.
Te size of the PCB has a signifcant impact on leaching efciency.When the size fraction is adjusted, the contact duration between the bioleaching contact material (species) and test samples is reduced.Te smaller the particle size, the higher the recovery rate.Te current investigation addresses the leaching of metals with diferent particle sizes, i.e., 4 mm, 2.3 mm, 0.6 mm, and 0.05 mm. Figure 5 depicts the variation in the particle size and the corresponding % metal recovery.Te rate of recovery is highest for a 0.05 mm-sized PCB metallic sample.Since the particles are so small, the bacteria can easily leach the metals, resulting in a process efciency of up to 95.34% for Cu 2+ , 67.30% for Sn 2+ , 85.80% for Pb 2+ , and 96.84% for Zn 2+ .As a result, 0.05-mm PCB samples are considered to be the best possible size for the experiment, in line with earlier research [38,39].

Modelling and Statistical
Analysis for Retrieving of Cu, Sn, Zn, and Pb.Te BBD-RSM method was used to investigate the interaction efects of parameters on the removal of Cu, Sn, Zn, and Pb from PCB samples using bioleaching techniques.Table 4 shows the coded and uncoded levels of independent factors from 17 experiments that correspond to BBD along with their responses.

Analysis of Variance (ANOVA
).An analysis of variance corresponding to the experimental results was presented in Tables 5 and 6.Te low probability (<0.05) with greater F-values implied that the model was accurate.Also, the acceptable and reasonable value of the lack of ft indicates the suitability of the method for good presentation of experimental data.As presented in Table 7, the model presents the high R 2 value for the metals as follows: 0.9917 for Cu, 0.9319 for Sn, 0.9713 for Zn, and 0.9659 for Pb indicate that there was a good agreement between the experimental and predicted results.Also, the predicted R 2 values were in reasonable agreement with the adjusted R 2 values.Te high values for the model's adequate precision (the signal to noise ratio) indicate that this model can be used to navigate the design space.

Development of the Model Equation.
A model equation is a representative equation that mathematically relates the response to factors.Te regression equations ( 6)-( 9) for the statistical analysis data plots for the metals Cu, Sn, Zn, and Pb from PCBs are as follows: Te fnal equations in terms of coded factors are as follows:   Figure 6 shows that the predicted value of the responses from the model was in agreement with observed values over the selected range of independent variables with reasonable higher values of the coefcient of determination (R 2 ).

3D Response Plots of Interaction Efects.
Figures 7-11 show 3D response surface plots of metal removal vs. leaching time, temperature, and pulp density interactions.3D surface plots can aid in the determination of response values [40].Each contour plot represents various combinations of two test parameters, with the other value set to zero.Te contour plot's shape reveals whether or not the variables' reciprocal interactions are signifcant.Te interactions between related variables in a circular contour plot are low but signifcant in an elliptical contour plot.Te efects and combinations of the variables' leaching time, temperature, and pulp density on Cu removal are depicted in Figures 7(a)-7(c).Te fgures show a signifcant interaction of time with temperature and pulp density (Figures 7(a) and 7(b)) but no interaction between pulp density and temperature (Figure 7(c)).Cu bioleaching performed best at high temperatures (28 °C) and for an extended period of time (28 days).Cu leaching is increased when pulp density rises (Figure 7(b)).
Te efects and combinations of the parameters on Sn removal are depicted in Figures 8(a

Journal of Chemistry
Te graphs demonstrate a strong link between time and temperature (Figure 9(a)), as well as between pulp density and temperature (Figure 9(c)).Te interaction between pulp density and time is not signifcant, as seen in Figure 9(b).Figures 10(a)-10(c) depicts how each of the parameters infuences Pb removal and how they interact.

Optimization of Parameters.
In numerical optimization, the desirability function helps people understand the multiresponse parameters better when the parameters are being optimized.Te current study's objective function is to maximize metal recovery percent.Figure 11 depicts the desirability profle for heavy metal removal percentage vs.factors.Te desirability scale ranges from 0.0 to 1.0, corresponding to the transition from an unpleasant to a much desired state [41][42][43][44].At a time interval of 28 days, a temperature of 31 °C, a pulp density of 23 g/L, and optimal removal of Cu 2+ of 95.62%, Sn 2+ of 96.27%, Zn 2+ of 95.6%, and Pb 2+ of 98.25% were reached with a desirability of 1. Te experimental tests were carried out at the optimal parameters specifed by the statistical technique, and the fndings produced were consistent with the RSM predicted values.Figure 12 provides the SEM-EDAX analysis of the PCB sample after leaching under optimal conditions.Te bioleaching process addresses the issues of high energy consumption, signifcant environmental contamination, and complex operation, and as a consequence, it is   Journal of Chemistry regarded as a potential strategy for metal recovery.In most situations, the known and recognized mode of PCB bioleaching is the indirect contact mechanism.Te metal dissolution from PCBs may be divided into two phases.In the frst phase, bacteria oxidize ferrous ions to produce ferric ions.After releasing the metal from the PCBs that it was linked to, the ferric ions are transformed to ferrous ions in the second phase of the process.Ferrous ions improve the leaching process by acting as an oxidizing agent.

Conclusions
Te fundamental advantage of bioleaching is that it produces no hazardous waste into the environment, resulting in safer waste disposal and paving the way for the development of a sustainable toxic metal recovery technology.Metal recovery by bioleaching is an efective method for handling the toxic metals present in PCBs.In this study, A. ferrooxidans and A. thiooxidans are used to leach metals from powdered waste PCBs.Using the RSM, the optimum removal of Cu 2+ of 95.62%, Sn 2+ of 96.27%, Zn 2+ of 95.6%, and Pb 2+ of 98.25% was reached with a desirability of 1 at a time interval of 28 days, a temperature of 31 °C, and a pulp density of 23 g/L.Te challenge that must be addressed is the scalability of bioleaching.Extensive research is required to develop suitable microorganisms for commercial activities.

Figure 1 :Figure 2 :Figure 3 :
Figure 1: Schematic diagrams of primary raw PCBs into stepwise size reduction under the various mechanical operations and metal composition analysis performed by SEM with EDAX (reproduced from Murugesan et al., [31] under https://creativecommons.org/licenses/ by/4.0).
)-8(c).Te variables had a signifcant infuence on tin bioleaching.Increasing cyanide production in response to increased pulp density will reduce bacterial growth.As shown in Figure8(b), Sn removal was high at low pulp density.Te efects and combinations of parameters on Zn removal are depicted in Figures 9(a)-9(c).

Figure 6 :
Figure 6: Te comparison plot between the experimental data and predicted data for the metals.

Figure 7 :
Figure 7: 3D interaction plots between the pulp density, temperature, and time for Cu removal.

Figure 8 :
Figure 8: 3D interaction plots between the pulp density, temperature, and time for Sn removal.

Figure 10 :
Figure 10: 3D interaction plots between the pulp density, temperature, and time for Pb removal.
e m p a r a tu r e ( C ) (c)

Figure 9 :
Figure 9: 3D interaction plots between the pulp density, temperature, and time for Zn removal.

Figure 11 :
Figure 11: Te desirability plot for the metal recovery rate with optimal conditions.

Table 1 :
Recovery data of metals with diferent microbes.

4
Journal of Chemistry tures ranging from 20 to 35 °C.As the temperature climbs over 20 °C, the rate of dissociation slows.It slows the rate of recovery.Te recovery rates are Cu 2+ of 97.19%, Sn 2+ of 96.23%, Zn 2+ of 93.67%, and Pb 2+ of 96.9% (Figure

Table 2 :
Levels of diferent process variables in coded and uncoded forms for biological leaching studies.

Table 3 :
Metal composition results in a sample.

Table 4 :
Te RSM-BBD experimental design: experimental and predicted values.

Table 5 :
Te ANOVA table for the model to predict percent of leaching of copper and tin.

Table 6 :
Te ANOVA table for the model to predict percent of leaching of zinc and lead.

Table 7 :
Quality of the quadratic model for the leaching of heavy metals.