Bioleaching of Lithium-Ion Battery Black Mass: A Comparative Study on Gluconobacter oxydans and Acidithiobacillus thiooxidans

Abstract

The growing demand for lithium-ion batteries (LIBs) requires efficient and sustainable recycling solutions. This study investigates bioleaching as an alternative to conventional hydrometallurgical methods, focusing on (i) organic acid-mediated leaching with Gluconobacter oxydans and (ii) sulfuric acid bioleaching with Acidithiobacillus thiooxidans. Experiments were conducted at 26 °C with leaching durations of one to three weeks, depending on the microbial system, at pH 1.35 for sulfuric acid treatments, and with liquid-to-solid ratios equivalent to 100 mL g⁻¹ (A. thiooxidans) or 100 mL g⁻¹ in culture medium (G. oxydans). Results show that indirect bioleaching with G. oxydans achieved high recovery rates for cobalt (96%), manganese (100%), nickel (65%), and lithium (68%), while the direct approach was less effective due to microbial inhibition by black mass components. Similarly, biologically produced sulfuric acid exhibited moderate leaching efficiencies, but chemically synthesized sulfuric acid outperformed it, particularly for nickel (93%) and lithium (76%) after one week of leaching. These findings suggest that bioleaching is a promising, eco-friendly alternative for LIB recycling but requires further process optimization to improve metal recovery and industrial scalability. Future research should explore hybrid approaches combining bioleaching with conventional leaching techniques.

1. Introduction

The increasing global demand for lithium-ion batteries (LIBs) is driven by their widespread application in electric vehicles and stationary energy storage systems. Since their commercial introduction in the 1990s, LIBs have gained prominence due to their high energy density, long cycle life, and relatively low self-discharge rates. However, as the transition towards sustainable energy solutions accelerates, concerns regarding the responsible management of end-of-life (EoL) LIBs are growing [1,2,3].

In response to the environmental and economic challenges associated with EoL batteries, the European Union (EU) has implemented regulations to enhance battery recycling and critical raw material recovery. The 2023 Battery Regulation (EU 2023/1542) [4] mandates that by 2030, 70% of an LIB’s total weight must be recycled, with specific recovery targets for cobalt, nickel, copper (95%) and lithium (80%) by 2031. These regulatory measures align with the objectives of the EU Green Deal [5] and the European Battery Alliance [6], which emphasize a circular economy approach to ensure a secure and sustainable supply chain for battery materials [3,7].

Current industrial recycling methods predominantly rely on pyrometallurgical and hydrometallurgical techniques. While these approaches enable the recovery of valuable metals, they pose significant environmental challenges due to high energy consumption, CO₂ emissions, and the generation of hazardous residues. As an alternative, biometallurgical processes–often referred to as bioleaching–have emerged as a promising method for metal extraction from secondary resources, including spent LIBs [8,9,10].

Bioleaching involves the use of microorganisms to catalyze the dissolution of metals through the production of organic acids or through redox reactions. This approach has been successfully applied in copper and gold mining and is now being explored for LIB recycling [11]. Compared to conventional methods, bioleaching offers several advantages, including lower energy demand, reduced environmental impact, and the potential for selective metal extraction. However, the process is still under investigation, and challenges related to microbial toxicity, reaction kinetics, and process scalability must be addressed [12,13].

This study investigates the effectiveness of bioleaching for LIB black mass, focusing on two bacterial species: Gluconobacter oxydans and Acidithiobacillus thiooxidans. The aim is to compare the leaching efficiency of microbially produced organic acids with direct bacterial activity and to evaluate the potential of bioleaching as a viable recycling approach for LIBs, with experiments conducted at 26 °Cover one- to three-week periods depending on the microbial system.

2. Scientific Background

2.1. Lithium-Ion Battery Composition and Recycling Challenges

A lithium-ion battery consists of several key components: a graphite anode, a cathode composed of lithium-metal oxides, an electrolyte facilitating ion transport, and a separator preventing short circuits. The cathode chemistry varies, with the most common compositions being lithium cobalt oxide (LCO), lithium iron phosphate (LFP), and lithium nickel manganese cobalt oxide (NMC). NMC batteries, in particular, have become increasingly popular and widely adopted in electric vehicle applications due to their high energy density and long cycle life [14,15,16].

The complexity of LIB materials presents a major challenge for recycling. Conventional processes involve mechanical pre-treatment followed by either pyrometallurgical and/or hydrometallurgical recovery of valuable metals. Pyrometallurgy employs high-temperature smelting to extract cobalt, nickel, and copper, often leading to the transfer of lithium in slag or flue dust. Hydrometallurgical methods use strong acids, such as sulfuric acid (H₂SO₄), to dissolve metal components, followed by precipitation or solvent extraction to separate individual elements. While hydrometallurgy offers higher metal recovery rates, it requires significant chemical input and produces large volumes of acidic wastewater [8,10,17,18,19].

As a result of the mechanical and/or thermal pre-treatment steps in LIB recycling, a fine-grained intermediate product known as black mass (BM) is generated. Black mass is a complex mixture of cathode active materials, graphite, and residual electrolytes. It is typically obtained after the shredding and vacuum distillation or pyrolysis of spent LIBs, where non-metallic components, such as electrolytes and binders, are removed or thermally degraded. The composition of black mass varies depending on the battery chemistry but generally contains lithium, cobalt, nickel, manganese, graphite, copper, and aluminum in varying proportions. Impurities, such as iron, silicon, zinc and residual organic compounds, may also be present [8,20,21,22].

Due to its high content of strategic or critical metals, black mass represents the most notable intermediate product in LIB recycling and serves as the primary feedstock for metal recovery processes. However, its heterogeneous composition and potential contamination with organic compounds pose challenges for both conventional and bio-based leaching methods. Understanding the interactions between microorganisms and the constituents of black mass is essential for optimizing bioleaching efficiency and ensuring selective metal recovery [20,21,22].

2.2. Biometallurgy as a Sustainable Alternative

Biometallurgy, or biohydrometallurgy, utilizes microorganisms to facilitate metal extraction from ore and secondary materials. Bioleaching can proceed either through direct microbial activity or via metabolites. Acidophilic bacteria such as A. thiooxidans oxidize elemental sulfur to generate sulfuric acid, thereby solubilizing transition metals. In contrast, G. oxydans oxidizes glucose to gluconic acid, which dissolves metals by complexation. However, elevated concentrations of dissolved transition metals or organic residues may inhibit microbial activity through enzyme binding or membrane disruption [12,23].

The bioleaching process can proceed via three primary mechanisms: [12,23]

• Redoxolysis: Microorganisms catalyze oxidation-reduction reactions to solubilize metal compounds.
• Acidolysis: Microbial production of organic or inorganic acids leads to metal dissolution.
• Complexolysis: Chelating agents produced by microbes form soluble metal complexes.

Despite the ecological advantages and growing interest in bioleaching, several inherent limitations still constrain its industrial implementation. A major challenge is the pronounced variability in black mass composition, which depends on battery chemistry, production methods, and aging. Differences in metal content, residual electrolytes, and binders directly influence process design and recovery efficiency. High concentrations of transition metals, for example, can enhance leaching thermodynamics but simultaneously inhibit microbial activity, while electrolyte residues may affect pH buffering and complexation behavior. Consequently, adaptive process parameters and, in some cases, pre-treatment steps are required to achieve consistent performance [24,25,26,27].

Beyond compositional variability, bioleaching faces broader technical, economic, and regulatory barriers. Slow kinetics, microbial sensitivity to toxic metals, and difficulties in scaling biological systems currently limit industrial feasibility. Moreover, longer residence times and higher reactor volumes increase costs, while regulatory uncertainties regarding microbial use further complicate implementation. Overcoming these challenges will require advances in strain engineering, reactor design, and hybrid process strategies to fully exploit the potential of bioleaching in LIB recycling.

In the context of LIB recycling, bioleaching offers an environmentally friendly alternative by eliminating the need for harsh chemicals and reducing energy input. However, the efficiency of bioleaching depends on multiple factors, including pH, temperature, metal toxicity, and microbial metabolism [12,23,24,25,26].

2.3. Microorganisms in LIB Bioleaching

Two microbial species have been identified as promising candidates for LIB bioleaching: [27,28]

• Gluconobacter oxydans: A Gram-negative, aerobic bacterium capable of oxidizing glucose into gluconic acid. This organic acid serves as a mild leaching agent, forming stable metal–organic complexes. G. oxydans is particularly effective in mobilizing lithium and cobalt, offering a potential route for selective metal recovery [12,29,30].
• Acidithiobacillus thiooxidans: A chemolithoautotrophic, acidophilic bacterium that oxidizes elemental sulfur and sulfide minerals to generate sulfuric acid. This metabolic process has been widely exploited in the bioleaching of copper and other sulfide ores and is now being investigated for its role in LIB metal dissolution [12,13,30,31,32].

Both microorganisms exhibit different leaching behaviors. While G. oxydans produces gluconic acid as a direct metabolic byproduct, A. thiooxidans indirectly enhances metal solubilization by lowering the pH through sulfur oxidation. The efficiency of these organisms in LIB recycling depends on their tolerance to metal toxicity, acid resistance, and the ability to maintain stable metabolic activity in the presence of black mass constituents [12,30].

Both direct and indirect bioleaching approaches were investigated in order to systematically assess the influence of microbial activity and acid generation pathways on metal recovery. The direct approach provides insight into the interactions between microorganisms and the black mass matrix, including potential inhibitory effects caused by high metal ion concentrations or organic residues. The indirect approach, in contrast, separates microbial growth from the leaching step, allowing the use of pre-produced metabolites under controlled conditions. This decoupling not only enables a clearer evaluation of acid-mediated dissolution mechanisms but also reflects a strategy commonly discussed for industrial applications, where acid production and leaching may be implemented as separate unit operations. Previous studies have reported higher extraction efficiencies for the indirect approach, particularly for cobalt and nickel, as well as improved process controllability and scalability under non-inhibitory conditions [25,28,33,34,35]. For these reasons, both approaches were included in this study to provide a comprehensive assessment of their performance and potential for industrial integration.

The key difference between the direct and indirect leaching approaches is the timing of the addition of black mass to the culture medium. In the indirect approach, this is delayed, allowing the bacteria time to metabolize the medium without the addition of foreign material. This approach separates the growth of the bacteria and the leaching of the black mass into two distinct process steps, thus enabling the optimization of both the growth conditions for the strain and the leaching parameters [28,33].

As illustrated in Figure 1, the indirect approach involves a distinction from the direct approach that is characterized by the aforementioned separation of the two sub-steps. The integration of the bacterium’s growth phase and the leaching of the black mass results in higher process efficiency, thus providing a notable advantage to this approach [28,33,34,35].

Both G. oxydans and A. thiooxidans exhibit specific physiological requirements that critically influence their bioleaching performance. Gluconobacter oxydans prefers slightly acidic to neutral conditions, with optimal growth observed at pH values between 5.5 and 6.5. Its acid production, however, can lower the pH to around 3.5–4.5, which still permits metabolic activity but may limit long-term viability depending on the medium composition. In contrast, Acidithiobacillus thiooxidans is an obligate acidophile, thriving in highly acidic environments with an optimal pH range between 1.5 and 3.0. The bacterium not only tolerates but actively maintains these conditions through sulfur oxidation, making it well-suited for bioleaching processes that require low pH for metal solubilization. These distinct pH preferences must be considered in bioleaching system design, especially in terms of microbial compatibility, metal solubility, and process integration [36,37].

In addition to microbial physiology, typical operating parameters reported in the literature provide an important context for evaluating bioleaching performance in LIB recycling. Bioleaching processes for LIB black mass typically operate under mild thermal conditions between 20 °C and 35 °C, depending on the microbial species employed. Organic-acid-producing bacteria such as Gluconobacter oxydans generally show optimal activity at pH 5.5–6.5, while acidophiles such as Acidithiobacillus thiooxidans thrive in highly acidic environments (pH 1.5–3.0). Reported leaching durations in laboratory-scale studies range from several days to three weeks, often with liquid-to-solid ratios between 50 mL g⁻¹ and 200 mL g⁻¹, depending on the targeted metals and the acidity of the medium. Sulfuric acid concentrations in biogenic or chemical form are usually adjusted to pH 1–2 to maximize solubilization of transition metals such as Co, Ni, and Mn. Extraction efficiencies vary widely across studies but can exceed 90% for cobalt and manganese under optimized conditions, while lithium recoveries typically range between 60% and 80% [12,19,23,29,30,31].

2.4. Research Objectives and Hypotheses

This study aims to compare the bioleaching efficiencies of G. oxydans and A. thiooxidans in extracting valuable metals from LIB black mass. The specific objectives include the following:

• Assessing the leaching performance of gluconic acid compared to direct bacterial activity: By analyzing the dissolution rates of lithium, cobalt, nickel, and manganese, we aim to determine whether organic acid-mediated leaching is more effective than microbial metabolism alone.
• Evaluating the effectiveness of biologically produced sulfuric acid: The study investigates whether microbially generated sulfuric acid differs in leaching performance compared to chemically synthesized sulfuric acid at an equivalent acidity (pH 1.35).
• Understanding microbial-metal interactions: Since black mass contains a complex mixture of metals, electrolytes, and carbonaceous materials, it is crucial to evaluate how these factors influence microbial growth, metabolism, and leaching efficiency.

The findings of this research will contribute to the development of sustainable LIB recycling processes, addressing key challenges in material recovery while minimizing environmental impact.

3. Materials and Methods

3.1. Black Mass Characterization

The black mass (BM) used in this study was obtained from an industrial lithium-ion battery (LIB) recycling plant. The batteries underwent a mechanical and thermal pre-treatment process, including sorting, discharging, and pyrolysis at 500 °C, followed by shredding and sieving. The BM fraction used for the bioleaching experiments had a particle size of <250 µm.

Elemental composition was determined using inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7500, Santa Clara, CA, USA). The major metallic components included Ni (22.0 wt%), Mn (7.3 wt%), Co (6.5 wt%), Li (5.2 wt%), Cu (4.8 wt%), and Al (3.7 wt%), with minor amounts of Zn (0.7 wt%). Aluminum in the investigated black mass occurs both as metallic aluminum (from current collector foils) and as aluminum oxide phases associated with cathode coating residues. Copper from current collector foils occurs predominantly as a metallic component. The heterogeneity of BM was confirmed through multiple analyses using ICP-MS, X-ray diffraction (XRD) (Bruker D8 Advance, Billerica, MA, USA), and scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS, Jeol JSM-IT300, Akishima, Tokio, Japan). Figure 2a shows the XRD pattern of the NMC-type black mass, with the dominant crystalline phases identified as layered Li(Ni,Mn,Co)O₂ (●), graphite (▲), and graphite (■). These phases are consistent with the elemental composition determined by ICP-MS. Figure 2b illustrates the heterogeneous particle morphology and size distribution as observed by SEM. Complementary EDX analysis confirmed the presence of Ni, Co, Mn, Li, Cu, Al, and C, in agreement with the bulk composition. The combination of XRD and SEM/EDX thus provides a comprehensive characterization of the black mass prior to leaching.

3.2. Microorganisms and Culture Conditions

Two bacterial species were used for bioleaching experiments:

• Gluconobacter oxydans (G. oxydans, DSMZ 3504, Leibniz Institute DSMZ, Braunschweig, Germany): A chemoorganotrophic, Gram-negative bacterium capable of oxidizing glucose to gluconic acid.
• Acidithiobacillus thiooxidans (A. thiooxidans, DSMZ 14887, Leibniz Institute DSMZ, Braunschweig, Germany): A chemolithoautotrophic, acidophilic bacterium that oxidizes sulfur to sulfuric acid.

Both strains were cultivated under sterile conditions in the following media:

G. oxydans was grown in Medium 105, consisting of 100 g∙L⁻¹ glucose, 10 g∙L⁻¹ yeast extract, and 20 g∙L⁻¹ CaCO₃. Agar (15 g∙L⁻¹) was added to the solid media.

A. thiooxidans was cultured in Medium 35, containing 3 g∙L⁻¹ KH₂PO₄, 0.1 g∙L⁻¹ NH₄Cl, 0.1 g∙L⁻¹ MgCl₂·6H₂O, 0.14 g∙L⁻¹ CaCl₂·6H₂O, and 10 g∙L⁻¹ elemental sulfur.

All cultures were incubated at 26 °C in an orbital shaker (Hydro H 20 S, Lauda, Lauda-Königshofen, Germany) at 40 rpm to ensure homogeneous mixing. G. oxydans cultures were maintained for one week, while A. thiooxidans required two weeks for sulfur oxidation. Elemental sulfur (10 g·L⁻¹) served as the sole electron donor and energy source for A. thiooxidans during acid generation; no sulfur is provided by the black mass in our experiments.

3.3. Bioleaching Experiments

Two different bioleaching approaches were investigated: (i) direct bacterial bioleaching, where BM was added directly to bacterial cultures, and (ii) indirect bioleaching, where pre-produced acids were used to leach BM.

For the bioleaching experiments, 0.5 g of black mass was used per 50 mL of culture medium in the G. oxydans tests, and 1.0 g of black mass per 100 mL of acid solution in the A. thiooxidans tests.

To ensure compatibility, the experiments were conducted under analogous conditions. The parameters of the two experiments are shown in

Table 1. Experiment parameters of the direct and indirect leaching approach.

Test Parameters	Direct Leaching Approach	Indirect Leaching Approach
Amount of culture medium	50 mL	50 mL
Amount of black mass	0.5 g	0.5 g
Incubator temperature	26 °C	26 °C
Stirred tank temperature	26 °C	26 °C
Incubation time	0 h	1 week
Leaching time in the stirred tank	1 week	1 week
Rotation speed of the stirred tank	40 U/min	40 U/min

and are the same for the three iterations of each test.

3.3.1. Direct Bioleaching with G. oxydans

In the direct bioleaching approach, the black mass was added to the culture medium immediately after inoculation, i.e., without a separate pre-incubation or growth phase. Therefore, the incubation time prior to contact between bacteria and black mass is indicated as 0 h in Table 1. This setup was chosen to simulate a scenario in which microbial activity and metal dissolution occur simultaneously from the start of the experiment. It also allows a direct assessment of the impact of black mass components on bacterial growth and metabolism, which is an important factor in evaluating the feasibility of single-step bioleaching processes.

3.3.2. Indirect Bioleaching with Gluconic Acid

In the direct bioleaching experiment, 50 mL of Medium 105 was inoculated with G. oxydans and supplemented with 0.5 g of BM. The mixture was incubated at 26 °C under continuous shaking (40 rpm) for one week. A control experiment without bacteria was performed under identical conditions.

3.3.3. Bioleaching with A. thiooxidans

A. thiooxidans was cultivated for two weeks in Medium 35, with elemental sulfur as the sole energy source. After incubation, the microbial culture was filtered to obtain sulfuric acid. Two variations in the sulfuric acid solution were tested:

• Non-sterilized cell-free sulfuric acid (containing active enzymes);
• Sterilized microbial sulfuric acid (autoclaved at 121 °C for 20 min).

Both sterilized and non-sterilized biogenic sulfuric acid were tested to examine whether residual microbial components (e.g., enzymes or metabolites) influence metal dissolution. The comparison allows differentiation between purely chemical leaching effects and potential bio-catalytic contributions to leaching performance.

For comparison, an inorganic sulfuric acid solution (pH 1.35) was prepared to match the acidity of the microbial sulfuric acid. In all cases, 100 mL of acid was used for leaching 1 g of BM over one week.

3.4. Analytical Methods

At the end of each leaching experiment, solid residues were separated by filtration (2–3 µm) and dried at 105 °C for 24 h. The metal content in the leachates was analyzed using Microwave Plasma–Atomic Emission Spectroscopy (MP-AES, Agilent 4210, Santa Clara, CA, USA). The pH and redox potential (E_h) were determined before the experiments.

The theoretical maximum soluble concentration (c_max) for each element was calculated based on the BM composition and the initial mass of active material used in the leaching experiments. By comparing these values with the experimentally determined metal concentrations (c_D for the direct approach and c_I for the indirect approach), the efficiency of metal extraction (η_D and η_I) was evaluated (see Equation (1)).

$$ƞ={\displaystyle \frac{c_{leached}}{c_{max}}}·100\%$$

(1)

where

• c_leached is the metal concentration in solution (mg∙L⁻¹);
• c_max is the theoretical maximum concentration based on BM composition.

3.5. Experimental Controls and Reproducibility

All experiments were conducted in triplicate, and control experiments were performed for abiotic leaching (without bacteria) and under sterile conditions. Statistical analysis was conducted using one-way Analysis of Variance (ANOVA), with a significance threshold of p < 0.05.

4. Results and Discussion

The following section presents the results of the bioleaching experiments and their discussion. First, the leaching performance of Gluconobacter oxydans is analyzed by comparing the direct and indirect bioleaching approaches. Subsequently, the focus shifts to the leaching efficiency of sulfuric acid produced by Acidithiobacillus thiooxidans, comparing its effectiveness with chemically synthesized sulfuric acid.

4.1. Leaching Efficiency of Gluconobacter oxydans

To compare the effectiveness of the two bioleaching approaches, the concentrations of dissolved metals in the filtrates were analyzed. The target elements for metal recovery were copper (Cu), nickel (Ni), cobalt (Co), lithium (Li), manganese (Mn), and aluminum (Al). Other elements such as silicon (Si), iron (Fe), and zinc (Zn) were excluded from the analysis, as they are considered impurities originating from the black mass (see Section 3.1).

A visual inspection of the leachates revealed a distinct color difference between the direct and indirect approaches. The direct bioleaching approach resulted in a pale-colored solution, whereas the indirect approach led to a distinctly darker filtration, suggesting a higher acid concentration in the indirect bioleaching process. Since G. oxydans produces gluconic acid, which enhances metal dissolution, the darker color indicates increased acid production and, consequently, a higher leaching efficiency.

The results of the leaching efficiency analysis are summarized in

Table 2. Comparison of the leaching efficiencies of Gluconobacter oxydans.

Element	cmax [mg·L−1]	cD [mg·L−1]	cI [mg·L−1]	ηD [%]	ηI [%]
Copper (Cu)	480	24	101	5	21
Nickel (Ni)	2200	186	1433	8	65
Cobalt (Co)	650	108	622	17	96
Lithium (Li)	520	183	352	35	68
Manganese (Mn)	730	390	730	53	100
Aluminum (Al)	370	130	228	35	62

c_max—maximum achievable concentration; c_D—achieved concentration of the direct approach; c_I—achieved concentration of the indirect approach; η_D—achieved efficiency of the direct approach; η_I—achieved efficiency of the indirect approach.

.

4.2. Comparison of Direct and Indirect Bioleaching

A detailed analysis of the extraction rates indicates that the indirect bioleaching approach was significantly more efficient than the direct approach in solubilizing most target metals.

High extraction efficiency in indirect bioleaching is detailed as follows:

• Cobalt (96%) and manganese (100%) exhibited the highest dissolution rates.
• Nickel (65%), lithium (68%), and aluminum (62%) also showed good solubilization efficiency.
• Due to its noble character, copper was only solubilized to 21%.

Lower efficiency in direct bioleaching after one week produced the following:

• None of the metals showed an extraction efficiency above 55%.
• Lithium and manganese were extracted at 35% and 53%, respectively—roughly half the efficiency observed in the indirect approach. Lithium extraction efficiencies of ~65% were achieved under the tested conditions, which is consistent with previous reports for LIB black mass using G. oxydans [26,31].
• The solubilization rates for cobalt and nickel were significantly lower (16.7% and 8.5%, respectively).
• For aluminum, the extraction efficiency was approximately 35%, which is half of that achieved in the indirect approach. The partial dissolution of Al observed in the experiments is explained by the oxidative action of gluconic and other organic acids produced by G. oxydans, which have been reported to solubilize aluminum phases in LIB residues [25].
• Copper extraction remained low in both approaches, with only 5% solubilized in the direct bioleaching test.

A graphical comparison of these results is shown in Figure 3.

The pronounced differences in leaching efficiency, particularly for cobalt and nickel, can be explained by their distinct chemical behavior and interactions with the leaching system. Cobalt often forms sparingly soluble spinel or oxide phases such as Co₃O₄, which are more resistant to acid dissolution than the corresponding nickel compounds [25,33]. Furthermore, cobalt ions are known to exert stronger inhibitory effects on microbial metabolism, leading to reduced acid production and leaching performance in direct bioleaching setups [35,37]. These phenomena have been consistently observed in previous studies, where nickel typically shows higher recovery rates than cobalt under comparable bioleaching conditions [28,36,38].

4.3. Influence of Black Mass on Bioleaching Performance

The significantly lower extraction rates observed in the direct approach can be attributed to the toxic effects of BM components on bacterial activity. The presence of high metal ion concentrations, residual electrolytes, and organic binders may have hindered bacterial growth and, therefore, acid production, thus limiting metal solubilization.

In the indirect approach, bacterial growth occurred in the absence of BM, allowing for unhindered acid production. The pre-produced gluconic acid was then applied to BM, enhancing leaching efficiency. The formation of organo-metallic complexes with gluconic acid may have further facilitated the dissolution of target metals, particularly cobalt and nickel. Such synergistic effects between microbial acid production and transition metal dissolution have likewise been described in biohydrometallurgical studies on LIB residues [25].

4.4. Interpretation and Implications of Bioleaching for Industrial LIB Recycling

The results clearly indicate that the indirect bioleaching approach is significantly more effective than the direct approach for extracting metals from black mass. The indirect process, in which gluconic acid was produced separately before being applied to the black mass, resulted in higher dissolution rates for all target elements, with particularly strong effects observed for cobalt (96%) and manganese (100%). In contrast, the direct bioleaching approach showed a notably lower extraction efficiency, with no metal exceeding a 55% recovery rate. These findings suggest that the primary limitation of the direct approach is the toxicity of black mass to microbial metabolism, which inhibits bacterial growth and acid production.

The differences in metal recovery observed in this study can be explained by several complementary mechanisms. First, organic acids produced by G. oxydans, particularly gluconic acid, promote metal dissolution through acidolysis and the formation of stable metal–gluconate complexes, which enhance solubility and prevent re-precipitation. Second, the presence of toxic metal ions, electrolyte residues, and binder components can inhibit microbial metabolism in the direct bioleaching approach, reducing acid production and slowing down dissolution kinetics. Third, passivation phenomena, such as the formation of sparingly soluble oxides or spinel-type phases, particularly for cobalt, limit the accessibility of reactive surfaces and thus decrease leaching efficiency. These combined effects explain the superior performance of the indirect approach and the observed differences in metal-specific recovery behavior.

In direct bioleaching, Gluconobacter oxydans is exposed to high concentrations of heavy metals, which can interfere with cellular metabolism. Many transition metals, including cobalt and nickel, are known to have antimicrobial properties at high concentrations, disrupting membrane integrity and enzyme function [25,37,38]. This effect likely explains the poor leaching efficiency for these metals in the direct approach, as the bacteria are unable to produce sufficient gluconic acid before being inhibited by metal toxicity. Additionally, the complex composition of black mass, which includes organic binders, residual electrolytes, and carbonaceous materials, may further stress bacterial populations, leading to a reduction in metabolic activity.

The indirect bioleaching approach circumvents these issues by allowing bacterial acid production to occur in a metal-free environment, thereby maximizing gluconic acid yield before exposure to black mass. This pre-produced acid then acts as a strong leaching agent, effectively dissolving cobalt, manganese, and nickel from the black mass. The color change observed in the leachates further supports this explanation: the darker solution in the indirect approach suggests a higher acid concentration, leading to increased metal dissolution. This observation aligns with the pH-dependent solubility of transition metals, which generally increases as acidity intensifies.

A particularly notable difference between the two approaches is the extraction efficiency of cobalt and nickel. In the indirect approach, cobalt recovery reached 96%, compared to only 17% in the direct approach, while nickel dissolution was 65% in the indirect and only 8% in the direct process. This vast discrepancy suggests that gluconic acid plays a crucial role in chelating these metals, facilitating their release into the solution. Given that G. oxydans primarily produces organic acids, these findings highlight the potential importance of organic complexation mechanisms in bioleaching, which differ fundamentally from the proton-driven dissolution mechanisms observed in inorganic acid leaching.

Lithium, which is typically present in lithium cobalt oxides (LCO) or lithium nickel manganese cobalt oxides (NMC), also exhibited a strong dependency on the bioleaching method. The indirect approach dissolved 68% of the lithium, whereas the direct approach extracted only 35%. This suggests that lithium dissolution is not solely acid-dependent but may also require oxidative conditions or metal–organic complex formation to achieve high recovery rates. The incomplete dissolution of lithium in both methods implies that additional leaching mechanisms, such as oxidation-reduction reactions or chloride-based complexation, may be necessary to fully recover lithium from black mass.

While continuous pH monitoring was not performed, endpoint measurements showed a stronger acidification in the indirect G. oxydans approach (final pH ~3.5–4.5) compared to the direct approach (>4.5). For the A. thiooxidans sulfuric acid experiments, the leaching solutions were adjusted to pH ~1.35.

The differential leaching behavior of aluminum and copper further supports the idea that the mechanism of metal dissolution is metal-specific. In the indirect approach, copper was only leached at a rate of 21%, while aluminum reached 62%, whereas in the direct approach, the rates dropped to 5% for copper and 35% for aluminum. The low extraction of copper is expected, as copper does not readily form soluble complexes in weak organic acids and typically requires stronger oxidative conditions for dissolution. In contrast, the partial dissolution of aluminum may be attributed to gluconic acid’s ability to form stable organo-metallic complexes, although the presence of a protective oxide layer on aluminum particles likely prevented complete dissolution.

From an industrial perspective, these findings strongly favor the indirect bioleaching process as the more viable approach for lithium-ion battery (LIB) recycling. The ability to pre-produce organic acids separately from the black mass leaching process allows for the following:

• Optimized acid production without microbial inhibition;
• Controlled acid application, leading to higher metal selectivity;
• Reduced microbial stress, potentially enabling repeated bioleaching cycles.

However, despite its higher efficiency, the indirect bioleaching approach is still slower than conventional hydrometallurgical methods, requiring longer leaching durations to reach maximum metal recovery. Additionally, the production of gluconic acid at an industrial scale would need to be optimized in terms of fermentation conditions, bacterial strain engineering, and process integration with existing LIB recycling workflows.

The observed inhibition is likely both concentration-dependent and partly strain-specific, as microbial tolerance to heavy metals and organic residues varies between species and even among strains. In principle, such inhibition can be mitigated through gradual adaptation or preconditioning of cultures to sub-inhibitory concentrations of black mass components, which has been reported to enhance microbial tolerance in similar systems [37,38]. However, no pre-adaptation experiments were performed in this study.

In conclusion, the indirect bioleaching approach with Gluconobacter oxydans is significantly more effective than direct bioleaching, particularly for cobalt, nickel, and lithium recovery. The data suggest that microbial growth inhibition due to metal toxicity is the primary limitation of the direct method, whereas pre-produced gluconic acid can act as an effective leaching agent when applied separately. While conventional inorganic acid leaching remains the most efficient method for LIB recycling, bioleaching presents a promising, environmentally friendly alternative that could be further optimized for industrial applications.

4.5. Leaching Efficiency of Different Sulfuric Acid Types

To evaluate the effectiveness of sulfuric acid in metal extraction, the concentrations of dissolved metals in the filtrates were analyzed. The elements nickel (Ni), cobalt (Co), lithium (Li), manganese (Mn), and aluminum (Al) were measured. The resulting data are summarized in

Table 3. Comparison of the leaching efficiencies of the experiments with sulfuric acid.

Element	cmax [mg·L−1]	corg [mg·L−1]	corg,S [mg·L−1]	cinorg [mg·L−1]	ηorg [%]	ηorg,S [%]	ηinorg [%]
Copper (Cu)	480	0.07	1.33	0.02	0.01	0.28	0.00
Nickel (Ni)	2200	122	64	446	26	13	93
Cobalt (Co)	650	67	189	207	14	39	43
Lithium (Li)	520	150	112	364	31	23	76
Manganese (Mn)	730	177	99	292	37	21	61
Aluminum (Al)	370	0.5	0.3	0.2	0.11	0.06	0.05

c_max—maximum achievable concentration; c_org—achieved concentration of the organic sulfuric acid; c_org,S—achieved concentration of the sterilized organic sulfuric acid; c_inorg—achieved concentration of the inorganic sulfuric acid; η_org—achieved efficiency of the organic sulfuric acid; η_org,S—achieved efficiency of the sterilized organic sulfuric acid; η_inorg—achieved efficiency of the inorganic sulfuric acid.

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4.6. Comparison of Sulfuric Acid Variants in Bioleaching

The leaching efficiencies were calculated using the method outlined in Chapter 3 and are illustrated in Figure 4, which compares the three sulfuric acid treatments:

• Biologically produced sulfuric acid from A. thiooxidans (green columns);
• Sterilized biologically produced sulfuric acid (red columns);
• Chemically synthesized sulfuric acid (blue columns).

Figure 4. Extraction rates of the used sulfuric acids.

Figure 4. Extraction rates of the used sulfuric acids.

The key findings are as follows:

• Poor solubility of copper and aluminum

In all three approaches, copper was barely solubilized, confirming that it can be effectively separated from the other valuable metals. Aluminum remained mostly undissolved, likely due to the presence of an oxide passivation layer, which requires a stronger acid concentration to dissolve.

2.

Superior performance of chemically synthesized sulfuric acid

Nickel (93%) and lithium (76%) showed the highest dissolution rates in the chemically synthesized sulfuric acid. This efficiency is significantly higher than in both biologically produced acid variants, suggesting that the acid strength and purity of industrial sulfuric acid enhance metal solubilization.

3.

Variability in cobalt and manganese extraction

The leaching rates for cobalt (Co) were similar in the inorganic (43%) and sterilized organic sulfuric acid (39%). However, the non-sterilized, biologically produced sulfuric acid extracted only 14% of the available cobalt. For manganese (Mn), the dissolution efficiency was: 40% with biologically produced sulfuric acid, 60% with chemically synthesized sulfuric acid, and 20% with sterilized biologically produced sulfuric acid. This suggests that the sterilization process negatively affected the acid’s leaching efficiency, likely due to the degradation of bio-catalytic components. This effect cannot be observed for cobalt. To determine how the interaction between Co and the bacterial-produced acid differs from Ni, Mn and Li, further experiments need to be conducted. Comparable trends of Co and Ni solubilization with sulfur-oxidizing bacteria have also been reported in earlier studies [26].

4.7. Interpretation of the Overall Results and Industrial Implications

The results demonstrate clear differences in the efficiency of the three sulfuric acid variants tested for leaching metals from black mass. The chemically synthesized sulfuric acid exhibited the highest extraction efficiency, particularly for nickel (93%) and lithium (76%), significantly outperforming the biologically produced acid variants. This superior leaching performance is likely due to the higher acid availability and purity of industrial sulfuric acid, which ensures optimal metal solubilization. However, despite its effectiveness, the use of synthetic sulfuric acid comes with inherent drawbacks. The production of industrial-grade sulfuric acid is energy-intensive [39,40] and associated with high CO₂ emissions (0.2 to 0.6 t CO_2e per t of H₂SO₄), making it a less sustainable option [41]. Additionally, handling highly concentrated sulfuric acid poses safety and environmental risks, particularly in large-scale battery recycling plants [10,21]

In contrast, the biologically produced sulfuric acid generated by A. thiooxidans achieved moderate leaching efficiencies, with manganese (40%) and cobalt (14%) exhibiting the highest recovery rates among the analyzed elements. While these rates are lower than those observed with inorganic sulfuric acid, biogenic acid production represents a more environmentally friendly alternative. Unlike chemically synthesized sulfuric acid, bioleaching relies on microbial metabolism, which operates under mild conditions, eliminates the need for additional chemical inputs, and reduces energy consumption. However, the relatively low extraction rates, particularly for cobalt and nickel, indicate that microbially produced acid may require longer reaction times or process optimization to reach competitive efficiency levels. Further investigations into microbial growth conditions, acid production kinetics, and pH regulation could improve the applicability of this approach in industrial recycling processes.

An interesting observation is the difference between sterilized and non-sterilized biologically produced sulfuric acid. The sterilized variant exhibited higher leaching efficiencies for cobalt (39%) and nickel (13%) compared to the non-sterilized variant, which only dissolved 14% of cobalt and 26% of nickel. This suggests that biologically active components in the non-sterilized solution may have an inhibitory effect on leaching, due to metal toxicity affecting residual microbial enzymatic activity or the presence of organic metabolites that interact with metal ions [37,38]. Conversely, for manganese, the sterilized solution showed a lower extraction rate (21%) compared to the non-sterilized variant (37%), indicating that certain biological factors may enhance manganese solubilization. These findings suggest that the presence of live bacterial cultures or microbial metabolites could modulate metal extraction efficiency in complex ways, warranting further research into their role in the leaching process.

The limited solubilization of copper and aluminum across all three approaches is also noteworthy. Copper extraction remained below 1.5%, while aluminum dissolution was minimal in all cases. The low copper leaching efficiency can be attributed to its noble character and limited reactivity in sulfuric acid solutions, which suggests that additional oxidative treatments (e.g., ferric ion oxidation or chloride-based leaching) may be required to enhance copper recovery. Similarly, the lack of aluminum dissolution is likely due to the formation of a stable oxide passivation layer, such as aluminum oxide (Al₂O₃), which requires a stronger acid or prolonged leaching duration for significant solubilization [18,42,43]

Compared to conventional hydrometallurgical processes, which typically achieve near-complete metal recovery (>95%) within a few hours under high-temperature and strongly acidic conditions [10], bioleaching operates at significantly milder parameters (ambient temperature, neutral to mildly acidic pH, low chemical input) and with substantially lower environmental impact. Although the slower kinetics and lower recovery for certain metals, particularly nickel and lithium, currently limit its competitiveness as a stand-alone process, bioleaching offers distinct advantages in terms of sustainability, safety, and reagent consumption. Therefore, its greatest potential lies in hybrid process concepts, where microbial acid generation complements chemical leaching or serves as a selective pre-treatment step.

From an industrial perspective, these results indicate that while bioleaching presents a viable and sustainable alternative to conventional hydrometallurgical processes, it currently lags behind chemical leaching in terms of metal recovery efficiency. The integration of biologically produced sulfuric acid into LIB recycling workflows could reduce environmental impact and chemical consumption, but process modifications—such as longer leaching durations, microbial adaptation to high-metal environments, or pH adjustments—may be necessary to achieve competitive extraction rates. Additionally, a hybrid approach combining biogenic acid production with selective chemical leaching could be explored to optimize metal recovery while maintaining environmental sustainability.

In practice, a hybrid process could involve the use of microorganisms primarily for acid or metabolite generation under mild conditions, followed by a conventional chemical leaching step to accelerate metal dissolution. For example, gluconic acid produced by G. oxydans could serve as a pre-leachant to selectively mobilize Co, Ni, and Li, after which a controlled inorganic acid treatment could ensure complete recovery. Alternatively, bio-generated sulfuric acid could partially replace chemical input in hydrometallurgical circuits, thereby reducing reagent consumption and environmental footprint. Such concepts combine the ecological advantages of bioleaching with the speed and completeness of chemical leaching, offering a more feasible route for industrial adoption.

In conclusion, bioleaching with A. thiooxidans provides an environmentally friendly, low-energy alternative to conventional sulfuric acid leaching, but its efficiency remains limited by factors such as metal toxicity, acid concentration, and microbial metabolic activity. While industrial sulfuric acid is currently the most effective option for LIB metal recovery, the development of optimized bioleaching strategies could significantly improve the feasibility of microbial approaches in large-scale recycling applications.

Bioleaching also contributes to the concept of sustainable mining by recovering critical metals from secondary resources under mild and environmentally friendly conditions. This reduces the need for primary ore extraction, lowers chemical consumption, and decreases the overall environmental footprint of the metal supply chain [44].

The pregnant leach solution (PLS) obtained from bioleaching can be further processed by conventional hydrometallurgical methods, such as selective precipitation, solvent extraction, or electrowinning, to recover individual metals in pure form [10,21].

5. Conclusions

The black mass (BM) investigated in this study originated from an industrial NMC-type LIB recycling process and consisted predominantly of nickel (22.0 wt%), manganese (7.3 wt%), cobalt (6.5 wt%), lithium (5.2 wt%), copper (4.8 wt%), and aluminum (3.7 wt%). Minor components included zinc (0.7 wt%) and various impurities. XRD analysis identified layered Li(Ni,Mn,Co)O₂, graphite, and traces of copper as the main crystalline phases, while SEM/EDX confirmed the heterogeneous particle morphology with sizes <250 µm. These compositional and structural characteristics formed the basis for evaluating bioleaching performance.

Indirect bioleaching with Gluconobacter oxydans at 26 °C achieved substantially higher metal extraction efficiencies compared to the direct approach. Cobalt and manganese were completely or nearly completely solubilized (96% and 100%, respectively); nickel reached 65%, lithium 68%, and aluminum 62%, while copper remained low at 21%. In contrast, direct bioleaching under otherwise identical conditions yielded ≤55% for all elements, with cobalt at 17%, nickel at 8%, lithium at 35%, and manganese at 53%. The superior performance of the indirect method is attributed to unhindered gluconic acid production in the absence of BM, mitigating microbial inhibition by metal toxicity and organic residues.

Bioleaching with Acidithiobacillus thiooxidans produced sulfuric acid that, when applied to BM at pH 1.35, dissolved manganese (40%) and cobalt (14%) most effectively, while nickel and lithium recoveries remained at 26% and 31%, respectively. Sterilization of the biologically produced acid increased cobalt extraction to 39% but reduced manganese to 21%. Chemically synthesized sulfuric acid under the same acidity conditions markedly outperformed both biological variants, extracting nickel at 93%, lithium at 76%, and manganese at 61%, indicating the influence of acid purity and strength on leaching efficiency.

Overall, indirect G. oxydans bioleaching demonstrated the highest efficiency among the microbial methods tested and appears promising for environmentally friendly LIB recycling, particularly for cobalt, manganese, and nickel recovery. Nevertheless, reaction times were longer than conventional hydrometallurgical processes, and copper extraction remained low for all approaches. Future research should focus on process optimization, microbial adaptation to high-metal environments, and integration with selective downstream hydrometallurgical steps such as precipitation, solvent extraction, or electrowinning to enhance overall recovery yields while maintaining the environmental benefits of bioleaching.