Biochemical treatment of leachates from hydrometallurgical recycling of spent alkaline batteries

Journal Pre-proof Biochemical treatment of leachates from hydrometallurgical recycling of spent alkaline batteries

Zhendong Yang, Witold Uhrynowski, Grazyna Jakusz, Jacek Retka, Joanna Karczewska-Golec, Klaudia DebiecAndrzejewska, Zbigniew Rogulski, Lukasz Drewniak PII:

S0304-386X(19)30281-6

DOI:

https://doi.org/10.1016/j.hydromet.2019.105223

Reference:

HYDROM 105223

To appear in:

Hydrometallurgy

Received date:

27 April 2019

Revised date:

14 November 2019

Accepted date:

29 November 2019

Please cite this article as: Z. Yang, W. Uhrynowski, G. Jakusz, et al., Biochemical treatment of leachates from hydrometallurgical recycling of spent alkaline batteries, Hydrometallurgy(2019), https://doi.org/10.1016/j.hydromet.2019.105223

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© 2019 Published by Elsevier.

Journal Pre-proof

Biochemical treatment of leachates from hydrometallurgical recycling of spent alkaline batteries Zhendong Yang1#, Witold Uhrynowski1#, Grazyna Jakusz1, Jacek Retka1, Joanna KarczewskaGolec1, Klaudia Debiec-Andrzejewska1, Zbigniew Rogulski2, and Lukasz Drewniak1* 1

Laboratory of Environmental Pollution Analysis, Faculty of Biology, University of Warsaw,

Miecznikowa 1, 02-096 Warsaw, Poland 2

Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw,

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Zwirki i Wigury 101, 02-089 Warsaw, Poland

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# equal contribution

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*Correspondence:

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Abstract

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E-mail: [email protected]

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Lukasz Drewniak

Recycling of waste electrical and electronic equipment including spent batteries focuses on maximizing the material recovery efficiency (of metals, polymers, or solvents) and decreasing the negative environmental impact of leachates that remain after the treatment (i.e. the secondary wastes). The major aim of this study was to develop a novel, low-cost biochemical treatment technology for the management of effluents generated during sulfuric acid–based hydrometallurgical recycling of spent alkaline batteries. We explored the use of various chemical reagents (40% NaOH, 20% Na2CO3, and 1% NH3(aq)) and biogenic ammonia produced by urea-degrading bacteria to increase the pH of the effluents (from pH 0.5 to pH

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Journal Pre-proof 5.0) and prepare them for further treatment by sulfate-reducing bacteria. Comparisons of the pretreatment efficiency and metal and sulfate removal yields, as well as the characterization of the neutralization products (sediments and effluents) showed that the most promising results were obtained when the raw leachates were treated with 40% NaOH (to reach pH 3.5), followed by the addition of biogenic ammonia (to reach pH 5.0). Sulfate-reducing bacteria (SRB) activity led to a further pH increase (up to ~7.3), almost complete (99%) sulfate removal and metal sulfide (ZnS, MnS) precipitation, as up to ~99% of Zn and Mn were

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removed in SRB cultures to which appropriately diluted pretreated leachates had been added.

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The presented study indicated that the pretreatment and neutralization of hydrometallurgical

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effluents based on the use of urea-degrading and sulfate-reducing bacteria could be an

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attractive alternative to conventional chemical treatment.

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Keywords: E&E waste, battery recycling, alkaline batteries, sulfate-reducing bacteria,

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1. Introduction

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leachate treatment, urea-degrading bacteria

Current hydrometallurgical technologies are increasingly adopting a circular economy strategy. They focus on the maximum recovery of primary and secondary raw materials while minimizing waste emission and energy leakage. The reduction of waste emission in hydrometallurgy aims mainly at managing leachates that remain after the treatment of raw materials. Depending on the raw materials and technology used, the leachates are acidic or alkaline solutions and may contain substantial amounts of metals and metalloids (Cui and Zhang 2008; Hyk and Kitka, 2017; Li et al., 2018). For example, in hydrometallurgical digestion of waste electrical and electronic equipment, sulfuric acid or hydrochloric acid are usually used (Rath et al., 2012; Tsydenova and Bengtsson, 2011; Tuncuk et al., 2012) and the

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Journal Pre-proof leachates remaining after the process may include the following metals: copper, iron, tin, nickel, lead, zinc, silver, gold, and palladium (Ficeriová et al., 2011; Kang and Schoenung, 2006; Mecucci and Scott, 2002; Namias, 2013). Thus, the primary aim of the treatment is to neutralize pH of the leachate and remove metals and other toxic elements or compounds from the solution. Several effective chemical methods, including neutralization (Akcil and Kodas, 2006), electrolysis (Sadoway, 1991), adsorption (Iakovleva and Sillanpää, 2013), ion-exchange

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(Dabrowski et al., 2004), membrane separation (Pospiech and Walkowiak, 2007), and

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photocatalysis (Ding et al., 2017) were developed to dispose acidic effluents from

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hydrometallurgical processes. Among them, chemical neutralization appears the most

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economically attractive (Chen et al., 2009; da Silveira et al., 2009). When bases are dosed, metals in the effluent can be removed as various forms of hydroxides by precipitation at

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different pH values, which allows for metal classification during recovery. Diverse reagents

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were proposed for alkalization. Precipitation efficiency, effluent chemical composition, and the cost are the main criteria for choosing a suitable reagent for the neutralization process.

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Inexpensive and safe to operate limestone (CaCO3) has been considered the preferred reagent in neutralization (Sibrell and Wattem, 2003). Other Ca-based materials, such as hydrated lime (Ca(OH)2) and quicklime (CaO), were also proposed (Brown et al., 2002; Tolonen et al., 2014). In contrast to limestone, their neutralization efficiency is high because of their high solubility (Brown et al., 2002; Skousen et al., 1996). However, when such reagents are applied in SO42-–containing acidic water, gypsum (CaSO4) is generated as an undesired byproduct in metal recovery processes (Feng et al., 2000). In turn, soda (Na2CO3) and caustic soda (NaOH) were reported to work more efficiently, but they are more expensive (Mirbagheri and Hosseini, 2004; Skousen et al., 1996).

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Journal Pre-proof The composition of acidic, metal-containing wastewater may be a difficult challenge to leachate neutralization. There are metals in the form of complexes, particularly oxyanions, that cannot be efficiently removed by neutralization, for instance vanadium and chromium (Sjöberg et al., 2012). In addition, some metal hydroxides have amphoteric properties, and thus when one metal precipitates at specific pH, the other returns into the solution (España et al., 2006; Fu and Wang, 2011). Finally, some metal ions, such as Cd2+, Zn2+, and Cu2, precipitate only when pH is over 7, which is due to the high solubility product constants (Ksp)

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of their hydroxides (Watzlaf and Casson, 1990; Wei et al., 2005). Not only does it cause

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alkaline effluent generation, but also creates a massive demand for the base volume when

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such a metal is being removed.

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To reduce the limitations of the aforementioned methods, combined biological and chemical treatment technologies are increasingly used. The biological treatment of acidic

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effluents is based on exploiting and enhancing the activity of microbial consortia that generate

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alkalinity. A number of biological processes generate alkalinity through the reduction of electron donors (mainly a carbon source), e.g., sulfate reduction, denitrification,

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methanogenesis, iron and manganese reduction (Rene et al., 2017). An alternative solution to the treatment of leachates that contain high concentrations of metal ions and sulfates is bioprecipitation of metal ions in the form of an insoluble precipitate after earlier reduction of sulfates to sulfides under anaerobic conditions (Rene et al., 2017; Sánchez-Andrea et al., 2014). Biological sulfate reduction for metal removal has several crucial advantages over the chemical (hydroxide) precipitation. The metal sulfide precipitate obtained after the reaction of hydrogen sulfide with heavy metals has very low solubility, is also more stable in a wide range of pH values. Separation of the precipitate and its further use as a raw material in metallurgical processes is therefore possible. If complexes and chelating agents are present in the leachate, sulfide precipitation in bioreactors could be more effective than chemical

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Journal Pre-proof precipitation (Huisman et al., 2006). In turn, the main disadvantages of biotechnologies are (i) their long retention time, (ii) performance fluctuations, and (iii) the need to adjust the microbial community composition to the various environmental conditions (Rene et al., 2017). Thus, these technologies are not linearly scaled and require individual optimization for a specific leachate. In this study, an innovative approach to leachate treatment from battery recycling processes has been explored. Our major aim was to combine chemical and biological methods

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to treat acidic effluents (pH 0-0.5) that contain a high concentration of sulfates and heavy

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metals (iron, zinc, and manganese). We focused on increasing the efficiency of the process

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and reducing its cost. We studied the use of various chemical reagents (NaOH, Na2CO3, and

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NH3(aq)) and bacterial metabolites containing biogenic ammonia to increase the pH of the effluents and prepare them for further treatment by sulfate-reducing bacteria. Specific

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objectives included: (i) determination of the leachate pretreatment efficiency and sediment

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productivity with the use of various neutralizing reagents, (ii) estimation of the toxicity of the leachates pretreated chemically (NaOH), or chemically (NaOH) and biologically (biogenic

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NH3) to sulfate-reducing bacteria, and (iii) comparison of sulfate and metal sulfide removal efficiency from various types of pretreated leachates. 2. Materials and methods

2.1 Electronic waste leachate

Leachate samples were obtained from a battery recycling company (BatEko Ltd. Poland). Leaching with sulfuric acid (2 M) was performed on crushed and sieved spent alkaline batteries at 80°C for 5 h. General scheme of the battery recycling process was presented in Figure 1. The raw leachate contained a high concentration of Fe, Zn, Mn, and sulfates and had very low pH at the level of 0.45. The metal composition of the raw leachate was determined by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-OES,

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Journal Pre-proof iCAP 6500 DUO, Thermo-Scientific) while sulfate concentration was assessed with ion chromatography (IC). A further description of the leachate is provided in the Results and Discussion section. 2.2 Production of biogenic ammonia Urease-synthesizing bacteria were used to convert urea into ammonia. For the experiment, nine ureolytic bacterial strains were selected from the culture collection of Laboratory of Environmental Pollution Analysis at the Faculty of Biology, University of

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Warsaw, Poland. These were: Ochrobactrum sp. C261, Providencia sp. FFAG, Bacillus

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aerius sp. RG230, Ochrobactrum sp. POC9, Solibacillus sp. LPSUB13, Lysinibacillus sp.

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LPSVB15, Bacillus sp. W21, Ochrobactrum sp. W86B, and Brevibacterium sp. LPMIX6.

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The strains were individually grown overnight in Lysogeny Broth (LB) (Sambrook and Russell, 2001). Then, bacterial cultures were mixed in a urea-containing growth medium

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consisting of the following compounds: peptone (1g/L), NaCl (5g/L), KH2PO4 (2g/L), and

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urea (20g/L) (Christensen, 1946). Before the addition of urea, pH of the medium was adjusted to 6.80-6.85 with 1 M NaOH and the medium was sterilized by autoclaving for 11 min at 15

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psi [1.05 kg/cm2] and 126°C on a liquid cycle. The urea solution was filtrated through a syringe filter with a pore diameter of 0.22 µm. One milliliter of each overnight bacterial culture was transferred to 100 mL of the urea-containing medium. Batch cultivations were conducted for 48 h. After that time, pH and ammonia concentration in the culture were measured. The pH was around 9.25, which is in agreement with previous reports (Abo-ElEnein et al., 2013; Whiffin, 2004). The concentration of ammonium ion (NH4+), determined photometrically with Nanocolor Ammonium 200 kit, was approx. 1% NH4+. 2.3 Chemical and biological pretreatment Chemical and biological pretreatment procedures were carried out in the same manner (Figure 2). The growth of sulfate reducing bacteria is pH dependent, thus, the raw effluent

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Journal Pre-proof was pretreated by partial neutralization to pH 5 for an effective biological sulfate reduction (Liu et al., 2018). Several pretreated leachates varying in the pH (from initial value of 0.45) were tested separately to evaluate and characterize the pretreatment process. The pH of the tested solutions were set as 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5. For each reagent, the pretreatment process was conducted in triplicate. For chemical pretreatment, the raw leachate was pretreated at 22°C with 40%, NaOH, 20% Na2CO3 or 1% NH3(aq) solutions prepared from analytical grade >98% purity reagents. Each alkaline solution was added to 100 mL of

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the raw leachate under stirring (300 rpm, magnetic stirrer). Each batch of the solution was

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added in about 30 s, followed by a 10-min interval for pH stabilization. For biological

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pretreatment, biogenic ammonia (synthesized as described above and contained in a post-

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culture medium) was added to 100 mL of the raw leachate under stirring, in batches, as described above. In the case of biogenic ammonia, the formation of foam resulted in the need

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to reduce the mixing and reagent dosing speeds by half. In this study, the concentration of 40%

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NaOH and 20% Na2CO3 refer to w/w, whereas 1% in NH3（aq） and biogenic ammonia refer to w/v.

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After pH adjustment, the pretreated effluents were separated from the precipitates. The obtained liquid and solid samples were then prepared for Flame Atomic Absorption Spectroscopy analysis (FAAS, SOLAAR M ATI Unicam). For this purpose, 4 mL of the effluent fixed with 1 mL of 69% HNO3 was used. The solid was dried to constant weight at 60°C for 48 h. After drying to dry matter, precipitates were weighed and mineralized in a closed microwave system (Milestone Ethos Plus) using 9 mL of 69% HNO3 and 1 mL of 30% H2O2 at 180°C for 25 min. Concentrations of Fe, Zn, and Mn in samples were determined by FAAS. The powder X-ray diffraction Debye-Scherrer-Hull method was carried out to characterize the geochemical composition of the precipitates obtained after pretreatment using NaOH and Na2CO3. Page 7 of 50

Journal Pre-proof 2.4 Analysis of sulfate-reducing bacteria activity in (bio)chemically treated leachates 2.4.1 Preparation of a mixed consortium of sulfate-reducing bacteria A mixed culture of sulfate-reducing bacteria (SRB) was prepared by subsequent passaging (10% v/v) of sewage sludge samples collected from several municipal wastewater treatment plants in Poland in 100-mL anaerobic bottles in modified Postgate B (MPB) medium [K2HPO4 0.5 g/L, NH4Cl 1.0 g/L, Na2SO4 1.0 g/L, CaCl2 • 2H2O 0.1 g/L, MgSO4

•

7H2O 2.0 g/L, 4 mL/L of sodium-D,L-lactate (50% solution in water), yeast extract 1.0 g/L, •

7H2O 0.5 g/L, resazurin sodium salt 1.0 mg/L, sodium thioglycolate 0.1 g/L,

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FeSO4

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supplemented with 10 mL/L of vitamin solution (VS) comprising (per liter): ascorbic acid 0.1

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g, biotin 2.0 mg, folic acid 2.0 mg, pyridoxine 10.0 mg, thiamine 5.0 mg, riboflavin 5.0 mg,

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nicotinic acid 5.0 mg, pantothenic acid 5.0 mg, vitamin B12 0.1 mg, p-aminobenzoic acid 5.0 mg, lipoic acid 5.0 mg), and 2 mL/L of trace element solution (TES; Tuovinen and Kelly,

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1973)]. The medium was autoclaved for 15 min at 15 psi [1.05 kg/cm2] and 121°C on liquid

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cycle, flushed with nitrogen to remove the dissolved oxygen and cooled to RT, before inoculation. After each passage, the cultures were incubated at 37±1°C for approx. 28 days.

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The presence (activity) of SRB in the cultures was indicated by a decrease in sulfate concentration (determined photometrically with Nanocolor Sulfate 200 and/or 1000 kits, Macherey Nagel), accompanied by the formation of a black precipitate (iron(II) sulfide) and/or hydrogen sulfide production (detected by Nanosens Gas Meter). Bacterial cell count in the cultures was assessed using the most probable number dilution assay (Postgate, 1984) in MPB medium, performed in duplicate. The microbial consortium obtained after ten subsequent culturing steps (passages) in MPB medium, containing approx. 107 cells/L of sulfate-reducing bacteria, was used as an SRB inoculum in further experiments. 2.4.2 Preliminary testing of the pretreated leachate toxicity to SRB

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Journal Pre-proof Cultures of SRB were carried out in MPB medium without the addition of sulfate salts, at 37°C for 21 days in 100-mL anaerobic bottles. The medium was supplemented with NaOH-pretreated leachates (pH 5.0), so as to reach the selected, increasing concentrations of Mn2+, Zn2+, and SO42- ions, as presented in Table 1 Inoculated MPB medium (with 1500 mg/L of sulfates and with supplements) was used as a positive control. The initial SRB count was 106 cells/L, which corresponded to the concentration of 10% (v/v) of the prepared inoculum. Sulfate-reducing bacteria activity was

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dilutions of the effluents, were used as negative controls.

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assessed as described in the section above. Uninoculated media, containing appropriate

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2.4.3 SRB activity and inorganic ion removal from (bio)chemically treated leachates

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Cultures of SRB were carried out 1-L anaerobic bottles in two main variants: (i) MPB medium without sulfate salts or supplements (vitamins and trace elements); (ii) MPB medium

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without the addition of sulfate salts but with the addition of 1% (v/v) of VS and 0.2% (v/v) of

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TES. Appropriate volumes of the (bio)chemically treated leachates were added to the media to set the initial concentrations of Mn2+, Zn2+, and SO42- ions to 125, 225, and 1500 mg/L,

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respectively, as in the Pre-1 variant of the preliminary toxicity experiment described in the Table 1. Inoculated MPB media with 1500 mg/L of sulfates, with or without the addition of supplements, were used as positive controls. The initial SRB count was 106 cells/L, which corresponded to the concentration of 10% (v/v) of the prepared inoculum. Uninoculated media were used as negative controls. Details on the individual variants are provided in the Results and Discussion section. The cultures were grown for 9 days at 37°C and gently mixed at the beginning of the experiment and on days 1, 3, 5, 7 and 9, before sample collection. The pH and sulfate concentration (Nanocolor Sulfate kit) were analyzed in the centrifuged (8000 rpm, 10 min) samples. At the end of the experiment, sediments from all the variants were collected by

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Journal Pre-proof centrifugation, followed by the removal of supernatants. The sediments were dried on air, and then mineralized in a 9:1 mixture of 69% HNO3 and 30% H2O2 in a microwave oven system, as described above. Manganese and zinc concentrations in the obtained mineralized-sample solutions and culture supernatants were analyzed by FAAS.

3. Results and discussion

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Sulfate-reducing bacteria (SRB) may be used for effective heavy metal removal from

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contaminated industrial wastewater. However, certain criteria must be met to support the

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activity of SRB. These include specific ranges of pH, oxygen content, heavy metal and other

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toxic compound concentrations, and redox potential. In this paper, we present an approach to preparation of leachates derived from alkaline battery recycling to be further treated by SRB

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and to effective removal of sulfates and residual heavy metals from the recycling process

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leachates. A variety of synthetic solutions (NaOH, Na2CO3, NH3(aq)) as well as biogenic ammonia were tested as agents for the initial pH increase (pretreatment). The efficiencies of

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pH increase and heavy metal precipitation were first analyzed. 3.1 Characteristics of the raw leachate In the last decades, new physical, pyrometallurgical, and hydrometallurgical processes have been employed to recycle batteries. Hydrometallurgical processes seem to be the most efficient methods and are thus most often used. Combined with mechanical pretreatment, hydrometallurgical technologies allow to recover not only metals and their compounds in various chemical forms, but also solutions and polymers. The EU legislation (Directive 2006/66/EC) defines a minimum efficiency of 50% for battery recycling processes. The composition of recycled waste depends both on the type of battery and its composition (Table 2) and on the chemical reagents used in the recycling process

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Journal Pre-proof In neutralization tests, we used industrial leachates from alkaline battery recycling. The leachates had low pH (0.5) and a high sulfate content (~130 g/L), as sulfuric acid (2 M) had been used as a leaching agent. Despite the application of appropriate separation methods (electrolysis), the leachates also contained substantial amounts of heavy metals (Table 3), including iron, manganese, nickel, and zinc. As mentioned earlier, the chemical composition of the batteries varies. Therefore, it is not surprising that the chemical composition of the raw leachate is different for different batteries (da Silva et al., 2010; Xará et al., 2013).

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3.2 Chemical and biological pretreatment of the raw leachate

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Most of the known SRB are neutrophiles that thrive in neutral pH environments. At

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low pH a higher energy input is needed to maintain the neutral intracellular pH and less

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energy would be left for bacterial growth and activity. Furthermore, at low pH, hydrogen sulfide can have a toxic effect on SRB, as in its undissociated/acid form it may diffuse

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through cell membranes. The same mechanism brings about a restraining effect of organic

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acids on the SRB growth (Sanchez-Andrea et al., 2014). Therefore, the most important stage of preliminary leachate treatment is to increase its

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pH. Although sulfate-reducing bacteria are active under both acidic and (slightly) alkaline conditions, to enable effective reduction of sulfates by most SRB strains, pH should be set between approx. 5 and 8 (Liu et al., 2018). For economic reasons, and as the SRB activity itself leads to an increase in the culture pH, the pretreatment procedure was aimed only at reaching pH 5. Following the solubility charts, at this pH level iron precipitation can be expected while other metals precipitate above pH 5 (Blais et al., 2008). Therefore, both the efficiency of pH increase and the amount of precipitated iron were monitored at various leachate/reagent ratios. Increasing the pH of the leachate was performed with NaOH, Na2CO3, and NH3(aq) solutions that are commonly exploited in industrial settings due to their low price among

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Journal Pre-proof chemical reagents. As a source of ammonia, we used a 1% solution of synthetic NH3(aq) (further referred to as S-NH3) and biogenic NH3 (further referred to as B-NH3) produced by ureolytic (urea-degrading) bacteria and contained in the post-culture medium. For the production of B-NH3 we used nine urease-synthesizing bacterial strains, which converted urea into ammonia. The raw leachate was gradually pretreated with the above-mentioned solutions at 22°C to reach the final pH 5, as described in the Materials and Methods section. 3.2.1 Addition of alkaline agents for pH increase

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Figure 3 shows neutralization efficiency of the four reagents used. The volumes of

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40% NaOH, 20% Na2CO3, 1% S-NH3, and 1% B-NH3 consumed to increase the pH of the

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raw leachate from the initial value to 1 were 6.15, 14.50, 55.21, and 46.00 mL, respectively.

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The B-NH3 increased the pH more efficiently than S-NH3 at the very beginning of the process, which may have resulted from the consumption of acidity by the additional metabolites

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(particularly carbonates) contained in the post-culture medium, alongside B-NH3.

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The total volumes of 20% Na2CO3 and 1% S-NH3 consumed to increase the initial pH to 5 are about 2 and 13 times higher, respectively, compared to that of 40% NaOH. The Kn

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refers to the overall pretreatment efficiency of each reagent, and is defined as follows: 𝐾𝑛 =

△ 𝑉𝑛 △𝑛

where Kn is the pretreatment rate at a specific pH value, △ 𝑉𝑛 is the differential of the consumed volume of a reagent at a specific pH value, and △ 𝑛 is the differential of a specific pH value. From the initial pH value to 3.5, the Kn of all reagents decreased throughout the pretreatment process. The Kn of 40% NaOH was lower than those of 20% Na2CO3 and 1% SNH3 before the pH increased to 3.5. After the pH reached the value of 3.5, the Kn for the three reagents was stable. While B-NH3 is considered an environmentally- and economically-

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Journal Pre-proof friendly reagent, 1% B-NH3 had the lowest Kn, indicating the lowest neutralization efficiency and a huge demand for reaction volume on a real industrial scale, which also means high cost. 3.2.2 Iron precipitation/depletion The relationship between Fe concentration and pH in the leachates treated with NaOH or Na2CO3 is shown in Figure 4A. The Fe concentration refers to the real Fe concentration, excluding the volume of the reagents added, and is calculated by the following equation:

c0 (Fe)×(V ' + 0.1) 0.1

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cR (Fe) =

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where cR(Fe) is the real Fe concentration (mg/L), c0(Fe) is the original Fe concentration

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determined in the analysis (mg/L), and V’ is the volume of the added base reagent (L). At pH 3.5, Fe was almost entirely removed from the NaOH- or Na2CO3-treated

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leachates, which is in agreement with the results of previous studies (España et al., 2006; Wei

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et al., 2005). Concentrations of Fe in leachates pretreated with 1% S-NH3 or 1% B-NH3 , at each of the tested pH levels, are shown in Figure 4B. In the case of the leachate pretreated

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with 1% B-NH3, Fe precipitated almost entirely at pH 3.0, whereas in that pretreated with 1%

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S-NH3 Fe precipitated almost entirely at pH 4.0. In general, a higher precipitation efficiency was observed in the solution treated with B-NH3. The final removal efficiency was at the same level for B-NH3 and the other three variants. Moreover, there was a similar trend in the Fe removal efficiency in the leachates treated with either 40% NaOH or 20% Na2CO3. The Fe removal started above pH 2.5. In the pH range of 0.48-2, Fe concentrations were 122.07-125.84 and 121.86-128.08 mg/L for leachates treated with 40% NaOH and 20% Na2CO3, respectively. In the leachate treated with 1% S-NH3, the Fe concentration was remarkably decreased above pH 1.5 until pH 2, then started to be stable. Above pH 3, up to pH 4, Fe concentration decreased again. As for the leachate treated with 1% B-NH3, the Fe concentration constantly decreased until pH 3. This suggests different mechanisms of the process with S-NH3 and B-NH3. One reason for the Page 13 of 50

Journal Pre-proof difference is that a low concentration B-NH3 was added in a large volume, which caused high dilution of the leachate, resulting in different complexation of Fe and other ions. Another possible reason for the observed differences in the Fe concentration pattern in the leachates was that the B-NH3 – obtained from a post-culture medium – was most probably accompanied by diverse secondary metabolites that had been synthesized by bacteria and could interact with Fe. 3.2.3 Sediment production

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Table 4 and Figure 5 show sediment productivities and Fe concentrations in

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sediments, respectively, at different pH values, following the pretreatment with 40% NaOH,

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20% Na2CO3, 1% S-NH3, or 1% B-NH3. Measurable amounts of precipitates were collected at

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pH 2.5 and 3.5 for NaOH- and Na2CO3-treated leachates, respectively. For the NaOHpretreated leachate, sediment productivity substantially increased with increasing pH,

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particularly above pH 4, whereas Fe concentration in the sediment increased until pH 3.5,

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then decreased. For the Na2CO3-pretreated leachate, there was a similar trend with regard to Fe concentration in the sediment and pH changes. Sodium hydroxide is a much stronger

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alkaline reagent compared to Na2CO3. Sediment productivity in the leachate pretreated with Na2CO3 remained quite stable below pH 4, indicating less intense co-precipitation. For 1% S-NH3 and 1% B-NH3, the precipitate emerged at pH 4 and 2.5, respectively. However, up to pH 4.0 and in the 4.0-5.0 pH range sediment productivities in leachates pretreated with 1% B-NH3 and 1% S-NH, respectively, were low. In NaOH- and Na2CO3pretreated leachates, more dissociated free hydroxyls that aggregate impurities were generated during the pretreatment process. The hydroxyls might have immediately reacted with ions in the leachate resulting in co-precipitation of oxides and hydroxides with not only Fe but also SO42- and cations, such as Zn2+, in the form of complex compounds, including jarosite and schwertmannite (Bigham et al., 1996; Dutrizac and Jambor, 2000). Zinc and manganese

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Journal Pre-proof concentrations were slightly reduced at pH from 1 to 5 (Table 7), which indicated that below pH 5 there was no obvious neutralization effect to Zn and Mn in the leachates, resulting from their higher solubility product constants. A previous study pointed out that most metal ions other than Fe3+, including Zn2+ and Mn2+, precipitate when pH is over 5 (Blais et al., 2008). However, at high concentrations of Zn and Mn, precipitation efficiency may be different. To summarize the results of NaOH-, Na2CO3- or NH3-based pretreatments of the raw leachate, each tested reagent precipitated Fe at pH 3.5-4. To remove iron, when 40% NaOH,

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20 % Na2CO3, or 1% B-NH3 are applied, the pH needs to reach 3.5. The highest Fe

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precipitation efficiency was achieved with 40% NaOH. The average volume of NaOH used to

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adjust the pH to 3.5 was 9.2 mL per 100 mL of the raw leachate. For 20% Na2CO3, the

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average volume was higher (22.8 mL per 100 mL of the raw leachate), but the reagent is less expensive. The average volume of 1% B-NH3 needed to reach pH 3.5 was 172.6 mL per 100

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mL of the raw leachate. When 1% S-NH3 was used, the pH needed to reach 4, and the average

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volume required was then 131.2 mL per 100 mL of the raw leachate. Thus, the use of

process.

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synthetic or biogenic NH3 is neither efficient nor economical at this stage of the treatment

Above the pH 3.5, the volumes of all reagents needed to reach pH 5.0 substantially decreased. The average volume of NaOH and Na2CO3 used was from 0.5 to 1.5 mL per 100 mL of the raw leachate. For B-NH3, the volume was much higher (16.6 mL per 100 mL of the raw leachate), but the biogenic reagent is environmentally friendly and relatively cheap. A simple comparison of purchase costs (Table S1), based on the prices of technical stock solutions, confirmed that the use of B-NH3 is economically attractive and the overall cost of its usage could be comparable with that of the corresponding synthetic reagents. The treatment of 1 m3 of the effluents with 40% NaOH will amount to around 46 USD, while the use of 20% Na2CO3 will amount to 30 USD. In turn, the costs of using S-NH3 and B-NH3 are

Page 15 of 50

Journal Pre-proof around 42 USD and 36 USD per 1 m3 of the neutralized leachate, respectively. If the investments and operational cost are included in the calculation, the cost of the treatment of 1 m3 of the leachate with various reagents to reach pH 5 becomes very similar and does not exceed 45-50 USD. Thus, the main criterion for selection of the reagent should be its neutralization efficiency and its potential influence on the activity of sulfate reducing bacteria. Considering both the price comparisons and the obtained results on pretreatment efficiency, the use of NaOH to reach pH 3.5 and biogenic NH3 in the 3.5-5 pH range seems the most

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optimal approach to raw leachate pretreatment.

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3.3 Bacterial sulfate reduction and metal removal from the pretreated leachates

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Although the increase of the pH to 5.0 during leachate pretreatment significantly

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decreases dissolved iron concentration, other metal cations (Zn, Mn, Ni, etc.) do not precipitate as readily at such pH. Thus, apart from sulfates, substantial amounts of zinc (~27.0

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g/L) and manganese (~11.4 g/L) still remained in the pretreated leachate, resulting in its high

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toxicity, potentially exerting an inhibitory effect on microbial activity. 3.3.1 Toxicity of the pretreated leachate to SRB

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In order to analyze the potential inhibitory effect of the pretreated effluents on the activity of SRB, toxicity tests in batch mode were carried out in modified Postgate B (MPB) medium without sulfates, with the addition of appropriate volumes of the leachates pretreated with 40% NaOH, so as to reach the desired final concentrations of Mn2+, Zn2+ and SO42- ions, as described in Table 1. After 21 days of incubation under optimal growth conditions, significant (>80%) reduction in sulfate concentration and formation of a black precipitate were observed only in Pre-1 variant that initially contained 125, 225 and 1500 mg/L of Mn2+, Zn2+ and SO42- ions, respectively. In Pre-2 variant (initially containing 250, 450 and 3000 mg/L of Mn2+, Zn2+ and SO42- ions, respectively), the activity of SRB was limited (sulfate reduction efficiency <50%), but not completely inhibited. In the other variants (Pre-3-6),

Page 16 of 50

Journal Pre-proof initially containing 400, 720 and 4800 mg/L or more of Mn2+, Zn2+ and SO42- ions, respectively, neither decrease in sulfate concentration nor the formation of a black precipitate were observed. This indicates that such high concentrations of zinc, manganese, sulfate or other inorganic ions in the pretreated leachates are toxic to SRB. 3.3.2 Treatment of the (bio)chemically-pretreated leachates using SRB Following the preliminary toxicity assessment, in order to further investigate SRB capabilities of sulfate reduction and thus the removal of metal ions from pretreated

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hydrometallurgical leachates, an experiment in 1-L bottles was carried out. In the experiment,

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two types of effluents were used: (i) hydrometallurgical leachates pretreated with 40% NaOH

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to reach pH 5.0 (further referred to as chemically treated leachates – Ch leachates) and (ii)

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leachates that had been treated first with 40% NaOH to reach pH 3.5 and then – with ureolytic bacteria post-culture medium containing biogenic ammonia to reach pH 5.0 (i.e. biologically

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and chemically treated leachates – BCh leachates). The Ch and BCh leachates were added in

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appropriate volumes to the MPB media without sulfates, to reach the same initial concentrations of inorganic ions as in the Pre-1 variant, in which the SRB activity had been

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confirmed in the preliminary experiment.

As mentioned earlier, BCh leachates were a part of an ureolytic bacteria post-culture medium that may serve as a source of vitamins and metabolites for microorganisms. Thus, to assess whether the use of a biological pretreatment step is beneficial for SRB activity, cultures in MPB medium without the addition of routinely used supplements (vitamin solution, VS and trace elements solution, TES) were also tested. The complete list of variants analyzed in the experiments is presented in Table 5. Uninoculated controls, corresponding to all culture variants listed in Table 5, were also analyzed. The kinetics of sulfate removal from SRB cultures that contained pretreated leachates is presented in Figure 6. The pH changes throughout cultivation are shown in Figure 7. Table

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Journal Pre-proof 6 summarizes the results of FAAS analysis of Mn2+, Zn2+, and SO42- concentrations in both culture supernatants and mineralized precipitates at the end of the SRB cultivation experiment. The results indicated that SRB can tolerate the addition of both types of the pretreated leachates, following their appropriate dilution. To support SRB activity, the concentrations of Mn2+, Zn2+ and SO42- (when these ions co-occur in a solution) should not be higher than 250, 450, and 3000 mg/L, respectively, but preferably lower, as in Pre-1 variant for which further

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optimization was performed. Within 9 days, SRB grown on the MPB medium containing

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pretreated leachates in the same concentration as in Pre-1 variant carried out (i) over 99%

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reduction in sulfate concentration (Figure 6), and (ii) substantial removal of manganese

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(>84%) and zinc ions (>99%) from the liquid phase of the cultures (Table 6). Moreover, SRB cultivation increased pH of the cultures to approximately 7.3 (Figure 7, Table 7).The observed

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pH increase proves that SRB are capable of complete neutralization of the pretreated (pH 5.0)

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leachates. In the light of these natural SRB capabilities, the use of chemical reagents to further increase the pH from 5.0 to 7.0 is unnecessary and uneconomic.

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Following a rapid decrease within the first 3 days of culturing, metal concentrations in the liquid phase of all experimental variants remained at the same level to the end of the experiment, indicating the stability of the metal sulfides formed. At the same time, metal concentrations in the negative control (without SRB) did not change significantly. As one example, Mn2+ and Zn2+concentrations in the negative control of the 2B variant (without SRB) were 126.9 and 193.3 mg/L, respectively. The maximum of 8.5 mg of Mn2+ and 40 mg of Zn2+ precipitated in the negative control variants, presumably due to the presence of phosphates in the medium, which may form insoluble salts with metal ions.” Taken together, the results demonstrated the advantages of using SRB to increase metal ion precipitation efficiency (Table 7). As demonstrated in the experiment, ZnS (Kso =

Page 18 of 50

Journal Pre-proof 1.1 ×10-24) precipitates before MnS (Kso = 5.6 × 10-16), and in several variants manganese removal was significant but not complete. Additional treatment steps (e.g., the addition of nutrients to the cultures) may therefore be needed for the complete Mn removal. Alternatively, if supplementation of the cultures does not result in the complete removal of manganese ions, oxidation of the remaining Mn2+ to insoluble MnO2 may be applied, a technique commonly employed in water purification systems (e.g., Elsheikh et al., 2018). The precipitate can be further separated from the treated water, just as in the case of metal sulfides

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obtained through SRB activity.

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The use of BCh- and Ch-leachates in the culture variants enabled the comparison of

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the effect of these pretreatment methods on SRB activity. The addition of vitamins and trace

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elements was shown to be crucial for efficient sulfate reduction in the variants that contained pretreated leachates, as the rate of the process in the 3B variant (Ch-leachate, no supplements)

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in days 0-7 was much lower than in the 2A and 3A variants (BCh and Ch-leachate,

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respectively) with the addition of supplements (Figure 6). However, the kinetics of sulfate removal in the 2B variant (BCh-leachate without supplements) is similar to those observed in

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the cultures with supplements, indicating that the BCh-leachate itself contained sufficient concentration of nutrients, vitamins and other metabolites to support SRB activity. Moreover, only in the variants with BCh leachate (and in positive controls) sulfate removal was complete (Table 6, Figure 6). The inclusion of supplements in the media containing the pretreated effluents (2A-3A) allowed, however, more efficient removal of manganese (>97%) compared to the variants (2B-3B) without VS and TES (approx. 84% removal) (Table 6). This was not the case for zinc, as almost complete removal of Zn2+ ions from the liquid phase was observed in all variants (Table 6). In large-scale, real-world applications, high concentrations of pretreated leachates are more likely to be used, and the overall cost of supplementation – also with nitrogen compounds, including ammonia produced by ureolytic bacteria – must be taken

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Journal Pre-proof into account. Therefore, the use of BCh-pretreated leachates containing growth-promoting metabolites seems to be preferable to extensive supplementation of chemically neutralized leachates with vitamins and trace elements. The need for effluent dilution before bacterial treatment may be addressed in a biological manner. Sulfide ions present in the SRB culture fluids following sulfate reduction immediately react with metal cations and form insoluble metal sulfides. The SRB culture fluids may therefore be used – similarly to those of ureolytic bacteria – to remove metal

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cations from metal-contaminated effluents. Alternatively, when no metals are present, sulfide

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ions produced by SRB react with water to produce volatile H2S that may also be used for

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preliminary removal of metal ions from other leachates (including both the pretreated and the

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raw ones). This metal removal technique was reported in the literature (Labrenz et al., 2000; Sanchez-Andera et al., 2014; Utgikar et al., 2002) and may help to further decrease the

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dilution rate needed to maintain the SRB activity at a high level.

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As explained above, the described biotechnology may be used for the treatment of leachates coming directly from hydrometallurgical processes, thus containing high

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concentrations of metal ions. In such case, due to the toxicity of metal ions (and especially Zn2+) to bacteria, including SRB, chemically pretreated leachates have to be diluted before they can be treated in a (micro)biological manner. To reduce the freshwater demand and reduce costs, the sulfate and metal-free water obtained as a result of the biological treatment process can be recycled and used to dilute subsequent portions of the pretreated leachates. This is especially beneficial, as sulfide ions present in the SRB culture fluids following sulfate reduction immediately react with metal cations and form insoluble metal sulfides. Therefore, SRB culture fluids may be used – similarly to those of ureolytic bacteria – to remove metal cations from metal-contaminated effluents.

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Journal Pre-proof Alternatively, when no metals are present, sulfide ions produced by SRB react with water to produce volatile H2S that may also be used for preliminary removal of metal ions from other leachates (including both the pretreated and the raw ones). This metal removal technique was reported in the literature (Labrenz et al., 2000; Sanchez-Andera et al., 2014; Utgikar et al., 2002) and may help to further decrease the dilution rate needed to maintain the SRB activity at a high level. Ultimately, the proposed method can be most beneficially applied for the removal (and recovery) of ions from waste effluents from which valuable

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metal ions cannot be efficiently removed by other, e.g. electrochemical methods. For such

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effluents, the dilution factor may be significantly lower than for loaded leachates, making the

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presented approach both economically and environmentally attractive.

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The experiments performed in this work in laboratory scale, i.e. pre-neutralization and sediment production, culturing of sulfate reducing bacteria on different pre-neutralized

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leachates combined with sulfate/metal ion removal, indicate an optimum combination of these

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unit processes to form a technological line for the treatment of leachates from the recycling of alkaline batteries (Figure 8). Based on the results, the increase of the pH should be

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beneficially performed in two steps, using first a chemical reagent (NaOH) to 3.5 and then, ureolytic bacteria culture fluids containing biogenic NH3 to reach the pH of 5.0. Apart from (partial) neutralization, the first step allows for the removal of most of the iron from the leachate (from 136.25 mg/l to 2.93 mg/l; Table 7) while the second step enables further (nearcomplete) removal of Fe ions (to below the limit of detection) and supplementation with nutrients facilitating the growth of sulfate-reducing bacteria. Due to the toxicity, the pretreated leachate has to be diluted approx. 80 times (to reach the concentration of Zn ions of ~225 mg/L, Mn ions of ~125 mg/L and 1500 mg/L of sulfates) before it can be effectively treated with SRB. As a result of the SRB activity, the sulfates contained in the pre-treated, diluted leachate can be completely reduced (Figure 6), and metal ions – removed (Table 7)

Page 21 of 50

Journal Pre-proof within 9 days. To increase the activity of SRB, additional supplements and vitamins may be provided. The remaining manganese ions, as discussed above, can be removed by, e.g., oxidation. Given the promising results of experiments in laboratory scale, we proposed a continuous-flow technology, which scheme is presented below (Figure 9). Following the biochemical treatment process, Fe sediments are reclaimed in a sedimentation tank, whereas the pre-treated, appropriately diluted leachate, enters the Upflow Anaerobic Sludge Blanket

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(UASB) bioreactor. There, microbial sulfate reduction, metal sulfide precipitation, and

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ultimate neutralization of the effuents take place. The generated metal sulfides can be

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separated in a second settling tank, while the excess of hydrogen sulfide can be used to

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precipitate metals from raw and/or pre-treated leachates. In the case the effluent from USAB still contains elevated concentration of manganese ions, additional oxidation (by air and/or

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facilitated by microorganisms) may be required. The obtained treated water may be released

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or re-cycled in the technology to dilute the effluents. Before full-scale application, the technology will be tested in pilot scale. This will allow to further investigate neutralization

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and dilution processes, bacterial sulfate and metal ion removal as well as the economic aspects of biological and chemical treatment of waste leachate from battery recycling. Conclusions

To conclude, the results confirmed the application potential of sulfate-reducing bacteria in the treatment of hydrometallurgical effluents, following their pretreatment to pH 5.0 and appropriate dilution that should be tailored to the specific concentrations of toxic metal ions. We also demonstrated that the combined biological and chemical pretreatment of acidic leachate is a robust alternative to purely chemical neutralization methods, as the biological pretreatment may facilitate bacterial growth. The mechanism of this enhancement will be further investigated. It is likely that the biological pretreatment increases the

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Journal Pre-proof concentration of beneficial growth supplements (especially nitrogen compounds and vitamins) in effluents. The lack of such compounds in chemically pretreated effluents adversely affects the growth and activity of sulfate-reducing bacteria, thus leading to a decrease in the efficiency of sulfate and metal removal. The laboratory-scale analyses yielded promising results, indicating that the combined biological and chemical treatment of hydrometallurgical effluents is effective. The proposed process should therefore be scaled up and tested in a (semi-)continuous mode in larger bioreactors.

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Authors’ contributions

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ZY performed chemical pretreatment experiments, contributed to the analysis of biological

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pretreatment data as well as figures and tables preparation and results interpretation, and was

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involved in manuscript writing.

WU performed experiments with sulfate-reducing bacteria, contributed to figures and tables

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preparation and results interpretation, and was involved in manuscript writing.

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GJ performed biological pretreatment experiments, contributed to physiological analyses of urea-degrading bacteria and preparation of preliminary data for chemical pretreatment, as well

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as to manuscript writing.

JR performed chemical analysis of raw effluents, contributed to results interpretation, and was involved in manuscript writing.

JK-G contributed to results interpretation, was involved in manuscript writing, and critically reviewed the manuscript. KD-A performed isolation and physiological characterization of urea-degrading bacteria. ZR contributed to chemical experiments design, delivered leachate samples, and critically reviewed the manuscript. LD is the project head, planned and directed the study, and critically read the manuscript. All authors read and accepted the final manuscript.

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Journal Pre-proof Acknowledgments: This work was funded by Team Tech programme of Foundation for Polish Science No. POIR.04.04-00-00-2053/16 (TEAM TECH 2016-2/9) as a part of Measure 4.4 of the 2014-2020 Smart Growth Operational Programme, EU. References Abo-El-Enein, S. A., Ali, A. H., Talkhan, F. N., Abdel-Gawwad, H. A., 2013. Application of microbial biocementation to improve the physico-mechanical properties of cement

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Watzlaf, G. R., Casson, L. W., 1990. Chemical stability of manganese and iron in mine

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drainage treatment sludge: effects of neutralization chemical, iron concentration, and sludge age. In Proceedings of the Mining and Reclamation Conference (pp. 3-9), West

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Virginia University, Morgantown, WV, USA

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Wei, X., Viadero Jr, R. C., Buzby, K. M., 2005. Recovery of iron and aluminum from acid mine drainage by selective precipitation. Environmental Engineering Science, 22(6),

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745-755. https://doi.org/10.1089/ees.2005.22.745 Whiffin, V. S., 2004. Microbial CaCO3 precipitation for the production of biocement (Doctoral dissertation, Murdoch University). Xará, S., Delgado, J., Almeida, M. F., Costa, C., 2013. Laboratory study on the leaching potential of spent alkaline batteries using a MSW landfill leachate. Journal of Material Cycles and Waste Management, 15(1), 61-72. https://doi.org/10.1007/s10163-0120091-8

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Figure 1. Scheme of the battery recycling based on acidic leaching.

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Figure 2. Scheme of the testing process of chemical and biological pretreatment in batch scale. Four reagents were used :40% NaOH, 20% Na2CO3, 1% synthetic NH3 (aq) (S-NH3), and

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1% biogenic NH3 (B-NH3).

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Figure 3. Pretreatment of the leachates with the use of various neutralizing agents: 40%

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NaOH, 20% Na2CO3, 1% synthetic NH3 (aq) (S-NH3), 1% biogenic NH3 (B-NH3).

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Figure 4. The real Fe concentration (cR(Fe)) in the leachates pretreated with (A) 40% NaOH or 20% Na2CO3, (B) 1% synthetic NH3 (aq) (S-NH3) or 1% biogenic NH3 (B-NH3).

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Figure 5. Iron concentration in the sediments formed after leachate pretreatment with (A) 40% NaOH or 20% Na2CO3, (B) 1% synthetic NH3(aq) (S-NH3) or 1% biogenic NH3 (B-NH3).

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1600 1400 1200 1000 800 600 400 200 0 1

3

5 Time [days]

7

1A (MPB, VS+, TES+)

2A (MPB, BCh+, VS+, TES+)

1B (MPB)

2B (MPB, BCh+)

9

3A (MPB, Ch+, VS+, TES+)

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Sulfates [mg/L]

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3B (MPB, Ch+)

Figure 6. Kinetics of sulfate removal in SRB cultures with or without the addition of the

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pretreated leachates. Data for negative controls (no SRB), where sulfate concentration

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remained constant (± 5%), was removed for the clarity of the figure. MPB – modified

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Postgate B medium, VS – vitamins solution, TES – trace elements solution, BCh –

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biologically and chemically treated leachates, Ch – chemically treated leachates.

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Figure 7. Changes of pH in SRB cultures with or without the addition of the pretreated

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leachates. Data for negative controls (no SRB), where sulfate concentration remained

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constant (± 5%), was removed for clarity of the figure. MPB – modified Postgate B medium,

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VS – vitamins solution, TES – trace elements solution, BCh – biologically and chemically

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treated leachates, Ch – chemically treated leachates.

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Figure 8. Laboratory scale technological line, based on the optimum variants of the unit

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processes tested in this study. Concentrations of ions are given in [mg/L]. B-NH3 – biogenic

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NH3, SRB – sulfate reducing bacteria.

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Figure 9. Scheme of the proposed technology.

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Journal Pre-proof Table 1. Concentration of Mn2+, Zn2+, and SO42- ions in the preliminary toxicity test. Control Pre-C

Pre-1

Pre-2

Pre-3

Pre-4

Pre-5

Pre-6

Mn2+

2 (trace) 5 (trace)

125

250

400

1000

2500

5000

225

450

720

1800

4500

9000

SO42-

1500

1500

3000

4800

12000

30000

60000

pretreated leachate dilution rate; (% v/v)

-

80x (1.25)

40x (2.5)

25x (4.0)

10x (10)

4x (25)

2x (50)

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Zn2+

Variant

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Initial ion concentration [mg/L]

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Mix battery (alkaline and zinc-manganese) powder (wt.%) (De Michelis et al., 2007)

28.3

15.46

Mn

26-45

26.3

33,59

K

4.53-7.3

–

3.26

Fe

0.07-0.36

3.4

0.50

Pb

0.005-0.03

–

–

Hg

<1

–

–

Cr

–

–

Cd

0.06

–

Al

–

–

Cl

–

–

Ti

–

–

Si

–

–

Ni

0.010

–

Other

30-41.80

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Zinc-manganese dry battery powder (wt.%) (Peng et al., 2008)

0.19

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–

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0.36 3.38

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0.27

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Element

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Zn

Alkaline battery powder (wt.%) (Salgado et al., 2003; de Souza et al., 2001; de Souza and Tenorio, 2004) 12-21

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Table 2. Typical metal composition of alkaline and zinc–carbon batteries.

0.49 –

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Journal Pre-proof Table 3. Element concentration (mg/L) in the raw leachate used in this study. As

B

Ba

Ca

Cd

Co

Cr

Cu

Fe

K

Li

62

<5

<3

<0.3

167

61.3

42.1

2.0

70.4

247

4036

4

Mg

Mn

Mo

Na

Ni

P

Pb

SiO2

Sr

Ti

V

Zn

39

18294 <0.9

249

409

<15

<3

96

1.8

1.1

<2

36049

Al.

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< below the limit of detection

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Journal Pre-proof Table 4. Sediment productivities and pH changes in the leacheates pretreated with the following agents: 40% NaOH, 20% Na2CO3, 1% synthetic NH3

(aq)

(S-NH3), or 1%

biogenic NH3 (B-NH3).

pH

40% NaOH

20% Na CO

1

ND

ND

ND

ND

1.5

ND

ND

ND

ND

2

ND

ND

ND

2.5

0.238

ND

ND

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3

0.554

ND

ND

3.5

0.650

0.446

0.116

4

0.629

0.462

0.191

0.429

4.5

1.319

0.532

0.225

0.912

5

1.668

0.687

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Sediment mass (g) obtained after neutralization with the following reagents

0.304

1.481

1% B-NH

3

3

ND

0.344

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1% S-NH

0.313 0.360

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ND – not detected

2

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Journal Pre-proof Table 5. Variants of SRB cultures grown on MBP medium with or without the pretreated leachates. BCh – biologically and chemically treated leachates, Ch – chemically treated leachates. Initial ion concentration [mg/L] Mn2+ Zn2+

Cultures with supplements MPB BCh Ch (no leachate) leachate leachate 2 125 125 (trace) 5 225 225 (trace)

Cultures without supplements MPB BCh Ch (no leachate) leachate leachate <0.001

125

125

<0.003

225

225

1500

1500

1500

1500

1500

1500

Variant label

1A

2A

3A

1B

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SO42-

2B

3B

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Journal Pre-proof Table 6A. Concentrations of inorganic ions after 9 days of negative control on MPB medium with or without the addition of pretreated leachates and with or without the addition of growth supplements. BCh -–biologically and chemically treated leachates, Ch – chemically treated leachates.

2A – BCh +

Mn2+

0.15

105.76

121.26

2+

0.01

140.37

SO42-

1464

1766

2+

0.06

4.78

2+

0.06 0.25

Zn

d.w. of the collected precipitate [g]

1.16

126.90

139.41

153.04

5.96

193.31

184.46

1737

1284

1560

1734

5.12

0.57

5.02

5.61

23.28

23.26

2.32

24.45

23.12

0.35

0.40

0.34

0.37

0.30

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Mn

precipitate [mg/g d.w.]

3B – Ch –

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Zn

2B – BCh –

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1A – – +

SRB Leachate Supplements Ion concentration (day 9)

supernatant [mg/L]

Negative control variant 3A 1B – – Ch – + –

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Table 6B Concentrations of inorganic ions after 9 days of SRB culturing on MPB

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medium with or without the addition of pretreated leachates and with or without the addition of growth supplements. BCh -–biologically and chemically treated leachates, Ch –

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chemically treated leachates.

Variant

1A

2A

3A

1B

2B

3B

–

BCh

Ch

–

BCh

Ch

+

+

+

–

–

–

Mn2+

2.39

2.86

3.22

0.30

19.27

20.12

2+

0.01

0.03

0.08

<0.003

0.01

<0.003

2-

<0.5

<0.5

115.0

<0.5

<0.5

159.0

2+

0.35

112.7

76.13

0.18

82.30

103.0

2+

Zn

2.38

153.6

166.9

1.93

134.3

155.5

d.w. of the collected precipitate [g]

0.66

1.25

1.23

0.57

1.32

1.30

Leachate Supplements Ion concentration (day 9) supernatant [mg/L]

Zn

SO4 precipitate [mg/g d.w.]

Mn

d.w. stands for dry weight; < below limit of detection

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Journal Pre-proof Table 7. Metal concentration and pH during the (pre)treatment. Raw leachate was treated with the following agents: 40% NaOH (to reach pH 3.5), ureolytic bacteria post-culture medium containing biogenic ammonia (to reach pH 5.0), and sulfate-reducing bacteria (to reach pH 7.25). Average values from the experimental variants were presented.

Original

First stage

Second stage

—

Chemical (NaOH)

Biological (Biogenic NH3)

Biological (SRB)

pH

0.48

3.5

5.0

7.25

Fe (mg/L)

136.25

2.93

<0.01

Zn (mg/L)

36049

31019

26960

Mn (mg/L )

18294

15426

11409

<0.01 0.03 11.37

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Treatment

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< below limit of detection

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Journal Pre-proof Table S1. Reagent cost of 40% NaOH, 20% Na2CO3, 1% S-NH3 and 1% B-NH3 to increase the pH of 1 m3 of the studied leachate to 5.

Stock preparation cost (USD/L)

Volume of the stock consumed (L/m3)

Reagent cost (USD/m3)

40% NaOH

0.460

100.0

46.00

20% Na2CO3 1% S-NH3

0.125 0.031

232.5 1368.3

29.06 42.41

1% B-NH3

0.019

1892.4

35.95

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Chemicals

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Journal Pre-proof 

Gradual acidic leachate pretreatment with sodium hydroxide and biogenic ammonia



Biogenic ammonia synthesized by urea-degrading bacteria



Combined biological and chemical pretreatment promotes the growth and activity of SRB

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Sulfates and metals were almost completely removed and metal sulfides precipitated

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

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Journal Pre-proof Conflict of interest statement The authors declare that they have no known competing financial interests or personal

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relationships that could have appeared to influence the work reported in this paper.

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Journal Pre-proof Author Contribution Statement ZY performed chemical pretreatment experiments, contributed to the analysis of biological pretreatment data as well as figures and tables preparation and results interpretation, and was involved in manuscript writing. WU performed experiments with sulfate-reducing bacteria, contributed to figures and tables preparation and results interpretation, and was involved in manuscript writing. GJ performed biological pretreatment experiments, contributed to physiological analyses of

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urea-degrading bacteria and preparation of preliminary data for chemical pretreatment, as well

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as to manuscript writing.

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JR performed chemical analysis of raw effluents, contributed to results interpretation, and was

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involved in manuscript writing.

reviewed the manuscript.

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JK-G contributed to results interpretation, was involved in manuscript writing, and critically

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KD-A performed isolation and physiological characterization of urea-degrading bacteria. ZR contributed to chemical experiments design, delivered leachate samples, and critically

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reviewed the manuscript.

LD is the project head, planned and directed the study, and critically read the manuscript. All authors read and accepted the final manuscript.

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