MSW incinerator fly ash was collected from the Waste-to-Energy Research Facility (WTERF) at Tuas South in Singapore. The dry ash was collected into sterile plastic containers, stored in a dry cabinet, and transferred to the lab. The fly ash was autoclaved at 121°C for 15 min for further studies.

Particle size and shape were analyzed using the particle size analyzer (Coulter LS 230), with particles in the range of 0.04 μm–2,000 μm, and scanning electron microscopy (SEM), respectively. The specific surface area was determined using a QuantaChrome Corporation Nova 3,000 Brunauer–Emmett–Teller analyzer based on the BET equation. The bulk density was calculated using the ASTM D1895-96 standard. A funnel positioned above a 10 mL measuring cylinder was filled with fly ash that was allowed to flow freely into the cylinder until it reached 10 mL. The bulk density was estimated after weighing the sample and the cylinder (Wu and Ting, 2006).

The elemental composition of the fly ash was analyzed using the US EPA SW 846 method 3050B. After acid digestion, the material was examined by inductively coupled plasma mass spectrometry (ICP-MS). Toxicity characteristic leaching procedure (TCLP) tests were performed in accordance with the US EPA SW 846 method 1,311 to determine its toxicity. During the leaching experiments, TCLP extraction fluid #2 (un-buffered acetic acid solution) was employed. The metal concentrations leached in TCLP were compared with the levels authorized by the US EPA and Singapore’s National Environment Agency (NEA) (Xu and Ting, 2004).

Acidithiobacillus was obtained from the DSMZ microorganism culture collection in Germany (A. thiooxidans, DSMZ 11478; A. ferrooxidans, DSMZ 3320). A. ferrooxidans was cultured in Silverman and Lundgren 9k nutrient medium, which contained ferrous sulphate as the energy source. The medium was prepared in two parts: 1) basal salt solution consisting of 0.1 g of KCl, 3.0 g of (NH4)₂SO₄, 0.5 g of MgSO₄.7H₂O, 0.5 g of K₂HPO₄, and 0.01 g of Ca(NO₃)₂ dissolved in 700 mL of de-ionized water; 2) iron sulphate solution (44.1 g of FeSO₄.7H₂O in 300 mL of water acidified by 1 mL of H₂SO₄ 10N. The basal salt solution was sterilized and autoclaved at 121°C for 15 min. The iron sulphate solution was sterilized by filtering the solution through a 0.04 μm syringe membrane as it decomposed upon heating and thus could not be autoclaved. The cultures were incubated at 30°C with shaking at 120 rpm (Zhang et al., 2013).

A. thiooxidans was prepared using a medium that consisted of 2.0 g of (NH4)₂SO4, 0.5 g of K₂HPO₄, and 0.25 g of MgSO₄7H₂O with powdered sulphur (10 g/L) in 1 L of de-ionized water; the medium was then sterilized. Finally, the cultures were incubated at 30°C with shaking at 120 rpm (Zhang et al., 2013).

The growth cycle of the bacteria is obtained by using three different methods and correlating them, e.g., growth pattern, pH reading (Narenkumar et al., 2019), and titration. It is important to obtain the growth cycle because the bacterium produces the most metabolites and acid near the end of the exponential phase. The experiment was conducted using the spent medium with the highest amount of metabolites and acid, as it was more economical.

A STAT Titrino auto-titrator was used with 0.1028M of NaOH to determine the amount and concentration of the acid produced. Inoculate (2 mL) was obtained from the culture and diluted to 20 mL for titration. From the auto-titration, the volume of NaOH used was obtained as follows:

Moles of NaOH used = volume of NaOH × 0.1028 M;

Moles of H₂SO₄ in 2 mL = moles of NaOH used × ½; and Concentration of H₂SO₄ of inoculate = moles of H₂SO₄ in 2 mL × ½ × 10³.

The electrokinetic cell was built using polycarbonate (three compartments) in the configuration, as shown in Figure 1. The volumes of the cathode and anode compartments were 200 mL each and the volume of the substrate compartment was 100 mL. The cells were covered to minimize loss through evaporation. A magnetic stirrer was placed in all substrate compartments to ensure a homogenous mixing of the contaminant and the leaching agents. Fly ash (20 g) was used for the electrokinetic experiments. Two separators with pores were used as support for the IONICS^® ion exchange membranes from GE water.

The anolyte and catholyte reservoirs were filled with different sets of solutions, such as the spent medium, sulphuric acid, and sterile H₂O as a working solution, and steel 316L served as the anode and cathode electrodes. The concentration of sulphuric acid was prepared to the same molarity as the spent medium to reduce the number of variables and observe any possible effects of the metabolites produced by the bacteria.

The experimental runs were conducted over a period of 14 days at a constant voltage of 20 V or 1 V/cm (Rodrigo et al., 2014). Samples (1 mL) was taken from the cathode and anode compartments every 2 days to monitor trends in the ionic movements. At the end of 14 days, the fly ashes were collected as sludge from the substrate compartment and dried using a drying oven to obtain the dry masses. The fly ashes were analyzed for their chemical composition and toxicity. The pH of the cathode site was controlled by adding 5% nitric acid and maintained at a constant low pH to prevent precipitation. When necessary, the loss of the liquid due to evaporation was compensated with the addition of water in the anode compartment. The residual fly ash absorption was analyzed during electrokinesis and was characterized through acid digestion, TCLP, and ICP-MS (Arul Prakash et al., 2021). ICP multi-element standard IV at a concentration of 1,000 mg/L was used to prepare the calibration standards, which were diluted to 2 ppm, 5 ppm, 8 ppm, 10 ppm, 15 ppm, and 20 ppm (1.99 mg/L, 4.99 mg/L, 7.99 mg/L, 9.99 mg/L, 14.99 mg/L, and 19.99 mg/L).

According to Xu and Ting (2004), the concentration of heavy metals in the fly ash (mg/kg) was calculated as follows:

Metal concentration (mg/kg fly ash) = concentration in solution (mg metal/liter solution) × dilution factor × liquid to solid ratio (liter solution/kg fly ash).

MSW incineration fly ash is defined as hazardous waste due to its high concentration of heavy metals, chloride salts, and dioxins (Dingwen and Anhui, 2020). The particle size of fly ash in this study was approximately 26.1 µm and within the size range commonly reported for these solids. The particle size is comparatively smaller then the range given in the literature. This might be because the fly ash sample originated from more effective incineration systems, which can consume huge char flakes and lower the size of fly ash particles. The bulk density was 0.7869 g/cm³ and the same order of magnitude as reported in the literature (Table 1). Likewise, the variance in density has been ascribed to the effectiveness of the cremation process and the efficiency of equipment collection (Ontiveros et al., 1989; Xu et al., 2014). Although they exist as fine particulates, the fly ash shows a low specific surface of 11.68 m²/g due to its non-porous nature. As shown in the SEM micrographs (Figure 2), clusters and non-porous structures were evident, with some showing cylindrical rod shapes.

The elemental composition of fly ash was analyzed using ICP-MS and SEM/EDX and the results are shown in Table 2. Significant constituents (>10,000 mg/kg) found in the MSW fly ash included mostly Al, K, Ca, Na, Cl, O, and C. The minor elements (1,000–10,000 mg/kg) in the fly ash were primarily heavy metals, such as Mg, Fe, Zn, and Pb. Some elements, such as Cu, Mn, and Sr, were found in trace amounts (<1,000 mg/kg). The MSW fly ash contained a high concentration of calcium (close to 13%). This is due to the lime spray tand flue gas scrubber treatments, which are critical for neutralizing acidic gases and reducing the degree of metal release in acidic circumstances. In addition, there was a high chloride concentration of approximately 9.5%, which might be due to the presence of polyvinyl chloride in the feedstock (Saikia, 2019). Oxygen was also detected by SEM/EDX, possibly due to the formation of metal oxides during the incineration process. Additionally, a higher amount of carbon was detected. This quantity of potentially unburned carbon is a significant source of organic pollutants. Owing to its huge adsorptive surface area, unburned carbon increases the amount of organic pollutants (Huang et al., 2003). The presence of Mn, Pb, and Zn in fly ash may be attributed to the presence of storage batteries and electrical devices in the feed source (Saikia, 2019). Toxic metals such as Ag were not detected in the fly ash.

A reasonably good agreement for fly ash elements was observed with ICP-MS and SEM/EDX. Nevertheless, there were slight differences due to the resolution limitation of the fine particles in SEM/EDX. Elements of low abundance (<0.5 %w) are less noticeable with SEM/EDX. In addition, the heterogeneity of the fly ash means there are differences between the bulk composition and surface components of the fly ash (Liu et al., 2009). The SEM/EDX technique, which is surface-sensitive, only evaluates the composition at the particle surface, whereas acid digestion provides bulk composition analyses. However, the digestion/ICP-MS approach is limited to the examination of metallic elements, whereas SEM/EDX can identify any elements with an atomic number greater than 4 (Xu et al., 2014).

The TCLP result revealed a higher Pb concentration (33 mg/L) than that authorized by the US EPA (5 mg/L) and Singapore’s National Environment Agency (NEA) (5 mg/L). As a result, fly ash might be deemed harmful. The significant leaching yield of Pb in the TCLP extract is attributable to the leachate’s high pH (above 11). Pb is more easily liberated in the acidic TCLP solution due to the alkaline ash’s strong acid neutralization capability. As a result of interaction with the extremely alkaline fly ash, the simulated acid rain TCLP fluid has become an alkaline leachate, and Pb, an amphoteric element, is leached when the pH exceeds 9.0 (Wu and Ting, 2006). This amphoteric metal forms complex ions and becomes soluble in alkaline conditions according to Equation 1: Pb 2 + + 3 OH − →  Pb OH 3 − aq (1)

As a result of this high leaching yield of Pb when the ash is in contact with highly alkaline leachate (Table 3), it is suggested that prior leaching of Pb in alkaline conditions should be considered (Xu et al., 2014).

The exponential phase of bacterial growth started on the sixth day of inoculation and ended on the seventh day of inoculation, after which it underwent the stationary phase until the 10th day, when the death phase started. In this experiment, it is more economically viable to obtain the spent medium at the end of the exponential phase when the bacteria would have produced the most amount of metabolites and sulphuric acid in the shortest possible number of days. This result was supported by pH reports too (Figure 3). In pH analysis, the pH of A. ferrooxidans and A. thiooxidans (pH 2 and 1.39 at day 14) was significantly reduced compared with the control (pH 2.59 and 2.2 at day 14) due to bacterial metabolites (Hallberg et al., 2010). It seems that cultures of this bacterium (A. ferrooxidans) often harbor heterotrophic contaminants, which are probably responsible for this anomaly (Schippers, 2007). Such contaminants live in symbiosis with A. ferrooxidans, and many others are members of the genus Acidophilium (Vera et al., 2013). It has been reported that most contaminants are recalcitrant to common purification techniques (Hallberg et al., 2010). Thus, it is apparent that much of the data accumulated on this bacterium should be interpreted with some caution.

During the electrokinetic analysis, the pH of the sample played a crucial role in the electrokinetic method. The anodic position shows an acidic condition due to the formation of H⁺ ions in the anodic reaction, whereas the cathodic sites show an alkaline condition (Arul Prakash et al., 2021). This condition might be favorable for the leaching of metal ions.

The average heavy metal leaching efficiencies in different bacterial media (A. thiooxidans/A. ferrooxidans) are presented in Table 4. This result reveals that K, Na, Ca, and Mg (A. thiooxidans, 85%, 47%, 92%, and 85%; A. ferrooxidans, 85%, 25%, 75%, and 74%, respectively) show higher leaching efficiencies than other metals, such as lead (Pd) and zinc (Zn). These results correlate with a previous report by Maini et al. (2000b). In the experiments using A. thiooxidans, the ACID and SM setups are more efficient in the leaching of all metals except lead (Pd) and selenium (Se) compared with the DI setup; this is due to the fact that the metals solubilize more easily in the acidic conditions created by the biochemically and chemically enhanced fly ash. Hence, it can be concluded that there is no added effect from the metabolites of A. thiooxidans.

However, A. ferrooxidans remarkably improved the leaching efficiency of Al, Zn, Pb, and Mg (46%, 67% 11%, and 55%) respectively (Figure 4), which might due to the higher concentration of sulphuric acid (0.0289M) produced by A. ferrooxidans. It can be concluded that bio-enhanced electrokinetic remediation is more efficient than chemically enhanced electrokinetic remediation (Mouna et al., 2021). Thus, it cannot be concluded that the bacterial metabolites of the spent medium have the ability to increase the efficiency of electrokinetic remediation. This integrated method can be applied to remediate the fly ash contaminated soil in an ecofriendly manner. Further studies are required to determine the cost-effectiveness of using bacteria-produced sulphuric acid, taking into account the cost of the medium, batch culture time, and any waste processing cost.

The TCLP results are shown in Table 5. In the presence of A. thiooxidans, the results showed that lead concentrations decreased for all three setups; the concentrations for the ACID and DI setups decreased below the US EPA and NEA requirements. However, the concentrations of Cr, Mn, Zn, Ni, and Cu increased due to the dissolution of the anode, which is made of steel, containing Cr, Mn, and Ni as its constituents. As for Zn and Cu, the dissolution of the copper wires and crocodile clips from the connections could have added to the anode compartment. These metals may have formed complexes and migrated toward the substrate compartment, passing through the anion exchange membrane. A more worrying fact is that Zn, Ni, and Cr far exceeded the US EPA and NEA requirements. In the presence of A. ferrooxidans, Pb toxicity decreased for the SM setups, possibly due to metal leaching (Mouna et al., 2021).