Acidithiobacillus sp. applied to sewage sludge bioleaching: perspectives for process optimization through the establishment of optimal operational parameters

Abstract

Using Acidithiobacillus sp. during bioleaching assays is a well-known biological approach to solubilizing metals within sewage sludge. However, sludge dewatering has also been reported as a secondary treatment benefit. Based on a literature review, the present work provides perspectives regarding the enhancement of bioleaching outcomes on a laboratory scale by establishing optimal operational parameters. Data from different studies suggest that greater bioleaching efficiency may be achieved using a 10% (v/v) mixed inoculum of Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans in a ratio of 4:1, supplemented with ferrous sulfate (FeSO₄) and elemental sulfur (S⁰), and an initial system pH near 6.0. However, operational parameters must be established according to the type of sludge being treated due to differences in their compositions. Bioleaching duration is also an aspect that must be considered since treatments conducted for longer than 48 h increased the concentration of Extracellular Polymeric Substances (EPS), a characteristic associated with reducing dewaterability performance.

Introduction

Sludge management is a global challenge because its volume, water content, and the presence of contaminants may hamper the handling, transportation, and final disposal options (Anjum et al. 2016; Gherghel et al. 2019; Geng et al. 2020). Different strategies have been developed to reduce sludge generation and eliminate pathogens, organic pollutants, and potentially toxic metals, thereby minimizing the risks imposed on the environment and living organisms (Bertanza et al. 2015; Anjum et al. 2016). Dewatering is an essential step of wastewater treatment, and it aims at decreasing the sludge water content to reduce operational costs associated with sludge transport and disposal. Dewatering methods based on flocculants and mechanical dewatering have limitations as this conventionally dewatered sludge cake retains up to 80% of moisture (Kurade et al. 2014; Zhang et al. 2017). Therefore, the establishment of more efficient alternatives is necessary.

Bioleaching is a natural process of metal solubilization through the direct action of sulfur-oxidizing and iron-oxidizing bacteria, usually Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. Besides being a well-known approach to favor the removal of sludge-borne metals (Liu et al. 2012b), bioleaching also enhances sludge dewatering by reducing the pH of the system and promoting the solubilization of extracellular polymeric substances (EPS) (Kurade et al. 2014). However, previous studies are still centered on metal removal instead of sludge dewatering (Liu et al. 2012a). Nevertheless, this approach has been successfully adopted to favor advanced dewatering processes on a commercial scale in China since 2010 (Hu et al. 2015). After the treatment, the moisture content of the sludge may reach 60% or below (Zhou 2012). On the bench scale, Capillary Suction Time (CST), Time to Filter (TTF), and Specific Resistance to Filtration (SRF) are the indexes commonly used to assess sludge dewaterability, and their reduction indicates an improvement in dewatering performance.

The inoculation of Acidithiobacillus sp. and biosurfactant-producing microorganisms has been considered a promising strategy to improve the efficiency of bioleaching processes. Unlike chemical surfactants produced from petroleum derivatives, biosurfactants have many advantages, such as high biodegradability, biocompatibility, and ecological acceptability (Ambaye et al. 2021). Furthermore, they are efficient at reducing surface tension, which allows them to be added at lower concentrations (Drakontis and Amin 2020). During bioremediation, biosurfactants may increase the solubility of pollutants, accelerate oil degradation, and improve the availability of poorly soluble compounds (Nitschke and Pastore 2002).

Considering that metal solubilization and sludge dewatering rates are associated with different factors, this study aimed to establish optimal operational parameters for bioleaching assays on a laboratory scale, based on a literature review, to improve the efficiency of the bioleaching process.

Methodological approach

Scientific literature adopted in this review was selected and analyzed using Science Direct, Scopus, and Web of Science databases, considering the following set of keywords: (i) Acidithiobacillus, sewage sludge, dewatering; (ii) Acidithiobacillus, sewage sludge, biosurfactants; (iii) Acidithiobacillus, sewage sludge, surfactants; (iv) Dewatering, sewage sludge, biosurfactants; (v) Dewatering, sewage sludge, surfactants; (vii) Acidithiobacillus, dewatering, biosurfactants and; (viii) Acidithiobacillus, dewatering, surfactants. The analysis was focused on the period comprising 2016 and 2021 since it totalizes 58% of all the publications on the theme. With the obtained results, a pre-selection was performed, discarding theses, dissertations, review articles, conference abstracts, book chapters, technical reports, and duplicated publications. A second refinement was carried out by reading the abstracts of each article or, in specific cases, the entire article. Those articles that jointly addressed bioleaching and sewage sludge were selected for further analysis, totalizing 17 papers (Fig. 1). Additional literature was also consulted when approaching concepts and perspectives already consolidated in the analysis field.

Fig. 1.

Methodological approach used to select articles for analysis

Metrics of scientific production

Searching for sets of keywords in the databases yielded 358 scientific papers published between 1989 and 2021. The difference between the results returned in each database was evident, as 295 articles of the total number were obtained through Science Direct, 36 through Scopus, and 27 through the Web of Science.

Regarding the chronology of scientific production, the period ranging from 1989 to 1999 was marked by only a few publications on bioleaching and sludge dewatering. However, the 2000s were marked by the increase in the research on these topics, reaching peaks of publications in 2001, 2006, and 2010. In 2013, a growth trend in these publications was observed, which remains until nowadays (Fig. 2).

Fig. 2.

Research articles about bioleaching obtained from the search for keywords in Science Direct, Scopus, and Web of Science databases—Historical series of publications

The period comprising 2016 and 2021 was selected as the focus of this analysis as it totalizes 58% of all scientific production published on the subject (208 references). Among the papers analyzed, studies focused not only on using Acidithiobacillus sp. during sewage sludge bioleaching but also on treating sludge from specific activities and treatments based on non-biological approaches. Due to differences in alignment compared to our perspectives, these studies were not considered. After excluding duplicated references and selecting only articles that address bioleaching and dewatering of sewage sludge using Acidithiobacillus sp., 17 papers were obtained. Although the increasing number of publications denotes greater prominence over the last few years, scientific production on the theme is still not very expressive and is concentrated in a few countries or regions. In this context, China stands out, producing around 70% of the publications selected for review, followed by Hong Kong, Australia, and Brazil, representing 18%, 6%, and 6%, respectively.

Sewage sludge

Sewage sludge is a wastewater treatment byproduct generated over different phases of a Waste Water Treatment Plant (WWTP). Although the composition of the effluent depends on its origin, sewage sludge can be characterized as a mixture of water, organic matter, microorganisms, and potentially toxic substances (Kacprzak et al. 2017) that can be adsorbed to its organic fraction or particulate matter. Among these substances, Cadmium (Cd), Copper (Cu), Chromium (Cr), Nickel (Ni), Zinc (Zn), Lead (Pb), and Arsenic (As) are the most commonly detected inorganic pollutants (Pathak et al. 2009), implying risks for secondary pollution. Furthermore, sludge management can comprise up to 50% of the total costs of a treatment plant (Neyens et al. 2004; Mahmood and Elliott 2006), as the volume and water content of the residue hamper its handling, transportation, and disposal options (Fig. 3).

Fig. 3.

Stages of wastewater and sewage sludge treatment, from water consumption until management—water consumption: urbanization process enhances water consumption and, consequently, the amount of wastewater generated; wastewater treatment: effluents are sent to Wastewater Treatment Plants (WWTP), and both liquid and solid phases are submitted adequately to specific treatment strategies; sludge generation: sludge is generated in several parts of a WWTP depending on the type of treatment implemented; sludge management: the generated sludge can be submitted to stabilization, thickening, dewatering, and another specific process before its disposal to reduce volume, water content and the presence of pathogens and toxic substances

Bioleaching is a biological approach applied to solubilize metals within contaminated residues using acidophile bacteria. A. thiooxidans and A. ferrooxidans, two obligately aerobic, chemolithoautotrophic, and gram-negative bacteria, are commonly used. Both bacteria can oxidize elemental sulfur and reduce inorganic sulfur compounds to favor their growth. However, A. ferrooxidans may also oxidize Fe²⁺ (Zhang et al. 2018; Yang et al. 2019). Additionally, the improvement of sewage sludge dewatering has been reported as a secondary benefit of bioleaching treatment (Gao et al. 2017; Ban et al. 2018; Cai et al. 2021; Li et al. 2022). Compared to other treatment routes, bioleaching is considered an environmental friendliness and low-cost option (Zhang et al. 2020). A further understanding of the conditioning aspects of bioleaching outcomes is needed to efficiently provide the solubilization of metals and the removal of water from sewage sludge.

Conditioning aspects of metal solubilization and sludge dewatering

Heavy metals migrate to the sludge during sewage treatment through physical–chemical and biological interactions (Geng et al. 2020). In this context, bioleaching is an effective alternative for reversing this process by transferring sludge metal content from the solid to the liquid phase, allowing subsequent recovery (Marchenko et al. 2018). Throughout bioleaching, A. thiooxidans and A. ferrooxidans solubilize metals through direct or indirect mechanisms, the first based on the physical contact between the bacterial cell and sulfide surface and the latter on the oxidation of sulfide minerals through the action of the bacterial secreted lixiviant (Bosecker 1997). Regardless of the process, acidophiles properly oxidize Fe²⁺ to Fe³⁺ and reduced sulfur compounds (S₈, S₂O₃²⁻, and H₂S or polysulphides) to sulfuric acid (H₂SO₄). Then, metals in sludge can be solubilized by the resulting ferric and protons from sulphuric acid (Srichandan et al. 2019). The following equations describe the direct and indirect mechanisms of heavy metals solubilization by Acidithiobacillus sp.:

Direct mechanisms:

$${\mathrm{FeS}}_2+3.5{\mathrm{O}}_2+{\mathrm{H}}_2\mathrm{O}\to {\mathrm{Fe}}^{2+}+2{\mathrm{H}}^{+}+2{\mathrm{SO}}_4^{2-},$$ 1

$$2{\mathrm{Fe}}^{2+}+\frac{1}{2}{\mathrm{O}}_2+2{\mathrm{H}}^{+}\to 2{\mathrm{Fe}}^{3+}+{\mathrm{H}}_2\mathrm{O}.$$ 2

Indirect mechanisms:

$${\mathrm{FeS}}_2+14{\mathrm{Fe}}^{3+}+8{\mathrm{H}}_2\mathrm{O}\to 15{\mathrm{Fe}}^{2+}+16{\mathrm{H}}^{+}+2{\mathrm{SO}}_4^{2-},$$ 3

$$M+2{\mathrm{Fe}}^{3+}\to M^{2+}+{\mathrm{S}}^0+2{\mathrm{Fe}}^{2+},$$ 4

$${\mathrm{S}}^0+\frac{3}{2}{\mathrm{O}}_2+{\mathrm{H}}_2\mathrm{O}\to 2{\mathrm{H}}^{+}+{\mathrm{SO}}_4^{2-},$$ 5

where M is an insoluble metal sulfide.

Medium pH is considered one of the leading conditioning factors of metal solubilization since the functional groups to which they are adsorbed become protonated in acidic media. This process negatively affects the adsorption and bioavailability of metals, favoring their mobility and release in the liquid phase (Peng et al. 2009). Thus, metals tend to be more insoluble or adsorbed to other compounds in alkaline media, while they have more excellent solubility in acidic environments (Camargo et al. 2018). To improve metal solubilization, besides low pH values, high Oxidation–Reduction Potential (ORP) values are also necessary (Lombardi and Garcia 2002). During bioleaching, microbial activity promotes a reduction in pH and an increase in ORP values (Wu et al. 2020). The increase in ORP results in higher rates of metal sulfides oxidation and organic compounds degradation, which accelerate the release of complexed or adsorbed heavy metals. The following equation describes the relationship between the oxidation rate and the release of metals:

$${M\mathrm{S}}_2+\frac{15}{4}{\mathrm{O}}_2+\frac{7}{2}{\mathrm{H}}_2\mathrm{O}\to M{(\mathrm{OH})}_3+2{\mathrm{SO}}_4^{2-}+4{\mathrm{H}}^{+},$$ 6

where M is an insoluble metal sulfide.

EPS content also has a vital role in metal transfer, mainly due to the presence of specific functional groups in the EPS matrix, such as carboxyl, hydroxyl, phosphoryl, and amide, which favor the establishment of connections between metal cations and microorganisms (More et al. 2014; Xu et al. 2017; Molaey et al. 2021). Geng et al. (2020) describe this process in two stages; the first is independent of cellular metabolism and is based on the adsorption of metal ions to the biomass via ion exchange, complexation, and inorganic precipitation reactions. The second stage relates to active uptake by cellular metabolism, where proteins transport heavy metals from the cell surface to the intracellular environment via biochemical reactions.

Regarding sludge dewaterability, CST, TTF, and SRF are the most commonly used parameters analyzed on a laboratory scale, although they are empirical methods that require greater precision (Wei et al. 2018; Wu et al. 2020). CST is a more straightforward and economical methodology than TTF and SRF due to not requiring an external pressure or suction source as the other mentioned methods. The extraction of EPS is also widely used to measure dewatering performance, mainly via the release of bound water content. However, these inferences depend on additional analyses to evaluate the content of proteins, polysaccharides, and humic substances. These are usually measured by colorimetric methods whose results may be underestimated or overestimated in wastewater (Wei et al. 2018).

Considering that bioleaching outcomes are associated not only with all these discussed aspects but also with the origin of the wastewater, the type of substrate used, and physicochemical aspects of the environment, perspectives to improve treatment efficiency on the laboratory scale are highlighted in the following.

Substrate usage to favor Acidithiobacillus sp. growth

Ferrous sulfate (FeSO₄) and elemental sulfur (S⁰) are the most widely used supplements as energy sources for species of Acidithiobacillus during bioleaching assays. The oxidation of these substrates increases the concentration of H⁺ ions, which acidifies the medium and promotes metal solubilization. Different studies have pointed out correlations between the amount of substrate added and the results of bioleaching so that the gradual increase in supplement concentration is often associated with improvements in metal solubilization rates and sludge dewatering (Wong et al. 2016; Gao et al. 2017; Ban et al. 2018; Cai et al. 2021; Zhao et al. 2021).

Cai et al. (2021) analyzed the efficiency of an A. ferrooxidans strain (ILS-2 from the Land and Water Business Unit of the Commonwealth Scientific and Industrial Research Organization) in the dewatering of digested sludge (Total solids—TS 28.3 g/kg, Volatile Solids—VS 19.6 g/kg, CST 339.1 s, pH 7.7, Zn 925.0 mg/kg, Mn 194.0 mg/kg, Ni 36.1 mg/kg, Sn 342.0 mg/kg, Ba 263.0 mg/kg, and Cr 44.1 mg/kg) collected from a WWTP in South East Queensland, Australia. The inoculum used was previously grown in a preculture medium (g/L) ((NH₄)₂SO₄ 0.8, MgSO₄.7H₂O 2.0, K₂HPO₄ 0.4, FeSO₄.7H₂O 20.0, and pH 1.8) and Wolfe's Mineral Solution 5.0 mL/L. The strain was subcultured monthly with a 10% inoculum cultivated at 28 °C for 2 to 3 days. Subcultures were made to adapt the strain to grow in the sludge: in the first subculture, 50 mL of digested sludge with 4.0 g/L of Fe²⁺ were mixed with 50 mL of preculture medium at 28 °C and 180 rpm until the system reached a pH of 2.0 ~ 3.0 and ORP near 600 mV; in the second and third subculture, the same process was repeated, mixing 30 mL of the first subculture and 70 mL of fresh digested sludge supplemented with 4.0 g/L of Fe²⁺ and 10 mL of the second sub-culture and 90 mL of fresh digested sludge supplemented with 4.0 g/L of Fe²⁺, respectively. Bioleaching tests were performed using 10 g of inoculum from the third subculture and 90 g of sludge supplemented with different concentrations of Fe²⁺ (10, 15, and 21%). The authors observed that the highest amount of Fe²⁺ resulted in the best dewatering conditions, with a reduction in CST from an initial 339–26 s after 48 h and a 41% reduction in moisture content in contrast with the control sample. The treatment with the best dewatering efficiency was also the one that corresponded to the maximum solubilization of the metals, with reductions of 93% Zn, 88% Mn, 80% Ni, 42% Sn, 24% Ba, and 23% Cr.

Wong et al. (2016) also indicate a correlation between substrate concentration and dewatering results, conducting bioleaching assays with bioflocculant produced by A. ferrooxidans ANYL-1 previously isolated from anaerobically digested sludge. The strain was maintained in a modified 9 K medium (g/L) ((NH4)₂SO₄ 3.0, MgSO₄.7H₂O 0.5, K₂HPO₄ 0.5, KCl 0.1, Ca(NO₃)₂.2H₂O 0.01, and FeSO₄.7H₂O 44.2 g/L). The culture was incubated at 30 °C and 180 rpm before being filtered. The filtrate, also called bioflocculant, containing bioferric iron and A. ferrooxidans cells in a density of ~ 10⁸ cells/mL, was used as inoculum for sludge conditioning. The tests were carried out using two types of sludge, Saline Activated Sludge (ACS) (TS 2.08%, ORP − 89.6 mV, CST 12.6 s, SRF 1.0 × 10¹³ m/kg, and pH 6.7) and Anaerobically Digested Sludge (ADS) (TS 2.10%, ORP − 117.1 mV, CST 19.5 s, SRF 8.3 × 10¹² m/kg, and pH 7.7), both collected from Sha Tin Sewage Treatment Works, in Hong Kong. During bioleaching experiments, 270 mL of sludge and 30 mL of the filtrate were mixed and supplemented with different ratios between Fe²⁺ and total solids (0:1, 0.01:1, 0.05:1, and 0.1:1). The best conditions of dewatering were obtained from the highest Fe²⁺ concentration, resulting in an SRF drop to approximately 1.0 × 10¹² m/kg in activated sludge and 3.0 × 10¹¹ m/kg in digested sludge. These results indicate that substrate concentration and bioleaching efficiency are the same for both activated sludge and digested sludge treatment. However, substrate oxidation in each sludge occurred at different rates. In the activated sludge, the results of the conversion of Fe²⁺ to Fe³⁺ were observed on the first day of treatment, while in the digested sludge, only after the second day. This aspect was possibly caused due to differences in the composition of the sludge in terms of total solids and EPS content, whose higher concentration implies resistance to the dewatering process. Thus, operational parameters aiming at bioleaching efficiency could be better established if evaluated for each type of sludge, considering its specificities (Cai et al. 2021), such as different origins and physicochemical aspects (Table 1).

Table 1.

Characterization of sewage sludge samples used in different studies based on origin and physicochemical parameters

Sludge origin	pH	Organic matter (%)	Solid content (%)	SRF (m/kg)	CST (s)	References
WWTP Wulogkou Zhengzhou, China	6.9	50.81	2.69	1.33 × 1013	N/A	Ban et al. (2018)
WWTP in South East Queensland, China	7.7	N/A	2.83	N/A	339.1	Cai et al. (2021)
WWTP in Porto Feliz, Brazil	7.4	N/A	10.40	N/A	N/A	Camargo et al. (2018)
WWTP Wulogkou Zhengzhou, China	6.9	50.81	3.28	1.33 × 1013	N/A	Gao et al. (2017)
WWTP in Guangzhou, China	6.6	N/A	N/Ac	3.79 × 1012	N/A	Huang et al. (2020)
STW Sha Tin, Hong Kong	6.8	N/A	2.40	3.29 × 1013	38.7	Kurade et al. (2016)
WWTP in Wuxi, China	6.4	N/A	4.00	2.86 × 1012	N/A	Liu et al. (2016)
STW Sha Tin, Hong Kong	7.5	42.50	2.05	8.62 × 1012	27.0	Murugesan et al. (2016)
STW Sha Tin, Hong Kong	6.7	N/A	2.08	1.00 × 1013	12.6	Wong et al. (2016)
STW Sha Tin, Hong Kong	7.7	N/A	2.10	8.30 × 1012	19.5	Wong et al. (2016)
WWTP Taihu New City, China	7.0	46.00	2.63	5.21 × 1012	23.2	Lu et al. (2019)
WWTP in Hewen Lake, China	6.7	28.00	4.20	8.47 × 1011	40.1	Zhang et al. (2018)
WWTP Wulogkou Zhengzhou, China	6.9	50.81	N/A	1.33 × 1013	N/A	Zhao et al. (2021)
WWTP Taihu New City, China	7.5	48.84	3.58	1.98 × 1013	N/A	Zheng et al. (2016a)
WWTP Taihu New City, China	7.4	N/A	3.42	6.31 × 1013	N/A	Zheng et al. (2016b)

WWTP Wastewater Treatment Plant, STW Sewage Treatment Works, N/A data not available

Although the applicability of S⁰ as an energy source in treatments with mixed cultures of Acidithiobacillus sp. is a well-established procedure, some studies signalize operational difficulties when using this substrate, given its low solubility in water. Hence, replacing it with a more soluble substrate would be a great strategy to increase the efficiency of the treatment, facilitating contact with bacteria present in the medium. For this reason, sodium thiosulfate (Na₂S₂O₃) has been approached as a promising alternative.

Ban et al. (2018), implementing a mixed culture of A. ferrooxidans (ATCC 23270) and A. thiooxidans (ATCC 53990) for the dewatering of sewage sludge, investigated the effects of different concentrations of Na₂S₂O₃ (0.5, 1.0, 1.5, and 2.0 g/L) and FeSO₄.7H₂O (4.0, 6.0, 8.0, and 10.0 g/L). The sludge used (Organic Matter—OM 50.81%, TS 2.69%, ORP 14 mV, SRF 1.33 × 10¹³ m/kg, pH 6.86, Cu 513.5 mg/kg, Zn 986.3 mg/kg, Pb 103.9 mg/kg, and Cr 206.6 mg/kg) was collect from a WWTP in Zhengzhou, China. A. ferrooxidans and A. thiooxidans were grown in modified 9 K and Waksman liquid medium (compositions not available), respectively. Before being supplemented with Na₂S₂O₃, Acidithiobacillus sp. were submitted to an acclimatizing procedure of mixing 90 mL of Waksman medium with 5 mL of A. ferrooxidans and A. thiooxidans each, maintained at 28 °C and 180 rpm until the system pH reached 2.0. Subsequently, 10 mL of the solution was withdrawn to the next acclimatizing step and repeated thrice. The inoculum used during the bioleaching assay consisted of 15 mL of acclimatized culture, 135 mL of fresh sludge, 1.0 g/L of Na₂S₂O₃, and 6.0 g/L of FeSO₄.7H₂O incubated at 28 °C and 180 rpm until pH dropped to 2.0. It was observed that the supplementation with the maximum analyzed quantity of Na₂S₂O₃ and FeSO₄.7H₂O, 2.0 g/L and 10.0 g/L, respectively, was the set that resulted in the most efficient dewatering performance. Furthermore, it implied that both A. ferrooxidans and A. thiooxidans could oxidize Na₂S₂O₃ properly.

When analyzing the effects of FeSO₄.7H₂O concentrations, although supplementing the medium with 10.0 g/L resulted in the lowest SRF value (6.24 × 10¹¹ m/kg) at the end of the assay, the difference of this parameter when compared to the 8 g/L concentration scenario is not significative (only graphical data available). Furthermore, using 8 g/L of FeSO₄.7H₂O faster declined the system’s pH, reaching near 2.5 after 1.5 days of assay—on the other hand, with 10 g/L, the system reached a 2.5 pH only after 3.0 days. Concerning the use of Na₂S₂O₃, the greater substrate concentration was associated with a sharp drop in SRF, denoting an improvement in dewatering. Regarding metal solubilization, the maximum addition of substrates did not result in better solubilization rates, obtained only by adding 10.0 g/L of FeSO₄.7H₂O and 1.5 g/L of Na₂S₂O₃—the maximum concentration of substrates resulted in solubilization of Cu, Zn, Pb, and Cr of 67.10%, 58.43%, 19.76%, and 24.36%, respectively. In contrast, the optimal concentration resulted in solubilization of 83.55%, 78.92%, 31.68%, and 37.95%. Regarding the substrate, the difference between the two conditions was related to the concentration of Na₂S₂O₃; complementary analyzes can contribute to new perspectives on the influence of this substrate on the solubilization of metals.

Zhao et al. (2021) also analyzed the combined use of Na₂S₂O₃ and Polysorbate 20, a commercial surfactant able to neutralize the hydrophobic nature of S⁰ (Huo et al. 2014), in bioleaching experiments. The sludge used (OM 50.81%, ORP 14 mV, SRF 1.33 × 10¹³ m/kg, pH 6.86, moisture content: 97.31%, Cu 513.5 mg/kg, Zn 986.3 mg/kg, Pb 103.9 mg/kg, and Cr 206.6 mg/kg) was directly collected from the thickening tank of Zhengzhou Wulongkou WWTP in Henan, China. The inoculum composed of A. ferrooxidans (ATCC 23270) and A. thiooxidans (ATCC 53990) were cultured in Modified 9 K and Waksman media (compositions not available), supplemented with 44.2 g/L FeSO₄.7H₂O or 10.0 g/L S⁰, respectively. Firstly, the cultures were maintained at 28 ºC and in an oscillation of 3 Hz for 3–4 days until reaching a cell density of around 108 colony forming units (CFU) per milliliter. Subsequently, they were submitted to an enrichment process of mixing 100 L of raw sludge, 10 L of A. ferrooxidans, and 10 L of A. thiooxidans in a continuous aeration sequence batch reactor. During the process, 2.0 g/L S⁰, Na₂S₂O₃, FeSO₄.7H₂O, or Polysorbate 20 were used as an energy source. The system was monitored until a pH drop near 2.0. Raw sludge was inoculated with 10% (v/v) of the sludge slurry, and the process was repeated until Acidithiobacillus sp. was the dominant strain in the medium. The assays outcomes showed the greatest dewatering efficiency from the use of a mixed culture of Acidithiobacillus sp. with 8.0 g/L FeSO₄.7H₂O, 1.0 g/L S⁰, 0.75 g/L Na₂S₂O₃, and 0.5 g/L Polysorbate 20—the SRF changed from initial 1.33 × 10¹³ m/kg to 1.90 × 10¹² m/kg and a 45% reduction in moisture content compared to the control condition was observed. This same treatment was appointed as the most efficient solubilization of Cu, Zn, Pb, and Cr, although numerical data was unavailable.

The effects of different concentrations of Polysorbate 20 (0.0, 0.5, 1.0, and 1.5 g/L), Fe²⁺ (4.0, 6.0, 8.0, and 10.0 g/L), and S⁰ (0.5, 1.0, 1.5, and 2.0 g/L) over sludge dewatering were also evaluated by Gao et al. (2017), when used as an energy source for mixed culture of A. ferrooxidans and A. thiooxidans. The sludge submitted to bioleaching (OM 50.81%, TS 3.28%, water content: 95.21%, SRF 13.32 × 10¹² m/kg, and pH 6.86) was collected from a thickening tank in a WWTP in Zhengzhou, China. A. ferrooxidans (ATCC 23270), and A. thiooxidans (ATCC 53990) were maintained in Modified 9 K and SM liquor media (compositions not available), respectively, supplemented with 44.2 g/L FeSO₄.7H₂O and 10.0 g/L S⁰. The culture was incubated at 28 °C and 180 rpm for 3–4 days until reaching cell density near 10⁸ CFU/mL. The inoculum was prepared by mixing 15 mL of each Acidithiobacillus sp. strain, 270 mL of sewage sludge, 10.0 g/L FeSO₄.7H₂O, and 2.0 g/L S⁰, being kept at 28 °C and 180 rpm until pH decreased to 2.0. The highest dewatering efficiency was obtained when using 10.0 g/L of FeSO₄.7H₂O and 2.0 g/L of S⁰; the maximum concentrations of these supplements, combined with 1.0 g/L of Polysorbate 20—SRF ranged from an initial 13.32 × 10¹² to 8.1 × 10¹¹ m/kg and the moisture content reduced by 50.5% when compared to the control. Whereas the acidification of the medium was accelerated when applied this supplement concentration, it was related to a more efficient consumption of nutrients by the bacteria. However, Polysorbate 20 concentrations greater than 2.5 g/L seemed to negatively affect the activities of Acidithiobacillus sp., as the substrate oxidation process was delayed.

As highlighted by Zhao et al. (2021), the type of substrate used to favor bacterial growth is directly associated with the predominant species present in the medium and, consequently, the efficiency of the bioleaching treatment. Thus, to establish optimal parameters to improve bioleaching outcomes, it is mandatory to comprehend the dynamics between Acidithiobacillus sp. and novel sources of nutrients.

Inoculum size and composition

Inoculum size and content are vital during bioleaching as they influence the substrate oxidation rate and the acidification of the medium, both necessary conditions for guaranteeing treatment performance. Different authors analyzed not only the effects of using mixed cultures of Acidithiobacillus sp. on sludge dewatering (Table 2) and metal solubilization (Tabel 3) but also the co-inoculation with microorganisms capable of neutralizing or attenuating harmful aspects of the medium, such as Rhodotorula mucilaginosa and Mucor circinelloides (Kurade et al. 2016; Zheng et al. 2016b; Zhang et al. 2017; Zhou et al. 2017; Camargo et al. 2018).

Table 2.

Bioleaching operational parameters used in different studies and resulting changes over sludge dewaterability

Microorganisms	Inoculum size	FeSO4 (g/L)	S0 (g/L)	Assay duration (hours)	Final system pH	Initial SRF (m/kg)	Final SRF (m/kg)	References
A. ferrooxidans + A. thiooxidans	10%	8.0	N/A	72	2.1	9.89 × 1012	1.03 × 1011	Ban et al. (2018)
A. ferrooxidans + A. thiooxidans	20%	10.0	2.0	72	2.0	1.26 × 1013	8.10 × 1011	Gao et al. (2017)
A. ferrooxidans + A. thiooxidans	10%	2.4	2.0	48	2.5	3.79 × 1012	8.51 × 1011	Huang (2020)
A. ferrooxidans	10%	N/A	N/A	1	5.9	3.29 × 1013	3.62 × 1012	Kurade et al. (2016)
A. ferrooxidans	4%	2.7	N/A	72	2.4	2.32 × 1012	5.31 × 1010	Liu et al. (2016)
A. ferrooxidans	10%	1.02	N/A	24	4.1	8.62 × 1012	5.25 × 1011	Murugesan et al. (2016)
A. ferrooxidans	10%	2.08	N/A	96	2.2	1.00 × 1013	1.00 × 1012	Wong et al. (2016)
A. ferrooxidans	10%	2.10	N/A	96	2.2	8.30 × 1012	3.00 × 1011	Wong et al. (2016)
A. ferrooxidans	10%	10.0	N/A	40	2.8	5.21 × 1012	3.15 × 1011	Lu et al. (2019)
A. thiooxidans + R. mucilaginosa	5%	N/A	8.0	48	2.3	2.60 × 1012	1.00 × 1012	Zhang et al. (2018)
A. ferrooxidans + A. thiooxidans	10%	N/A	8.0	48	2.5	1.33 × 1013	1.91 × 1012	Zhao et al. (2021)
A. ferrooxidans	10%	10.0	N/A	24	3.1	1.82 × 1013	1.35 × 1012	Zheng et al. (2016a)
A. ferrooxidans + M. circinelloides	20%	10.0	N/A	48	2.5	1.89 × 1013	1.00 × 1012	Zheng et al. (2016a)
A. ferrooxidans + A. thiooxidans	10%	10.0	2.0	48	2.6	6.31 × 1013	5.16 × 1012	Zheng et al. (2016b)
A. ferrooxidans + A. thiooxidans	15%	N/A	N/A	144	2.0	1.96 × 1009	2.69 × 1008	Zhou et al. (2017)

N/A data not available

Table 3.

Bioleaching operational parameters used in different studies and resulting changes over sludge metal solubilization

Microorganisms	Inoculum size	FeSO4 (g/L)	S0 (g/L)	Final pH	Cu (%)	Zn (%)	Pb (%)	Cr (%)	Cd (%)	References
A. ferrooxidans + A. thiooxidans	10%	8.0	N/A	2.1	83.5	78.9	31.7	37.9	N/A	Ban et al. (2018)
A. ferrooxidans	10%	30.0	N/A	2.9	35.0	93.0	N/A	23.0	N/A	Cai et al. (2021)
A. ferrooxidans + A. thiooxidans	5%	10.0	2.0	2.1	37.4	70.3	8.1	12.6	17.9	Camargo et al. (2018)
A. ferrooxidans + A. thiooxidans + R. mucilaginosa	11%	10.0	2.0	2.2	22.0	76.5	7.1	9.8	9.8	Camargo et al. (2018)
A. ferrooxidans + A. thiooxidans	15%	N/A	N/A	2.0	89.6	72.8	39.4	N/A	N/A	Zhou et al. (2017)

N/A data not available

Zhou et al. (2017) evaluated how different Acidithiobacillus sp. inoculum sizes (0, 5, 10, 15, and 20%), with varying proportions of A. thiooxidans and A. ferrooxidans (4:1, 2:1, 1:1, 1:2, and 1:4) are related to the efficiency of bioleaching. The sludge used (OM 46.4%, TS 5.3%, pH 6.94, SRF 1.96 × 10⁹ s²/g, Cu 288.0 mg/kg, Zn 473.0 mg/kg, and Pb 147.0 mg/g) was collected from a WWTP in Zhengzhou, China. A. ferrooxidans LX5 (CGMCC No. 0727) and A. thiooxidans TS6 (CGMCC No. 0759), both obtained from China General Microbiological Culture Collection Center, were cultivated in a Modified 9 K medium with 10.0 g/L of energy source substrate – only S⁰ for A. thiooxidans and A. ferrooxidans, S⁰ and FeSO₄.7H₂O in a 1:9 proportion). The inoculum used during bioleaching assays was obtained after submitting Acidithiobacillus sp. to a three-phase acclimation process. During this, 100 mL of 9 K modified medium and 20 mL of bacterial suspension were transferred to a 250-mL Erlenmeyer flask and incubated at 15 °C and 180 rpm until reaching a cell density in the range of 10⁸ cells/mL. Afterward, batch experiments were carried out with 100 mL of raw sludge and different inoculum sizes and compositions to determine which would perform better concerning bioleaching. For inoculum sizes ranging from 0 to 15%, the acidification of the medium was accelerated proportionally to the number of bacteria. In this sense, the 15% inoculum was the optimum condition analyzed.

This same condition was the most efficient in dewatering and solubilizing metals, leading to a reduction of approximately 87% of SRF and solubilization of Cu, Zn, and Pb of 79.5%, 73.8% and 34.6%, respectively. When assessing the 20% inoculum size, less favorable results were observed compared to those obtained with the 15% inoculum test group, which may indicate a shortage of nutrients in the medium, resulting in competition between the microorganisms and negatively affecting the bioleaching process. Regarding the inoculum composition, the greater presence of A. thiooxidans was associated with more efficient nutrient consumption, leading to a rapid pH drop. At the end of the treatment, the lowest pH (approximately 2.0) was observed when using an A. thiooxidans and A. ferrooxidans inoculum ratio of 4:1. This proportion also resulted in the best dewatering conditions with a reduction of 86.3% in the SRF and reductions in Cu, Zn, and Pb of 89.6%, 72.8%, and 39.4%, respectively, 10% more solubilization than when using a pure culture of A. ferrooxidans.

Zhang et al. (2017) reinforced the relationship between A. thiooxidans concentration and its effects on the acidification of the medium. Bioleaching assays were carried out using an inoculum composed of A. thiooxidans JJU-1 and R. mucilaginosa JJU-2, previously isolated from sewage sludge. A. thiooxidans was cultured in Waksman medium (composition not available) supplemented with 5.0 g/L S⁰ at 28 °C and 180 rpm until reaching a cell density of 10⁸ cells/mL, and R. mucilaginosa was maintained in potato dextrose agar (PDA) at 28 °C and 120 rpm until reaching a cell density of 10¹² cells/mL. The inoculum was obtained by mixing 2.1 L of A. thiooxidans, 700 mL of R. mucilaginosa and 14.2 L of sludge supplemented with 8.0 g/L S⁰ in a bioreactor at 28 °C and 200 rpm until reaching a pH near 2.0. The ratio between bacteria and yeast was 3:1, and thus, the higher the inoculum size, the greater the amount of A. thiooxidans. This inoculum was mixed at different proportions (2, 5, 10, and 20%) with raw sludge (15.68, 15.2, 14.4, and 12.2 L, respectively) and 8.0 g/L of S⁰ during bioleaching tests. Assays were carried out with a sludge collected from Hewen Lake WWTP in Jiujiang City, China (TS 4.20%, CST 40.10 s, OM 28%, pH 6.72, and SRF 8.47 × 10¹¹ m/kg).

Although the aspects regarding the presence of R. mucilaginosa were not fully explored, the influence of the inoculum size over ORP and pH was observed. In the 2% inoculum experiment, the pH decreased from 4.97 to 2.52, and ORP ranged from 110 to 253 mV after 4 days. However, the 20% inoculum size changed pH and ORP from 4.80 to 0.76 and 125 to 346 mV, respectively. In terms of dewaterability, however, the greater concentration of the inoculum resulted in an unexpected increase in CST and SRF values, negatively affecting sludge dewatering. Applying a 20% inoculum size, CST and SRF ranged from an initial 153.2 s and 1.19 × 10¹² m/kg, respectively, to 885.4 s and 10.64 × 10¹² m/kg after four days. Meanwhile, using a 5% inoculum decreased CST and SRF from 151.0 s and 2.60 × 10¹² m/kg to 65.0 s and 1.00 × 10¹² m/kg after 3 days, respectively. Despite the 5% inoculum also showing an increase in those parameters after day 4, that concentration was associated with the optimum dewaterability results, in addition to setting a new perspective concerning the optimum duration limit to favor the treatment.

Zheng et al. (2016b) investigated the use of M. circinelloides, R. mucilaginosa, and A. ferrooxidans as inoculums for the bioleaching of sewage sludge samples (OM 48.84%, TS 3.58%, pH 7.54, and SRF 1.98 × 10¹³ m/kg) collected from the Taihu New City WWTP thickening pond, in Wuxi City, China. The authors also highlighted the effects of sequential and simultaneous inoculation. A. ferrooxidans LX5 (CGMCC No. 0727) was grown in a modified 9 K medium (composition not available) supplemented with 44.2 g/L FeSO₄.7H₂O, maintained at 28 °C and 180 rpm until a cell density of 10⁸ cells/mL was reached. M. circinelloides ZG-3, previously isolated from sewage sludge, was grown in potato dextrose broth (PDB), where 1% (v/v) of a spore suspension at a density of 10⁵ spores/mL was kept at 28 °C and 120 rpm until reaching a concentration of 3.78 mg/mL mycelium dry weight. R. mucilaginosa R30, also isolated previously from sludge, was cultivated in PDB at 28 °C and 120 rpm until a cell density of 10¹² cells/mL. 90 mL of primary sludge DOM obtained by filtering sewage sludge supernatant was inoculated with 10 mL of the fungus to investigate the degradability of DOM by M. circinelloides. After being kept at 28 °C and 120 rpm for three days, DOM concentration decreased from an initial 755.56 mg dissolved organic carbon per liter (DOC/L) to 680.00 mg DOC/L.

Furthermore, the time required to completely oxidize Fe²⁺ used as an energy source by an inoculum in a 9 K medium supplemented with primary sludge DOM decreased from 11 to 4 days when the medium was inoculated with M. circinelloides, demonstrating great efficiency. Sequential inoculation of microorganisms, which consisted of the inoculation of both 30 mL of A. ferrooxidans and 30 mL of M. circinelloides in 240 mL of raw sludge supplemented with 10.0 g/L FeSO₄.7H₂O was more effective in terms of dewatering than simultaneous inoculation, which occurred by mixing 35 mL of M. circinelloides and 315 mL of raw sludge on day 0, followed by inoculation of 10% (v/v) A. ferrooxidans and 10.0 g/L FeSO₄.7H₂O on day 1. The differences were probably due to restrictions imposed by both microorganisms—co-inoculation with A. ferrooxidans rapidly reduced the pH of the medium (approximately 3.0 after one day of treatment) and was harmful to M. circinelloides, whose ideal pH range for growth is 5.0. Considering the development was hampered, the fungus is less effective in degrading DOM, not favoring the activity of Acidithiobacillus sp. Thus, although simultaneous inoculation has resulted in improvements in dewatering, with a reduction in the SRF from 1.82 × 10¹³ to 3.38 × 10¹² m/kg, the sequential inoculation of M. circinelloides and A. ferrooxidans with an interval of 24 h was even better, with a variation in the SRF from 1.89 × 10¹³ to 1.00 × 10¹² m/kg, after two days of treatment. This strategy also proved to be more efficient than sequential inoculation of A. ferrooxidans and R. mucilaginosa in dewatering, resulting in an SRF 34% higher than that obtained in treatment with A. ferrooxidans and M. circinelloides.

Although using biosurfactants is a promising strategy to favor bioleaching, few studies on the subject were carried out in the analyzed period—R. mucilaginosa and M. circinelloides, for instance, are microorganisms that produce biosurfactants having great potential for sludge dewatering. However, this aspect was not highlighted in the analyzed data. Camargo et al. (2018) made the only contribution from this perspective when investigating the effects of the inoculation of Acidithiobacillus sp. with Meyerozyma guilliermondii, a non-conventional biosurfactant producer yeast, on the solubilization of metals from anaerobic sludge (TS 10.4%, pH 7.4, Cd 20.7 mg/kg, Cr 619.9 mg/kg, Cu 2201.0 mg/kg, Ni 356.0 mg/kg, Pb 629.2 mg/kg, and Zn 8216.8 mg/kg) collected from Porto Feliz WWTP in São Paulo, Brazil. A. ferrooxidans (ATCC 23270) and A. thiooxidans (ATCC 19377) were respectively maintained in modified 9 K medium (g/L) ((NH₄)₂SO₄ 3.0 g, KCl 0.1 g, K₂HPO₄ 0.5 g, MgSO₄.7H₂O 0.5 g, and pH 2.8) supplemented with 44.2 g/L of FeSO₄.7H₂O and modified T&K medium (g/L) ((NH₄)₂SO₄ 2.5, K₂HPO₄ 0.45, MgSO₄.7H₂O 2.5, and pH 1.8) supplemented with 10.0 g/L of S⁰. The inoculum used during the assays was prepared by mixing Acidithiobacillus sp. with 100 mL of 9 K or T&K medium at 30 °C and 150 rpm until reaching a 10⁸ cell/mL cell density. M. guilliermondii, previously isolated from soil contaminated with diesel, was obtained from the Laboratory of Environmental Microbiology Collection at the Federal University of São Carlos and was grown in Czapek medium at 30 °C and 150 rpm until reaching a cell density of 10⁷ cells/mL. Sludge bioleaching was carried out using 222.5 mL of autoclaved sewage sludge, 12.5 mL of A. ferrooxidans, and A. thiooxidans, 10.0 g/L of FeSO₄.7H₂O, 2.0 g/L of S⁰ and 15 mL of M. guilliermondii. The presence of the yeast was associated with difficulties in the medium acidification process, delaying the beginning of bioleaching. Furthermore, its inoculation with Acidithiobacillus sp. was less efficient in terms of solubilization of Cr, Cu, Ni, and Zn, only showing better results for Cd, when compared to the inoculum containing just Acidithiobacillus sp.—with the presence of M. guilliermondii, the solubilizations obtained were 76.5% Zn, 59.8% Ni, 22.0% Cu, 9.8% Cd, 9.8% Cr, and 7.1% Pb. At the same time, without yeast, they were 70.3% Zn, 53.1% Ni, 37.4% Cu, 17.9% Cd, 12.6% Cr, and 8.1% Pb. Given the lower solubilization efficiencies, the hypothesis was that the yeast acts as a bioaccumulator of metals, decreasing their availability in the liquid phase of the sludge.

Kurade et al. (2016) analyzed the use of A. ferrooxidans bioflocculants applied as a sludge conditioner, providing other perspectives on the inoculum composition. The bioleached sludge (TS 2.4%, pH 6.79, ORP − 213 mV, CST 38.7 s, and SRF 3.29 × 10¹³ m/kg) was anaerobically digested and collected from Shatin Sewage Treatment Works in Hong Kong. A. ferrooxidans ANYL-1 was previously isolated from sewage sludge and was maintained in a 9 K medium (pH 2.5) supplemented with 44.2 g/L FeSO₄.7H₂O. The bioflocculant used to condition sludge was obtained by transferring 10% (v/v) of a 72 h old culture to a fresh medium, filtered through Advantec No. 1 filter paper after reaching a cell density of 108 cells/mL. During dewatering assays, 270 mL of sludge and 30 mL of A. ferrooxidans bioflocculant were incubated at 30 °C and 180 rpm, with samples collected at regular intervals to analyze dewaterability changes. Bioflocculant acidified the medium after just 1 h of the application, reducing pH from 6.79 to 5.89, showing better results than the commercial flocculant, Cationic Polymer FLOPAM, added at a concentration of 0.2%, resulting in a pH drop to 6.28 after 0.5 h, followed by an increase to 6.52 after 1 h. Regarding dewatering, the rapid variations in CST from an initial 38.7 to 16.25 s with commercial flocculant and 10.1 s with the bioflocculant after 1 h denoted 38% more efficiency when using flocculant of microbiological origin. The efficiency of the bioflocculant was reinforced by analyzing the implied variations on the SRF, whose reduction was 25% greater than that obtained from the synthetic equivalent.

Extracellular Polymeric Substances (EPS)

Extracellular Polymeric Substances (EPS) are macromolecules originating from cell lysis or secreted by microorganisms as a protective response to harmful environmental aspects, such as sudden pH changes, the presence of toxic substances and pathogens, or other extreme conditions imposed by the medium (Liu and Fang 2003; Wu et al. 2020). They predominantly consist of polysaccharides and proteins, although nucleic acids, lipids, and humic substances can also be in varying concentrations (Wingender et al. 1999).

In sludge flocs, EPS may be divided into slime EPS (S-EPS), loosely bound EPS (LB-EPS), and tightly bound EPS (TB-EPS) (Wang et al. 2015). S-EPS is soluble and available in the supernatant after the sedimentation of the sludge, but TB-EPS and LB-EPS form a double layer around the flakes. While the former is an internal layer with a defined shape, bonded more stably to the cell surface, the latter does not show a well-established coverage and is more dispersed and weakly bonded to particles (Guo et al. 2016). After EPS extraction, residual cells are called pellets (Yu et al. 2009) (Fig. 4).

Fig. 4.

Stratification of Extracellular Polymeric Substances (EPS) layers—Slime EPS (S-EPS): soluble and commonly available in the supernatant after sludge sedimentation; loosely bound EPS (LB-EPS): weakly bound to particles, does not provide well-established coverage; tightly bound EPS (TB-EPS): well-defined shape and very stable linked to the cell surface; and pellet: residual cell obtained after EPS extraction

According to Vesilind and Hsu (1997), the water within the sludge may be divided into four forms: free water, which is not bounded to sludge solids and may be separated by gravitational settling; interstitial water, trapped inside cells or sludge flocs, mostly released by cell disruption or when the floc is broken but might be removed by mechanical dewatering; vicinal water, associated with solid particles, cannot be removed by any mechanical means, and; water of hydration, chemically bound to sludge particles, being removed only through thermomechanical destruction of the flocs. The bound water content, comprising interstitial, vicinal, and hydration waters, imposes significant difficulties for sludge dewatering since its removal demands more energy than the removal of free water (Mowla et al. 2013).

EPS molecules, within or near sludge surfaces, can bind a large amount of water through electrostatic interactions or hydrogen bonds, forming a gel-like, three-dimensional, and extremely hydrated matrix (Neyens et al. 2004) that contributes to the formation of microbial aggregates in biofilms and allows their adhesion to the surfaces (Flemming and Wingender 2001). Excess amount of EPS results in poor sludge dewaterability, especially due to this gel-like matrix because it prevents water seepage through the pores of the sludge flocs (Mowla et al. 2013). Nevertheless, since EPS molecules are negatively charged, higher concentrations increase the repulsive forces between cells, reducing sludge flocculation and settleability (Mowla et al. 2013).

In this scenario, the degradation of EPS and the consequent release of EPS-bond water is a key aspect concerning the enhancement of the dewatering efficiency (Neyens et al. 2004; Chen et al. 2016). Sludge conditioning methods are used before the dewatering phase to improve sludge dewaterability (Praharaj et al. 2022). Despite the method's nature, whether physical, chemical, or biological, these approaches aim to favor coagulation or flocculation of sludge particles, reducing sludge solids' compressibility or rupturing flocs to release trapped water molecules (Mowla et al. 2013).

Although high EPS content is detrimental to dewatering processes, lower EPS concentrations improve sludge dewaterability (Houghton et al. 2001). Analyzing the influence of EPS content over activated, digested, and raw sludge, Houghton et al. (2001) observed that each type of sludge presented different optimum EPS concentrations to favor its dewaterability. In activated sludge, the best dewatering condition was associated with a concentration of 35 mg EPS/g SS, which improved sludge flocculation due to the reduction of small particles in the medium. Further concentrations, however, were detrimental because of excess water retention. On the other hand, 20 mg EPS/g SS and 10 mg EPS/g SS were the optimum concentrations observed when assessing raw sludge and digested sludge dewatering, respectively, both being more negatively affected by increasing concentrations of EPS in comparison to activated sludge.

Concerning the more recent research, Zheng et al. (2016a) conducted bioleaching assays and related its outcomes with changes observed over EPS levels throughout the process. Acidithiobacillus sp. used, A. ferrooxidans LX5 (CGMCC No. 0727) and A. thiooxidans TS6 (CGMCC No. 0759) were cultured in medium 9 K pH 2.5) and SM liquid (composition not available, pH 3.0) media supplemented with 44.2 g/L FeSO₄.7H₂O and 10.0 g/L S⁰, respectively, at 28 °C and 180 rpm until reaching a density of 10⁸ cells/mL. The sludge treated (TS 3.42%, Volatile Suspended Solids—VSS 51.87%, pH 7.36, and SRF 6.31 × 10¹³ m/kg) was collected from the thickening pond of Taihu New City WWTP in Wuxi City, China, and bioleaching assays were conducted by mixing 540 mL of fresh sludge, 60 mL of bioleached sludge as inoculum, 5.98 g FeSO₄.7H₂O, and 1.20 g S⁰. Submitting sludge samples (TS 3.42%, VSS 51.87%, pH 7.36, SRF 6.31 × 10¹³ m/kg) to a centrifugation process (14.000 g during 20 min) to mechanically strip EPS, the authors reinforced the idea that the EPS content plays a fundamental role in improving dewatering since their removal was the only factor associated with the improvement in dewaterability after sludge centrifugation—SRF decreased to approximately 1.08 × 10¹³ m/kg. Furthermore, Acidithiobacillus sp. was also related to a reduction of 70% of EPS content in the first 48 h of treatment. However, with a sudden increase after this period—the EPS content ranged from an initial 49.73 mg/g VSS to 15.14 mg/g VSS in 48 h. However, it reached 126.15 mg/g VSS at the end of the essay. The same trend was observed for SRF and water content in the sludge—both parameters ranged from 6.31 × 10¹³ and 47.18%, respectively, to 5.16 × 10¹² m/kg and 25.83% in 48 h, but at the end of treatment, presented final values of 3.29 × 10¹³ m/kg and 49.88%. These results indicate that the treatment conduction time is crucial for its efficiency, given the influence on the EPS content. In the analysis in question, the best dewatering condition coincided with the lowest EPS concentration at 48 h of treatment.

This same variation in EPS levels during bioleaching was also investigated by Zhang et al. (2017) based on an experiment with a 5% inoculum composed of A. thiooxidans and R. mucilaginosa. In terms of suspended solids (SS), the EPS content decreased from 21.78 to 6.27 mg/g SS at 48 h, increasing to 8.37 mg/g SS at 96 h of treatment. As observed by Zheng et al. (2016a), the behavior of the CST and SRF throughout the treatment coincided with the variations observed over the EPS. It was found that the increase in EPS concentration and the deterioration of dewatering conditions were associated with a reduction in pH to values below 2.3. Analyzing the initial decrease in the EPS content, a relationship with the concentration of TB-EPS was observed, which, until then, was the predominant fraction of the total EPS, reduced from 12.10 to 0.38 mg/g SS. After the deterioration of dewatering conditions, there was a new change in the proportion between the EPS components, in which S-EPS became the predominant fraction. Thus, while the reduction in TB-EPS was identified as the main factor in the sludge dewater improvement, the increase in S-EPS was considered the main factor in the dewatering deterioration.

Using bioflocculants produced by A. ferrooxidans was also pointed out as a factor capable of causing changes in EPS layers. Murugesan et al. (2016) submitted samples of ADS (TS 2.05%, OM 42.5%, ORP − 183 mV, pH 7.45, CST 27 s, and SRF 8.62 × 10¹² mg/kg) collected from Sha Tin WWTP in Hong Kong through a conditioning process using bioflocculants produced by A. ferrooxidans ANYL-1, also previously isolated from ADS. The bacteria were routinely maintained in modified 9 K with FeSO₄.7H₂O 44.2 g/L and pH 2.5 at 30 °C and 180 rpm. The bioflocculant was extracted after filtrating a 3-day-old culture (~ 10⁸ cells/mL) through a Whatman No one filter. Experiments were accomplished by mixing 270 mL of ADS, 30 mL of A. ferrooxidans culture, and FeSO₄.7H₂O at iron to sludge solid ratio of 0.05:1. During sludge conditioning, a sharp reduction in protein (EPSP) and carbohydrate (EPSC) concentrations were observed within the first 8 h of treatment. Variations in CST and SRF followed the reduction in the extractable content of LB-EPS, so the improvement in dewatering was justified by the interaction between the layer of proteins and Fe³⁺. In TB-EPS, the reduction in extractable EPSP content was taken as an indication that they are more related to dewatering than the EPSC content. Analyzing these results, the authors proposed a possible Acidithiobacillus sp. mechanism to promote dewatering concerning the binding of Fe³⁺ to EPS layers, which become non-extractable and more compacted on the surface of the flakes. In this process, the negative charges are neutralized by the bioflocculant, allowing greater aggregation between the particles and favoring the dewatering of the sludge.

Evidence that the bioreplacement of heterotrophic bacteria by acidophilic autotrophic bacteria during bioleaching is the main determinant of the EPS content reduction during treatment was investigated by Liu et al. (2016). Samples of raw sludge (TS 40.00 g/L, VS 19.97 g/L, pH 6.44, and SRF 2.88 × 10¹² m/kg) were collected from the gravity thickener tank in a WWTP of Wuxi, China was submitted to bioleaching by mixing 240 mL of sludge, 10.0 mL of inoculum, 6.7% (w/w dry weight ratio) of Fe²⁺, and 2.0 g/L S⁰ at 28 °C and 100 rpm for 72 h. A. ferrooxidans (CGMCC 1.6369) used during the assays was cultured in 9 K medium, and the inoculum was obtained after acclimation process with 50 mL of raw sludge and 250 mL of 9 K medium, being kept at 28 °C and 100 rpm for 144 h. As the medium became acidic, the diversity of microorganism species reduced, as environmental conditions restricted the survival possibilities of non-acidophilus microorganisms. On the other hand, the concentration of A. ferrooxidans gradually increased. Considering that A. ferrooxidans was the predominant species in all samples at the end of the treatment, regardless of the treatment adopted and that the presence of the bacteria was associated with a reduction in the EPS content, the authors concluded that the change in the microbiological community is a decisive factor in improving dewatering.

Medium pH and the impacts on the bioleaching process

During bioleaching, the oxidation of the substrate by bacteria leads to the gradual acidification of the medium, causing the sludge flakes to break and neutralize the negative charges of the particles present. This process reduces repulsive forces between these particles, favoring dewatering due to greater aggregation of the flakes and the release of water contained within them (Kurade et al. 2016). Throughout the bioleaching process, changes observed over pH values reflect Acidithiobacillus sp. activities, so the faster the variation, the greater the microbiological activity (Gao et al. 2017). Thus, pH analysis during the treatment is a good guide for the current treatment phase. However, excessive acidity can increase EPS concentration due to the production of secretion by microorganisms as a strategy to survive the extremely acidic conditions of the environment, as well as the cell lysis process of these microorganisms that are not adapted to survive under such conditions, releasing polysaccharides, proteins and genetic material (Liu et al. 2016; Zheng et al. 2016a).

The relationship between pH and other conditioning factors of bioleaching was frequently addressed in the analyzed references from different perspectives. Thus, the selection of Acidithiobacillus sp. more adapted to acidic environments through previous chemical acidification of the medium was investigated by Zhou et al. (2017). It was found, however, that the substrate oxidation process occurred more efficiently in samples whose initial pH of the system was 6.0, also resulting in better dewatering and metal solubilization conditions—in the sample with an initial pH of 3.0, for instance, the rates of solubilization of Cu, Zn, and Pb were 70.3%, 50.5%, and 27.4%, respectively. In contrast, samples with an initial pH of 6.0 were 86.3%, 68.4%, and 36.3%. Thus, prior acidification did not result in an enhancement to the efficiency of the treatment. Regarding the final pH, the solubilization of metals was optimized the lower the pH of the medium—the solubilization of Cu, Zn, and Pb were around 80%, 60%, and 30%, respectively when the pH of the medium was approximately 2.0. However, the more acidic conditions of the medium at the end of the treatment did not result in the greatest reductions in SRF in all the cases evaluated, reinforcing the relationship between the acidification of the medium and an increase in EPS concentration.

When investigating the relationship between pH and EPS concentration, relevant contributions regarding the optimal duration time for bioleaching treatment were provided by Zheng et al. (2016a) and Zhang et al. (2017). An excessively long treatment did not benefit the dewaterability of the sludge because as the medium gradually became more acidic, there was an increase in the EPS concentration, deteriorating the dewatering conditions—in both references, the best dewatering efficiency coincided with the lowest EPS concentration with pHs ranging between 2.3 and 2.6 at 48 h of treatment. Continuing the treatment for longer periods than the one associated with the best dewatering conditions increased the concentration of EPS in the system and reduced pH to values below 2.0.

The pH was also pointed out as an influencing factor on the dissolved oxygen concentration (DO) in the medium (Zhao et al. 2021), as the oxidation of nutrients by the Acidithiobacillus sp. and the resulting acidification of the medium leads to the inhibition of heterotrophic bacteria, reducing the consumption of O₂ and increasing the concentration of DO (Liu et al. 2016). The relationship is especially important as the aeration rate is also a determinant of bioleaching. In the analysis in question, the best dewatering condition was obtained from an aeration rate of 1.6 m³/h. Evaluating the effects of DO on dewaterability, Li et al. (2021) also highlighted that the higher the dissolved oxygen concentration, the faster the substrate oxidation and acidification of the medium, reflecting better growth conditions for the bacteria acting in the process. Higher aeration intensities were also related to a decrease in EPS content, indicating that the DO facilitated the disintegration of the sludge flakes.

Conclusion

Although the optimum conditions for bioleaching treatment must be considered for each type of sludge given the differences in their compositions, using 10% of mixed inoculum comprising A. thiooxidans and A. ferrooxidans, at a ratio of 4:1, supplemented with 10.0 g/L of FeSO₄.7H₂O and 2.0 g/L of S⁰ showed to be an efficient setting to improve bioleaching results in laboratory-scale assays. Inoculating Acidithiobacillus sp. with fungi, yeasts, and organic matter-degrading bacteria is a promising strategy to favor the bioleaching process. However, more knowledge is required about these microorganisms' dynamics during treatment. Even though metal solubilization increases as the medium becomes more acidic, this condition does not necessarily contribute to the dewaterability of the sludge, as excessive acidification induces an increase in the EPS content and consequently deteriorates the dewatering rates. Therefore, carrying out the treatment for a limited period of 48 h and adjusting the initial and final pHs of the system at approximately 6.0 and 2.0, respectively, may optimize the bioleaching outcomes.

Author contributions

Conceptualization: ICD and LPN. Methodology: ICD and LPN. Formal analysis and investigation: ICD, LPN, and JG. Writing—original draft preparation: LPN; Writing—review, and editing: ICD, LPN, and JG. Funding acquisition: LPN. Resources: ICD. Supervision: ICD. All authors have read and approved the final manuscript.

Declarations

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Research involving human participants and/or animals

No human participant and/or animals were involved in this research.