Bioremediation of Organic Compounds and Trace Metals from Landfill Leachate Using Lactobacillus kefiri Strains: A Sustainable Approach Based on Facultative Treatment

Yasmin Cherni ^1,*, Ridha Elleuch ², Cristian Botta ³, Luca Cocolin ³, Amjed Kallal ⁴, Ismail Trabelsi ¹, Lobna Elleuch ⁵

1. Laboratory of Treatment and Valorization of Water Rejects Water Researches and Technologies Center, Borj-Cedria Technopark, University of Carthage, 8020 Soliman, Tunisia

2. Laboratory of Ceramic Composite Materials and Polymers, Sciences Faculty of Sfax, University of Sfax, Tunisia

3. Department of Agriculture, Forest and Food Sciences, University of Torino, Torino, Italy

4. Laboratory of Water, Energy and Environment (3E), Sfax National School of Engineering, University of Sfax, Sokra Road, Sfax, 3038, Tunisia

5. Wastewater and Environment Laboratory, Water Research and Technologies Center CERTE, P.O. Box 273,8020 Soliman, Tunisia

* Correspondence: Yasmin Cherni

Academic Editor: Maria del Carmen  Márquez

Special Issue: Advances in Treatment of Leachate from Solid Wastes

Received: November 09, 2023 | Accepted: September 09, 2025 | Published: September 16, 2025

Recent Progress in Materials 2025, Volume 7, Issue 3, doi:10.21926/rpm.2503014

Recommended citation: Cherni Y, Elleuch R, Botta C, Cocolin L, Kallal A, Trabelsi I, Elleuch L. Bioremediation of Organic Compounds and Trace Metals from Landfill Leachate Using Lactobacillus kefiri Strains: A Sustainable Approach Based on Facultative Treatment. Recent Progress in Materials 2025; 7(3): 014; doi:10.21926/rpm.2503014.

© 2025 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited.

1. Introduction

Landfill leachate (LFL) production poses significant environmental challenges due to its complex and highly variable composition [1,2]. These effluents often contain a mixture of mineral, organic, and toxic substances, which can lead to the contamination of surface waters and soils if not properly managed [3]. Consequently, thorough assessment and treatment of LFL are essential before environmental discharge. The appropriate treatment strategy largely depends on the specific characteristics and composition of the leachate [4,5]. Numerous studies have confirmed the hazardous potential of landfill leachate, emphasizing the environmental risks associated with uncontrolled discharge [3]. Typically, LFL contains high concentrations of persistent compounds, including ammonia, which impair the performance of biological treatment processes, accelerate eutrophication, and increase toxicity in aquatic ecosystems [6]. To address these challenges, a variety of treatment technologies, both physicochemical and biological, have been explored [7,8,9].

Among them, combined approaches involving both biological and physicochemical methods have proven particularly effective for removing toxic pollutants from LFL [10]. One widely employed physicochemical method is coagulation-flocculation, commonly used in rural and industrial wastewater treatment [11]. This process neutralizes or reduces the electrical charge of colloidal particles, promoting their aggregation into flocs [12]. Typical coagulants include metal salts such as aluminium sulfate, ferrous sulfate, and ferric chloride [11]. Coagulation-flocculation is often applied as a pre-treatment step in LFL treatment to remove heavy metals and refractory organic compounds [5,13]. It effectively decreases COD by targeting high-molecular-weight organics. The process produces a significant amount of sludge, which is made up of metal hydroxides and adsorbed contaminants, posing handling and disposal issues. While coagulation-flocculation is successful at improving water quality, it is often employed in conjunction with other treatment methods, such as biological or advanced oxidation processes, to complete leachate treatment.

It is a relatively cost-effective and straightforward process, but optimization is essential. Factors such as coagulant type, pH, and dosage must be carefully evaluated to maximize treatment efficiency [14]. In 2021, Djeffal and co-workers [14] tested the coagulation-flocculation process for treating landfill leachate from the Souk-Ahras Technical Center. Three coagulants, ferric chloride, aluminum sulfate, and ordinary alum, were studied with both mechanical and ultrasound agitation. Outcomes showed that pH adjustment was essential for effective treatment. Optimal conditions included a coagulant dose of 15%, stirring at 250 rpm for 5 minutes (15 minutes for ferric chloride), and a coagulant-to-leachate volume ratio of 1:1. Turbidity reductions reached 99.4% with ferric chloride, 98.9% with aluminum sulfate, and 98.6% with ordinary alum. Ferric chloride and aluminum sulfate effectively eliminated bacteriological contaminants, unlike ordinary alum. Ultrasound agitation (37 kHz, 30 W) improved turbidity reduction further, achieving 0.19 NTU with ferric chloride. Optimal clarification occurred at 20°C, yielding a BOD₅ of 100 mg O₂/L for ferric chloride and aluminum sulfate, and 200 mg O₂/L for ordinary alum. Biological treatment alternatives are gaining increased attention due to their reduced chemical requirements, cost-effectiveness, and environmentally friendly nature. These methods rely on complex biochemical interactions between microorganisms and organic pollutants. Bacteria play a vital role in facilitating biodegradation, bioaugmentation, and biosorption of contaminants, converting them into less toxic or inert compounds [15,16]. In 2020, Cherni et al. [16] evaluated the potential of microbial strains isolated from kefir grains for LFL treatment. Molecular techniques (Rep-PCR and 16S rRNA sequencing) revealed that kefir grains contained high populations (>10⁷ CFU/mL) of lactic acid bacteria and yeasts, predominantly Lactococcus lactis, Lactobacillus kefiri, Bacillus spp., and Kluyveromyces marxianus. A co-culture of L. lactis and K. marxianus at 1% inoculum demonstrated the highest efficacy in pollutant degradation. The treatment achieved removal efficiencies of 75.8% for chemical oxygen demand (COD), 85.9% for ammonium nitrogen, and 75.13% for salinity. Additionally, heavy metal removal reached 75% for Ni and Cd, and 73.45%, 68.53%, and 58.17% for Cu, Pb, and Fe, respectively. These findings support the use of kefir-derived microorganisms as a promising bioremediation strategy for landfill leachate. Despite its presence in traditional fermented foods, Lactobacillus kefiri, commonly found in kefir grains (KGs), has not been widely explored for industrial wastewater treatment applications. The present investigation aims to evaluate the effectiveness of L. kefiri inoculation in the biological treatment of landfill leachate. The primary objective is to develop an integrated treatment strategy by coupling coagulation-flocculation with biological treatment for LFL characterized by low biodegradability and elevated inorganic content, such as high NH₃-N concentrations.

This research examines the effects of key operational parameters, specifically pH and coagulant dosage, on the efficiency of landfill leachate treatment. By optimizing these parameters, the study aimed to maximize the removal of chemical oxygen demand (COD), ammoniacal nitrogen (NH₃-N), turbidity, and trace metals. Experimental trials were conducted to identify the conditions under which coagulation-flocculation processes exhibit peak performance.

In the subsequent biological treatment phase, Lactobacillus kefiri, isolated from kefir grains, was evaluated for its capacity to enhance the biodegradation of organic matter and facilitate biosorption of trace metals. The integration of L. kefiri into the treatment process demonstrated notable improvements in pollutant reduction, indicating its potential as a bioaugmentation agent. The findings contribute to the development of a more sustainable, biologically driven treatment strategy for high-strength wastewater such as landfill leachate.

2. Materials and Methods

2.1 Study Area and Leachate Sampling

Leachate samples were collected from the Jebel Chakir landfill, situated in the southwest of Tunis City (36°44′21″ N, 10°04′10″ E). This site receives approximately 2,000 tons of municipal solid waste (MSW) daily, of which about 68% is organic matter. The landfill generates an estimated 270 m³ of leachate per day. 20-liter samples were taken from the leachate collection system manually. The samples were stored in polyethylene containers at 4°C in the dark to minimize microbial activity. The properties of the leachate analyzed for use in the experiment are presented in Table 1.

Table 1 Physico-chemical characterization of raw Jebel Chakir LFL.

2.2 Experimental Procedure

Coagulation-flocculation experiments were performed using a standard jar test apparatus to assess the removal efficiency of organic matter, ammonia nitrogen, and turbidity from the leachate. All experiments were conducted in triplicate to ensure reproducibility. A total volume of 100 mL of raw leachate was introduced into each beaker of the jar test unit. Aluminum sulfate (Al₂(SO₄)₃·18H₂O) was used as the coagulant with a concentration of 1000 mg/L. The coagulant solution was prepared fresh before each test. The jar test was carried out in two sequential phases: rapid mixing and slow mixing, followed by a sedimentation (decantation) period. The operational parameters were as follows:

• Rapid mixing at 150 rpm for 5 minutes to ensure uniform dispersion of the coagulant throughout the leachate.
• Slow mixing at 70 rpm for 30 minutes to promote particle aggregation and floc formation.
• Settling for 60 minutes to allow for the separation of flocs by gravity.

After the sedimentation period, supernatant samples were carefully withdrawn from a fixed depth using sampling ports to avoid resuspension of settled solids. The collected samples were examined for various water quality parameters. Experiments and analytical measurements were conducted in triplicate (Table 2).

Table 2 Operational conditions for coagulation process.

2.3 Isolation and Identification of LAB Strains Isolates from Kefir Grains

In our previous study [15,16], a LAB strain was isolated from kefir grains and identified using biochemical and molecular methods. For inoculum preparation, L. kefiri was reactivated on Man, Rogosa, and Sharpe (MRS) Agar plates and incubated at 30°C for 24 hours. The bacterial cell density was estimated at 600 nm. The culture was subcultured on MRS broth medium and incubated for 24 hours at 37°C with agitation at 150 rpm. Then, the activated LAB was fermented in aerobic environment for 6, 12, and 24 hours of incubation.

2.4 L. kefiri Batch Fermentation on Landfill Leachate

The process was carried out in batch, with an Erlenmeyer flask (50 ml) containing 20 ml of pretreated samples (mainly the resulting supernatants). The series of runs was duplicated. The selected LAB strains (L. kefiri) were added separately to the test samples at different inoculum sizes (1%, 3%, and 5% [v/v]) and incubated at room temperature in an orbital shaker with a rotation speed of 150 rpm. The fermentation process runs for 24 hours. A blank experiment with non-inoculated, pre-treated LFL was conducted under the same conditions as the test samples to assess the impact of endogenous flora.

2.5 Elemental Analysis

The increase in the removal rates of Chemical Oxygen Demand (COD), turbidity, and trace metal was measured to evaluate the effectiveness of the combined treatment of landfill leachate. pH, Total Dissolved Solids (TDS), and Electrical Conductivity (EC) were determined using a multiparameter (Consort C 860). COD was measured according to the standard methods described by Rodier and Legube [17]. Similarly, total nitrogen (TN) and ammonium nitrogen (NH₄⁺-N) were also determined following the procedures outlined by Rodier and Legube [17]. Turbidity was measured using a turbidimeter (WTW Turb 555). Trace metal concentrations were analyzed using the flame atomic absorption spectroscopy method (Analytic Jena AG, Spectrometer AAS Vario 6). Cation and anion concentrations were determined using an ion chromatography system equipped with an anion exchange column (AS4A-SC, 150 mm × 4 mm), coupled with a conductivity detector and controlled by IC NET software (Metrohm, France). Bacterial cell biomass was assessed by measuring the optical density of samples at 600 nm. The removal efficiency of pollutants after treatment was determined using the following Equation:

\[ \mathit{\pmb{Removal\,efficency}}\,(\%)=\mathbf{\frac{{\mit{C}}_0-{\mit{C}}}{{\mit{C}}_0}}*\mathbf{100}, \]

which C₀ and C represent the measured parameters, respectively, in initial and final sample.

3. Result and Discussion

In this section, the characterization of raw leachate, the investigation of operational conditions for coagulation, and then a biological process are presented.

3.1 Physico-Chemical Characterization of LFL Wastewater

Table 1 represents some of the characteristics of leacahte. The raw LFL was brownish in color, with a pH of 7.73 on average. The presence of a significant number of COD (26.200 mg O₂/L) in leachate indicates that the organic matter content is high. Furthermore, significant levels of salinity (3.62 g/L) and trace metals such as Cd (2.73 mg/L), Pb (1.78 mg/L), Fe (9.23 mg/L), Ni (3.52 mg/L), and Cu (1.62 mg/L) were found in leachate samples. Furthermore, the turbidity and levels of salinity are high, owing to the presence of mineral ions and colloidal particles. The values exceed the maximum allowed by current legislative guidelines for the discharge of treated wastewaters (NT 106.02), making direct biological treatment an inadequate alternative. As a result, a prior physicochemical process, such as coagulation/flocculation, would be highly beneficial in terms of reducing pollutants and improving the biological treatability of this effluent.

3.2 Assessment of Operational Parameters for the Coagulation Process

Landfill leachate contains various suspended particles like biopolymers and colloidal particles, which are responsible for its turbidity. In light of the information gathered from the literature; it has been confirmed that pH plays vital role in the performance of the leachate coagulation process. It could affect coagulant efficiency and the hydrolysis behavior of leachate [18,19]. According to literature, several researchers (e.g. [19,20]) have reported that the treatment is better at 1 and 1.5 g/l as concentrations of aluminum sulfate.

3.2.1 Optimum pH

Figure 1 illustrates the removal efficiency of COD, NH₃-N, and turbidity using Alum as the coagulant at various pH levels. Figure 1(a) shows that pH 4 has a slightly higher tendency to remove COD, NH₃-N, and turbidity. The pH of the LFL has effectively determined the coagulation effect. The best COD removal performance (50%) was achieved at pH 4. The highest removal efficiency of NH₃-N and turbidity was found to be 25% and 70%, respectively. According to the literature, this pH corresponds to a region where positively charged species, particularly Al(OH)₂ and Al₂(OH)₂, and insoluble Al(OH)₃ species were dominant. It also increases the positively charged environment in this acidic condition by increasing the density of positive charges (H⁺) around the coagulant’s hydrolysates. The coagulation process is dominated by the neutralisation of charge and complex reaction between alumine sulphate and dissociated organic compounds of leachate, which could confirm the production and precipitation of insoluble Al-Organic compound complexes [21]. Thus, the hydrolysis of aluminum sulfate in water can be represented as:

\[ \mathrm{Al}_2(\mathrm{SO}_4)_3\,\cdot\,18\mathrm{H}_2\mathrm{O}\,\to\,2\mathrm{Al}^{3+}\,+\,3\mathrm{SO}_4^{2-}\,+\,18\mathrm{H}_2\mathrm{O} \]

Followed by the hydrolysis of aluminum ions, forming aluminum hydroxide:

\[ \mathrm{Al}^{3+}\,+\,3\mathrm{H}_2\mathrm{O}\,\to\,\mathrm{Al}(\mathrm{OH})_3\downarrow\,+\,3\mathrm{H}^+ \]

Figure 1 Effect of pH (a) and coagulant dose (b) on COD, NH₃-N, and turbidity removal rate from landfill leachate during the coagulation process.

At pH 7 and 10, however, there was no significant increase in COD, NH₃-N, or turbidity removal rate. The finding demonstrated that the chemical coagulation process was pH dependent. At neutral pH, H⁺ competes for organic ligands with metal hydrolysis products, resulting in poor removal efficiency and some of the produced organic acids dropping to precipitate [18]. The COD removal efficiency has decreased to 40% at pH 10, which could be explained by the presence of negatively charged species, such as Al(OH)₄, which reduces their attraction to anionic organic compounds. Furthermore, at basic pH, hydroxide ions compete with organic compounds for metal adsorption sites, resulting in co-precipitation [22]. The work of other researchers supports all our findings. Djeffal et al. [14] recently demonstrated that at acidic pH, the maximum turbidity removal was 98.9%. The pH was found to be the most critical parameter in the coagulation process by Zainol et al. [23]. COD removal was 50.5% at pH 4 using Al₂(SO₄)₃ at a concentration of 5000 mg/L. Turbidity and suspended solids, on the other hand, achieved maximum removal at a pH of 4, with values of 85% and 75%, respectively. Several studies have shown that under acidic conditions, all organic compounds are completely oxidized to carbon dioxide [24]. Even though anaerobic organisms are not oxidising agents, organic content degradation can occur via dissolved microorganisms in the leachate sample. This finding is supported by TUNÇ [25], who stated that the optimum pH for using aluminium sulphate in coagulation is between 3 and 6.

3.2.2 Optimum Coagulant Dosage

One of the most critical parameters in removing pollutants from LFL is coagulant dosage [14]. According to Mohd-Salleh and colleagues [11], the optimum coagulant concentration is defined as the maximum value at which there is no significant increase in removal efficiency by further coagulant addition. The ideal pH for aluminium sulphate was 4. The coagulant dose ranges between 500 and 2000 mg/L.

This range was chosen to determine the best dose of aluminium sulphate. The results obtained are shown in Figure 1(b). The findings clearly show that the COD elimination patterns increase progressively as the dose of aluminium sulphate is increased. Nonetheless, trends have exceeded an optimal dose. According to the results, the optimal coagulant dose is 1000 mg/L, with removal rates of 50% for COD, 26% for NH₃-N, and 75% for turbidity, respectively. This could be due to the presence of too much cationic coagulant, which causes re-dispersion in the leachate sample [25,26]. It is worth noting that there is significant pollutant removal that has favorable trends at very low coagulant doses. The pattern for different parameters was increased from the starting point until the optimum coagulant concentrations were achieved, and then it was reduced as the coagulant concentrations increased. This phenomenon could be explained by the presence of a proportion between the negative charges of the leachate and the positive charges of the coagulant, resulting in a strong attraction of charges that improves floc production. Furthermore, the decrease in removal rates has been attributed to colloidal destabilization caused by an overdose of aluminum sulfate [26]. Similar studies that tested the treatment of leachate by the coagulation-flocculation process using polyaluminum chloride (PAC) and tapioca starch (TS) can confirm all of our findings [27]. They demonstrated that a high coagulant dose has the opposite effect of neutralizing all positively charged particles, causing repulsive forces to be exerted between them.

Furthermore, the effectiveness of Al₂(SO₄)₃ in removing mineral salts is well established. Additionally, the effectiveness of coagulation in removing mineral salts from raw LFL has been evaluated. The results revealed that the maximum removal efficiency in pH 4 is 44.53%, 45.03%, 26.01%, and 25.64% for nitrate, phosphate, calcium, and magnesium removal, respectively. In summary, the coagulant dosage clearly plays a vital role in the decolorization of effluent and the removal of several pollutants. Under these conditions, the determination of sludge volume is dependent on the operating conditions and the coagulant used during the coagulation process [28]. The sludge volume (SV) was determined in this study after 1 hour of settling under optimal conditions (pH = 4 and coagulant dose of 1000 mg/L). According to the results, aluminium sulphate produces 42.5 ml of SV. During the coagulation process, mass transfer occurs primarily through diffusion and adsorption, where dissolved pollutants such as metal ions and nutrients migrate from the bulk liquid to the surface of aluminum hydroxide flocs formed by alum hydrolysis. These flocs facilitate the removal of contaminants via charge neutralization and sweep flocculation, resulting in a dense physico-chemical sludge containing adsorbed metals, nutrients, and organic matter. Given its complex and potentially hazardous composition, this sludge requires appropriate treatment before disposal. Standard treatment methods include stabilization and solidification to immobilize contaminants, thermal treatment to reduce volume and destroy organics, chemical extraction for selective metal recovery, and, less frequently, biological treatment for organic degradation. These approaches help mitigate environmental risks and enable safer handling or reuse of the sludge by-products. The coagulant dosage of 1000 mg/L was determined to be the most appropriate for LFL treatment. It is also essential to highlight the effectiveness of alum in removing metal ions [29]. Figure 2 demonstrates an interesting removal of Cu (23%), Ni (45%), Cr (41%), Pb (33%), and As (20%). Recently, Jaradat and colleagues [30] demonstrated that the removal mechanism of metal ions by organic compounds through the coagulation process is complex, primarily dependent on the hydrolyzed species of each coagulant in wastewater. In this context, colloids in water absorb metal ions on the surface through two primary mechanisms: ion exchange and charge attraction. As a result, the charge neutralisation of hydroxide complexes plays a critical role in colloidal aggregation. Furthermore, the resulting flocs adsorb a few metal ions during the precipitation process, and co-precipitation contributes to their removal in this process [31]. As a result, the coagulation process using aluminium sulphate was demonstrated to be technically feasible and effective for removing organic matter and trace metals from LFL. The obtained results are consistent with the findings of other studies that discussed the performance of the coagulation process for the removal of various pollutants from LFL [18,32].

Figure 2 Effect of optimum condition of Alum coagulation on trace metals removal from landfill leachate.

3.2.3 Optimum Variable Condition

In assessing the optimum conditions for alum coagulation, the evaluation of the preliminary run was a prerequisite. The selection of the variables was based on the highest removal of pollutants. Table 3 presents the optimum conditions of the Alum coagulation. It can be observed that the optimum condition of the Alum is achieved at pH 4 and a coagulant concentration of 1000 mg/L to reduce various contaminants. The final concentrations of the pollutants are: COD: 13.100 mg O₂/L; Turbidity: 82.7 NTU; NO₃^-: 4.102 mg/L; PO₄^3-: 15.560 mg/L; Ca²⁺: 9.10 mg/L; Mg²⁺: 11.606 mg/L; NH₃-N: 575,64 mg/L; Cu²⁺: 1.247 mg/L; Ni²⁺: 1.936 mg/L; Cr²⁺: 2.655 mg/L; Pb²⁺: 1.192 mg/L; As: 0.96 mg/L. The final concentration of the treated leachate sample still exceeded the existing legislative guidelines for the discharge of treated wastewater (NT 106.02). Additionally, the LFL was treated with a biological process using the L. kefiri strain.

Table 3 Optimum variable of alum coagulation and percent removal.

3.3 Bioremediation Using Lactobacillus kefiri

In this study, the bioremediation of landfill leachate (LFL) was carried out using the bacterium L. kefiri, under aerobic conditions, to reduce key pollutants including COD, NH₃-N, turbidity, and trace metals. The biological treatment process focused on optimizing the inoculum size of L. kefiri to enhance pollutant removal efficiency within 24 hours. The use of an exogenous microbial inoculum was compared with a control that had no bacterial addition to highlight the role of L. kefiri in leachate degradation. According to the literature, it is important to note that bioremediation is a cost-effective and environmentally friendly technology, increasingly applied to reduce pollutant concentrations in wastewater and soils [12,14,33,34]. However, to better understand the bioremediation potential and ecological compatibility of L. kefiri, future experiments should include evaluations using the autochthonous microbial community through in situ bioremediation approaches. This would allow a direct comparison between indigenous microorganisms and the introduced inoculum, providing deeper insight into competitive interactions and overall treatment efficiency. The primary objective of this study was to determine the optimal conditions for the application of Lactobacillus kefiri in the treatment of landfill leachate (LFL). L. kefiri was evaluated for its effectiveness in reducing chemical oxygen demand (COD), ammonia nitrogen (NH₃-N), turbidity, and trace metals in leachate. As shown in Figure 3, the removal of COD and NH₃-N was largely proportional to the inoculum size of L. kefiri, up to an optimal concentration. The findings demonstrated that L. kefiri significantly enhanced the removal efficiency of key leachate parameters. After 24 hours of treatment with a 5% (v/v) inoculum, COD and turbidity removal efficiencies reached 63% and 90%, respectively. Lower inoculum sizes of 1% and 3% also showed significant COD reductions of approximately 42% and 53%, respectively, after 24 hours. A treatment based on L. kefiri has several potential benefits over traditional anaerobic digestion (AD). Although AD is frequently used to treat leachate and is well-known for its capacity to turn organic matter into biogas in completely anaerobic settings, it usually necessitates longer hydraulic retention periods. It is susceptible to changes in temperature, pH, and the concentrations of harmful compounds. Furthermore, AD systems can perform effectively at low pH levels and may need several weeks to start up. On the other hand, the lactic acid bacterium L. kefiri performs well in slightly acidic environments and acts rapidly, removing a significant quantity of pollution in a day. In addition to lowering COD, L. kefiri's synthesis of organic acids, such as lactic acid, encourages the development of viable microbial populations while suppressing infections. By inhibiting the growth of unwanted bacteria, these acids and antimicrobial substances improve the biodegradation process overall [35]. Furthermore, compared to AD's demanding needs, L. kefiri can survive in a wider variety of conditions. Its use could eliminate the need for extended retention periods or substantial infrastructure, making it a viable additional or substitute approach to AD, particularly when immediate assistance is required or when leachate properties restrict AD performance. As a result, the use of L. kefiri offers an immediate and more reliable approach under particular operating conditions, even though anaerobic digestion remains the fundamental component of biological leachate treatment. To assess the scalability, cost-effectiveness, and long-term stability of L. kefiri-based systems in comparison to conventional AD methods, more investigation and pilot-scale testing are advised. On the other hand, the removal efficiency of NH₃-N is being investigated (data not shown). After 24 hours, the treatment process consistently achieves a high efficiency of NH₃-N of 67% at 5% (v/v) inoculum size. Despite this, after 24 hours of treatment, L. kefiri at a 3% inoculation size removed 48% of the NH₃-N. After 24 hours of contact time, adding 1% L. kefiri to the leachate resulted in an NH₃-N removal rate of approximately 46.6%. According to the literature. When the NH₃-N concentration is high, high pH values increase the free ammonia concentration and inhibit nitrifying activity [36]. To demonstrate the efficacy of the used bacterial strain, pre-treated LFL without bacterial inoculation was used as a control run. The control demonstrated a COD removal rate of only 18%, which is considered lower than that of the inoculum. Thus, the results confirmed that the inoculum can be proven to be the main key in enhancing LFL biodegradation [14,15]. Based on information gathered from the literature, LAB degrades complex compounds into simple products and produces additional biomass. It improves carbohydrate metabolism by producing lactic acid and other antimicrobial compounds. Those antimicrobial products with antibacterial properties inhibited the growth of pathogens and other unsuitable microorganisms during the biodegradation of organic particles in leachate samples [34]. The results of the experiment demonstrated the ability of our strain to survive under high specific environmental pressures. Those findings appear to be more intriguing than those reported by Reis et al. [37], who demonstrated the ability of Saccharomyces cerevisiae in a membrane bioreactor to degrade organic compounds during leachate treatment. Thus, COD, color, and NH₄⁺-N degradation were attained 69, 54, and 34%, respectively, after 2 days of incubation. To the best of our knowledge, this is the first study to investigate the performance of L. kefiri in the LFL treatment over a long period of time (only 24 hours). The success of bioremediation is dependent on the ability of the introduced strain to perform its activities and survive in stressful environmental conditions [38]. Furthermore, numerous studies have shown that inoculation size is a critical factor in the biological treatment of LFL with high levels of contaminants [5].

Figure 3 Effect of inoculum size on COD (A) degradation and turbidity (B) removal during the combined process.

According to the literature, trace metals and organic matter are a global environmental issue in terms of polluting water due to their toxic effects, high mobility, accumulation, and persistence throughout ecosystems. Several studies have recently been conducted to develop less expensive and more effective wastewater treatment alternatives. In this context, biosorption has been demonstrated to be an effective technique for removing pollutants from wastewater [39]. Various biosorption studies for metal removal have developed the use of microorganisms or biomass generated by food processing industries or wastewater treatment units [39]. Several organisms, including bacteria, yeast, and algae, can absorb dissolved metals from their surroundings and have been successfully used to remove trace metal ions [38].

Table 4 indicates the results of an investigation into the removal of various trace metals using the L. kefiri strain. After 24 hours, the removal efficiencies for Cu, Ni, Cr, As, and Pb were 32, 53, 47, 35, and 40%, respectively, with 5% (v/v) as the inoculum size. However, there are no reports in the environmental field on the biological treatment of landfill leachate using L. kefiri. To the best of our knowledge, this is the first study to look into the ability of L. kefiri to remove various pollutants from LFL. Several studies have demonstrated that the process is more complicated with living biomass because its metabolic activity is incorporated into the passive mechanisms. Pollutants can be actively transported through the membrane into the cell interior as a result of this metabolic activity. Pollutants can build up inside the cell in this manner (bioaccumulation). When using living biomass as a biosorbent, it has the potential to remove a greater number of pollutants, which is a significant advantage [40,41].

Table 4 Trace metals removal rate using coagulation pre-treatment and different inoculum sizes of L. kefiri after 24 hours of treatment.

3.4 Combined Pathway for LFL Treatment

The main objective of this work is to investigate a powerful and cost-effective treatment alternative for highly polluted effluent, such as landfill leachate. Overall, the removal efficiencies of COD, NH₃-N, and turbidity were 63, 67, and 90%, respectively. Similarly, there is a significant reduction in trace metals in LFL, with about 50% removal of Ni and Cr. Furthermore, removal rates for Cu, As, and Pb were 32, 35, and 40%, respectively. Currently, as reported by several scientists, combined treatments have proven to be more effective and have emerged as the treatment of choice for landfill leachate. In 2021, El Mrabet et al. [10] investigated landfill leachate treatment using a combined Fenton and adsorption onto natural local bentonite clay. The Fenton process reduced 92% of the color and 73% of the COD from the leachate under optimal conditions (2,000 mg L^-1 of Fe²⁺ and 2,500 mg L^-1 of H₂O₂ at pH = 3). Following that, the Fenton process was combined with adsorption (3 g L^-1 bentonite concentration, pH 5, 5 h reaction time, and T = 35°C) to achieve total COD and color removal of 84% and 98%, respectively. As a result, the combined process would be a viable option for leachate treatment. Talalaj and colleagues [42] investigated the efficacy of landfill leachate treatment using a combined sequencing batch reactor (SBR) and reverse osmosis (RO). As a result, they demonstrated that the combined process had a high removal rate (up to 80%) for all the analysed parameters (COD, BOD₅, ammonia nitrogen, etc.). In 2018, Tezcan [43] investigated the simultaneous treatment of LL with an EC procedure as a pre-treatment, followed by anaerobic treatment. The EC procedure was adjusted to have a maximum COD elimination efficiency of 69%. The effluent was subsequently exposed to anaerobic treatment, which was further optimized using a factorial design technique. The most excellent COD elimination rate with anaerobic treatment was 74%. The combined EC and anaerobic treatment achieved an overall COD elimination effectiveness of 92%. The study stresses the efficacy of the combined treatment strategy over the process alone for leachate wastewater. In the same vein, Colombo and colleagues [44] researched the use of physical-chemical and biological processes to reduce contaminants in Leachate. They used a regular biological therapy first, then a photo-Fenton (PF) procedure, and finally a combination of the two. The PF process was enhanced with a central composite rotatable design (CCRD). After a decantation process, the biological treatment lasted 40 hours. When the upgraded PF process was combined with the biological process, COD and BOD₅ removal efficacy was 98%, which met the requirements for releasing treated wastewater into bodies of water. This study is unique in that it investigates the combination of coagulation and bioremediation processes using L. kefiri isolated from KGs in removing trace metals and COD from the LFL. Thus, the treated effluent would be reused as a source of water for plant growth in agricultural fields, which is the subject of ongoing and future research.

4. Conclusion

This study established the viability of biologically treating landfill leachate with Lactobacillus kefiri under ideal conditions. The combination strategy of coagulation pre-treatment followed by L. kefiri bioremediation greatly improved pollutant removal. Optimal coagulation conditions (pH 4, 1000 mg/L coagulant) resulted in removal efficiency of 50% for COD, 26% for NH₃-N, and 75% for turbidity. Trace elements like Cu, Ni, Cr, Pb, and As were decreased by up to 23%, 45%, 41%, 33%, and 20%, respectively. Adding L. kefiri bioremediation enhanced efficiency, resulting in 63% COD, 67% NH₃-N, 90% turbidity, and trace metal reductions of up to 53% for Ni and 47% for Cr. While these findings emphasize the combined process's potential as a long-term and cost-effective option for leachate treatment, certain constraints must be addressed before field deployment. The investigations were carried out on a laboratory scale and in batch mode, with no long-term performance evaluation or testing under dynamic leachate compositions, and several metal removal rates (e.g., As and Cu) were relatively low. Pilot-scale trials are required for real-world implementation to evaluate system performance while in continuous operation. The optimization of hydraulic retention duration, aeration, and nutrient supplementation should be investigated, and incorporating L. kefiri into microbial consortia may improve stability and broaden treatment capacity. To meet regulatory standards, the system may also require post-treatment procedures such as activated carbon or accelerated oxidation. A thorough economic and environmental study, including a biosafety analysis, will be necessary to ensure the feasibility and safety of large-scale deployment.

Acknowledgments

This work was supported by the Laboratory of Treatment and Valorization of Water Rejects (LTVRH), Water Researches and Technologies Center (CERTE). We thank members for great assistance with experiments and reagents.

Author Contributions

Y.C, C.B: methodology; Formal analysis, investigation, data curation: Y.C, C.B, L.E; Validation, writing and editing: Y.C, A.K, R.E; Supervision and funding acquisition: I.T, L.C.

Funding

This work was supported by the Tunisian Ministry of Higher Education and Scientific Research under grant [LR15CERTE05].

Competing Interests

The authors have no competing interests to declare that are relevant to the content of this article.