The leachate sample was collected at the final drainage point of an active municipal landfill located in the southern region of Minas Gerais, Brazil. The landfill has been in continuous operation for 13 years and serves a population of approximately 137,000 inhabitants. The sample was obtained at the base of the landfill’s drainage system, ensuring that the material had percolated through all waste layers—approximately 50 meters in vertical height—thus representing a mature leachate with advanced degradation stages. Collection was performed manually using a clean, high-density polyethylene (HDPE) container with a total capacity of 20 liters. The sample was transported under cooled conditions to the laboratory within two hours of collection. Upon arrival at the Water Quality Laboratory of the Center for Environmental Quality Studies (CEQUAM) at Universidade Federal de Itajubá (UNIFEI), the material was immediately refrigerated at −4˚C until further analysis, in accordance with standard procedures outlined in ABNT NBR 9898:1987.

For the pretreatment experiments, the leachate was homogenized and then aliquoted into 100 mL borosilicate glassware for each experimental condition.

Leachate characteristics are highly variable, influenced primarily by factors such as the landfill’s age, the composition of the disposed waste, seasonal rainfall, and local climatic conditions. To ensure that the collected sample was representative of typical landfill leachate for this specific site, sampling was conducted immediately after the rainy season in June, a period when leachate generation is generally at its peak due to increased infiltration and percolation of rainwater through the waste mass. Sampling in this period captures the leachate composition under realistic operational conditions, including the mixture of organic compounds, nutrients, and potential contaminants [6] [5] .

In order to determine the physicochemical parameters of the landfill (LF) leachate, pH value analyses were performed (ASTM E70-20), acidity index (ISO 660-2020), alkalinity (ISO 99631-:1994) and TOG (AOCS 920.39-2012.). The analyses and calculations of these acidity and alkalinity parameters were also based on the literature on physical-chemical methods for food analysis from the Adolfo Lutz Institute [16] .

To characterize the lipid material from the leachate, determinations on the acidity index were made [16] as well as additional tests measuring free fatty acids were also performed (ASTM D 5555-95, 2001) and also saponification index tests (AOC D 3-25, 1995). The analyses of the lipid material had to undergo a dilution process in distilled water because the product after extraction of raw material had high viscosity, which would be an impediment to measuring the parameters mentioned.

The pretreatments applied were intended to improve the extraction capacity of lipid material from the sample, using three techniques: thermal treatment [17] , by acidification [18] and by ultrasound [19] . The lipid extraction efficiency for each process was evaluated, using the Soxhlet process, by TOG measurements (AOAC 920.39-2012). All pretreatment and extraction experiments were performed in triplicates.

In order to investigate the effect of the heat treatment as a pretreatment for lipid extraction, the furnace (SSA 30 L Model) was heated until the temperature stabilized at 200˚C. After reaching this temperature, the 25 mL leachate sample was inserted into the oven and heated to 200˚C for 5 minutes. The sample was then cooled in a thermal oven (Solid Steel 40 L Model) at a temperature of 20˚C, for 5 minutes. The same procedure was performed for other leachate samples, applying a temperature of 250˚C, with both samples being stored in beakers in a thermal oven at 21˚C for later analyses. These temperatures were chosen based on the work of CHOI [17] .

To evaluate the effects of pretreatment by acidification for the extraction of lipid material from LF leachate, 40 mL of leachate sample was used, and the pH value of the sample was measured (pH meter PHS3BW). Then, a 0.1 N HCl solution was gradually added to the sample using a pipettor until the pH value reached 2. The sample was stored in a thermal oven at 21˚C for later analysis.

The procedure began with the separation of 100 mL of leachate into 500 mL beakers. The samples were placed in an ultrasonic bath, and three 120-minute intervals were conducted, at a frequency of 40 Hz [19] .

The lipid extraction process was carried out using the Soxhlet technique (AOAC 920.39-2012). The procedure adopted was characterized by the analysis in triplicate of the pretreatment samples stored in a thermal oven at 21˚C. In the analysis of the preheat and ultrasound pretreatment samples, the posttreatment liquid raw material was transferred to 100 mL evaporation porcelain for drying in a thermal oven at a temperature of 100˚C for a drying period of 12 hours. In the analysis of the pretreatment by acidification, due to the lipid material having accumulated in the liquid phase of the solvent, direct drying of the solvent was carried out instead of the liquid raw material, with the following steps being the same as those previously mentioned, and all samples (thermal, ultrasound and acidification) having been collected in solid state. After carrying out this preprocedure, the Soxhlet technique applied was based on the gravimetric difference of beakers without material and after the passage and drying in an oven of the liquid hexane solvent over the solid raw material, in which the raw material was retained in Styrofoam tubes with the continuous passage of refluxing solvent.

The total pretreatment cost per kilogram of lipids extracted was calculated by combining the energy cost, chemical input cost, and any additional operational expenses:

C_total = ((E × C_energy) + C_chem + C_oper)/M_lipid (1)

where:

- C_total = total pretreatment cost per kg of lipid (USD/kg);

- E = energy consumed (kWh);

- C_energy = cost of electricity (USD·kWh⁻¹);

- C_chem = cost of chemicals used (USD);

- C_oper = other operational costs (USD);

- M_lipid = mass of lipid extracted (kg).

The data obtained for the characterization of the raw leachate were summarized in Table 1 .

The analyses of the raw leachate sample served as an indication of its quality for its capacity to be used in the removal of lipid material, considering the possible variations that the material may present depending on the origin and physical-chemical composition of the leachate coming from it.

That is considering that a landfill, over the course of its operation, can go

Source: Own authorship.

through different phases, such as hydrolysis, acidogenesis, acetogenesis and methanogenesis, in which the complex material is progressively transformed into simpler compounds [3] . The first characteristic we can evaluate in the material is its pH and alkalinity, which helps us determine what stage of degradation it is in [20] .

In the context of energy production, it is desirable that the leachate is mainly in the initial stages of degradation [21] . This is due to the fact that for the viability of biodiesel production from this material, the presence of high quantities of fatty acids, which can be used in the esterification process, and triglycerides, in the transesterification process, is of utmost importance, as demonstrated in Figure 1 .

On the other hand, when the landfill is older, the leachate contains free fatty acids in high concentration, acting as undesirable components because they favor saponification when basic catalysts are used [22] , and damaging subsequent biodiesel extraction processes.

Therefore, the alkalinity test can be useful in assessing the maturity of the material, calculating the concentration of bicarbonate ions ( ${\text{HCO}}_3^{-}$) and carbonate ( ${\text{CO}}_3^{2-}$) in the middle. As evidenced in research by Tchobanoglous, Theisen and Vigil [24] , the results indicate that recent landfills, with less than 2 years of operation, have an average alkalinity in the range of 1000 to 10,000, with a pH between 4.5 and 7.5. In contrast, older landfills, with more than 10 years of operation, have an alkalinity range of 200 to 1000 and a pH between 6.6 and 7.5.

Therefore, obtaining a referentially low alkalinity value of 250.00 mg·L⁻¹ of CaCO₃ may indicate that the leachate analyzed comes from leachate that is already in the methanogenic phase, corroborated by the obtaining of a pH that is alkaline, a pH of 8.9, characteristic of the environment with a majority of methanogenic bacteria.

When analyzing the acidity index, relevant information is obtained about the presence of acids in the medium, which come from the acidogenic phase. A high value may indicate that, even when the material is in the methanogenic phase, there is still the presence of non-hydrolyzed acids, preferably free fatty acids, although with the presence of others in smaller quantities, such as carboxylic and fatty acids [3] .

In the analyses of oil and grease content (OGC), obtaining 8 ppm per sample is an indication that the leachate contains lipid materials of animal, vegetable and other sources. This high value differs from the results obtained by Moura [25] for landfills of the same time period; however they can be attributed to the fact that the sanitary landfill from which the material studied comes, receives waste from urban and rural areas and with the deposition of type II A waste, therefore also having the disposal of general organic materials from food industries (NBR 10004) unlike the industrial waste previously studied by Moura [25] . Therefore, in addition to conventional urban organic waste, such as food scraps and waste, there are also contributions of organic waste of animal origin from farms and rural land.

Leachate, being a percolate with variations in the amount of volumetric formation according to the region's rainfall and the capacity to penetrate the landfill layers, has the capacity to drag and retain organic materials with variations in relation to the location of the landfill and its climatic conditions [26] .

If we consider the purpose of biodiesel production, it is possible to extend this concept of leachate entrainment to the retention of biodegradation microorganisms from the landfill in the material under study. Thus, it is possible to use not only the free lipid fraction present in the material, but also the lipids coming from microorganisms and complexes from the environment [19] .

Cellular lipids may include diglycerides, triglycerides, phospholipids, monoglycerides, sterols, and free fatty acids (FFAs) [27] . Besides, fatty acids, sterols and some aliphatic components can be found in up to 36.8% cellular lipids [28] . These quantities can be used in the biodiesel production process, especially with the use of pretreatments that mainly aim at cell disruption and the breakdown of complexes.

The choice of the organic solvent to be used for the removal of these specific components from the leachate also affects the efficiency of the process. For the removal of materials released by cell rupture, such as phospholipids, the use of polar organic solvents, such as methanol, is more suitable [29] . Other possibilities, such as the use of mixtures of polar and nonpolar organic solvents, may also prove effective for the comprehensive extraction of lipid materials in the leachate [30] . However, when the main objective is the recovery of the fraction of triglycerides released by cell rupture, the use of a nonpolar organic solvent, such as hexane, becomes more advantageous considering the specific application for the polarity of the target lipid [31] .

Furthermore, the high affinity for neutral lipids, low polarity, and excellent lipid solubility, particularly for triacylglycerols and free fatty acids. Hexane is widely recognized in standard methods for lipid extraction due to its efficient recovery of nonpolar lipid fractions and ease of evaporation at relatively low temperatures (boiling point ~69˚C), which minimizes thermal degradation of the extracted compounds [32] [33] .

In this study, the lipid extraction process was preceded by three different cell disruption pretreatment techniques: thermal, ultrasound, and acidification. These methods were selected based on their ability to enhance cell lysis and improve the release of intracellular lipid content into the extraction medium.

The efficiency of each pretreatment was subsequently evaluated using the Soxhlet extraction method with hexane as the solvent, as previously justified. The lipid extraction yields were quantified and expressed as the amount of lipid material (in milligrams) extracted per gram of raw leachate sample. The comparative results for each pretreatment method are presented in Figure 2 .

The use of acidification as a pretreatment technique for lipid extraction obtained the results of lower extraction capacity when compared to thermal and ultrasound pretreatment. However, a satisfactory result was obtained, 73.33 ± 4.03 mg·g⁻¹, because it was higher than the lipid extraction capacity of the raw material only by applying the Soxhlet technique, 13.93 ± 1.46 mg·g⁻¹.

The acidification technique was chosen based on its ability to, in addition to acting on the cellular disruption of microorganisms present in the leachate, also potentially be capable of breaking the bonds between lipids in emulsions, linked to carbohydrates and proteins in the medium, increasing the efficiency of the extraction of lipid material [19] . The low efficiency compared to other techniques can possibly be attributed to the high severity of cell disruption associated with this technique, indicating greater lipid degradation and resulting in lower extraction yields.

As highlighted in research by Olkiewicz et al. [18] and Kech et al. [34] , the acidification as a pretreatment may increase the presence of free fatty acids at the expense of esters for transesterification. However, this technique may be advantageous in allowing liquid-liquid extraction as demonstrated in Figure 3 , with consequent reduction in costs related to subsequent steps, such as drying of the raw material by Soxhlet extraction, which normally represents a significant part of the process costs.

Furthermore, a quantitative cost analysis reinforces the economic viability of the acidification pretreatment method. Based on experimental data, the lipid yield obtained through acidification was 55.7 ± 3.01 mg·g⁻¹ of dry leachate, corresponding to approximately 2.23 g of lipids extracted from a 40 mL sample. The total cost of processing this volume—factoring in electricity consumption, chemical reagents (HCl and NaOH), and operational costs—was calculated at US$ 0.0595, leading to an estimated cost of US$ 26.71 per kilogram of lipid extracted using formula 1.

This cost is significantly lower than the estimated US$ 35 - 50/kg for Soxhlet extraction, which demands higher energy input due to prolonged heating cycles and solvent reflux. Acidification, on the other hand, relies primarily on pH adjustment and liquid-liquid separation at ambient temperature, resulting in minimal electricity consumption (~0.2 kWh per batch) and reduced solvent loss, especially when hexane is recovered through low-temperature distillation with >90% efficiency [33] [31] . A simple model is presented in Figure 4 .

In consideration of the environmental benefits and drawbacks of using acid pretreatment on a large scale, studies by Agarwal et al. indicate that the main challenge is the disposal of the acidic material, which can alter the pH of water and soil and pose potential environmental risks that must be carefully managed [35] . Conversely, the ability to perform lipid extraction at ambient temperature reduces the formation of volatile compounds, minimizing air emissions. Furthermore, recent research on green solvents for large-scale liquid–liquid extraction suggests that process optimization can further mitigate environmental impacts by improving biodegradability and reducing toxicity [36] .

In the case of the ultrasound technique, which is based on cell rupture through cavitation of microbubbles in the cell membrane, this process has a gentler approach due to the possibility of controlling frequency and retention time, with potentially less loss of lipid material when compared to acidification [37] . However, the ultrasound pretreatment presents significant economic limitations under the tested conditions. The procedure involved subjecting 100 mL of leachate to three 120-minute cycles at 40 Hz in an ultrasonic bath (300 W), totaling 6 hours of operation. This resulted in an energy consumption of approximately 1.8 kWh, corresponding to an energy cost of US$ 0.27 (based on US$ 0.15/kWh). No chemical reagents were employed; however, operational expenses, including labor and equipment wear, were estimated at US$ 0.50 per batch. The lipid yield from this treatment was approximately 111.4 mg, resulting in a calculated cost of US$ 24.33 per kilogram of lipid extracted using formula 1.

These values highlight that, although ultrasound can be effective in disrupting particulate organic matter then acidification, the method currently demands high energy inputs and extended processing time, as well as long retention periods to avoid damage to the component of interest considering the non-application of frequencies higher than 80 Hertz [37] , which sharply reduce its economic feasibility. Pilot-scale implementation would require substantial investment in high-capacity ultrasonic equipment and further optimization of frequency and exposure time. Therefore, while ultrasound remains a promising tool for enhancing lipid extraction efficiency but not for up scaling, its viability in leachate valorization depends on future process intensification strategies that reduce energy consumption and improve yield per cycle.

Thermal pretreatment showed a lipid extraction efficiency similar to that from ultrasound pretreatment, with a value of 104.67 ± 5.04 mg·g⁻¹ at 250˚C. However, at 200˚C, the efficiency was lower, with 73.33 ± 4.03 mg·g⁻¹, but still higher than that obtained with acidification pretreatment. The gradual increase in temperature in two ranges, 200˚C and 250˚C, resulted in greater efficiency in the cell rupture process, considering that the gradual variation in the amplitude of the thermal shock established greater cellular stress for rupture. This statement is corroborated by studies of Lee et al. 17], which indicate that the gradual increase in temperature has such an effect, although there are limitations above 300˚C, resulting in consequences similar to those of acidification in lipid degradation.

Thermal pretreatment at 250˚C was the most effective method, yielding 104.67 ± 5.04 mg·g⁻¹ of lipids, followed closely by ultrasound-assisted extraction (101.17 ± 0.59 mg·g⁻¹). Both methods significantly outperformed acidification (55.7 ± 3.01 mg·g⁻¹). At 200˚C, the yield was lower (73.33 ± 4.03 mg·g⁻¹), but still superior to the acidification method. The gradual increase in temperature between 200˚C and 250˚C enhanced the cellular rupture process due to progressive thermal stress, as supported by Lee et al. [17] . However, the literature also cautions against temperatures above 300˚C, which may induce lipid degradation effects similar to acid hydrolysis.

From an operational standpoint, thermal treatment offers favorable economics. Each 250 mL sample required approximately 5 minutes of heating in a laboratory oven operating at 1.5 kW, totaling 0.125 kWh per sample. This corresponds to an energy cost of US$ 0.019 (at US$ 0.15/kWh). No chemical reagents were required (Laboratory scale), and additional operational costs (e.g., handling, maintenance) were estimated at US$ 0.10 per batch. Given the lipid yield per 100 mL sample of 10.47 g, the total cost per kilogram of lipid extracted was calculated using formula 1, with result of US$ 11.71 per kg of lipid.

This result indicates that thermal pretreatment is significantly more cost-effective than ultrasound, with a similar extraction efficiency and a markedly reduced processing time (5 min vs. 6 hours). Furthermore, the scalability of heat-based systems—through the integration of autoclaves, heat exchangers, or continuous thermal flow reactors—makes it a technically feasible and economically sustainable strategy for lipid recovery from leachate on a pilot or industrial scale. These findings support the thermal method as a robust and efficient alternative in waste-to-biodiesel valorization schemes, as illustrated in Figure 5 .

Table 2 presents a comparative summary of four pretreatment methods applied

to leachate samples—acidification, ultrasound, and thermal treatments at 200˚C and 250˚C—with respect to lipid extraction efficiency, energy and operational costs, scalability, and key technical considerations. The economic analysis was based on direct measures of energy consumption and estimated market prices for chemicals and operational inputs. This integrative assessment highlights the trade-offs between extraction yield and total cost, offering insights into the most suitable methods for large-scale applications.

As shown in Table 2 , the lipid yield obtained from landfill leachate (55 - 105 mg·g⁻¹ dry solids) is within the same order of magnitude as sewage sludge (80 - 200 mg·g⁻¹), demonstrating that this challenging waste stream can serve as a viable lipid source. Although conventional feedstocks such as vegetable oils (200 - 400 mg·g⁻¹) and animal fats (>700 mg·g⁻¹) exhibit higher yields and lower market costs [38] - [40] , they are also associated with sustainability concerns, including competition with food production and land-use impacts. In contrast, valorizing landfill leachate contributes to both waste management and pollution mitigation, transforming an underutilized residue into a renewable energy feedstock. While the current processing costs (US$ 5.0 - 24.3 per kg lipid) remain higher than those of established feedstocks (soybean oil: ~US$ 0.70 - 1.00 per kg; animal fats/waste cooking oil: ~US$ 0.30 - 0.60 per kg) [38] - [40] , advances in pretreatment, purification, and process integration are expected to reduce these gaps, reinforcing the potential of landfill leachate as a complementary and environmentally strategic biodiesel feedstock.

The data obtained for the characterization of the raw leachate were summarized in Table 3 .

Source: Own authorship.

The first point to be observed in the analysis of Table 3 data is the fact that the acidity index value drops and the similarity with the free fatty acid index value.

Obtaining approximate values between the number of free fatty acids and the acidity index, derived from the analysis of the lipid material, suggests that the leachate sample primarily presents a degradation of the lipid material into simpler forms, such as free fatty acids. This result is in line with the pH and verification of the maturity of the percolate, which indicates that the material has already passed through the initial phases of hydrolysis, acidogenesis and acetogenesis. However, for the production of biofuels, these values are not effective due to the tendency to favor the saponification reaction, when alkaline catalysts are applied, to the detriment of transesterification in the environment in question. In addition, they are also not in accordance with the values defined by Brazilian standards [41] .

A possible advantage of the system is that the lipid extraction process using hexane in a Soxhlet apparatus resulted in the purification of the acidic components in the medium, yielding a lipid material composed of approximately 98% Free Fatty Acids (FFAs). This high FFA content, although initially a challenge for biodiesel production due to risks of soap formation and corrosion in alkaline transesterification, can be addressed through acid-catalyzed pretreatment. Specifically, the application of sulfuric acid (H₂SO₄) in the presence of methanol enables the conversion of FFAs into fatty acid methyl esters (FAMEs) via esterification. This strategy not only prepares the feedstock for subsequent base-catalyzed transesterification but also aligns with established methodologies in the literature. For instance, Sharma et al. [42] achieved 97.3% ester content after applying acid pre-esterification to animal fats with high FFA levels, while Supeno et al. [43] demonstrated 96.1% ± 0.4% conversion at room temperature in a pilot-scale setup. Moreover, Nakpong et al. [44] reported 98.4% biodiesel yield from coconut oil (12 wt% FFA) using similar two-step strategies. These findings support the feasibility of converting leachate-derived lipids—despite their high acidity—into biodiesel-compatible materials through scalable and cost-effective esterification techniques, aiming at the occurrence of esterification, as shown in Figure 6

Studies have already been carried out using two main techniques regarding the application of transesterification with acid catalyst and pre-esterification and transesterification with basic catalyst [45] . The results suggest that, with the application of acid-catalyzed pre-esterification, it is possible to obtain a material with over 97% ester content, thereby meeting European standards for biodiesel production. Therefore, it is possible to consider future analyses to obtain a material suitable for biodiesel production, even with high concentrations of FFAs compared to studies by Moura [25] .

In saponification index analysis, which consists of the complete hydrolysis of triacylglycerols in alkaline solutions, results in the formation of fatty acid salts and can be used for a rapid assessment of the ester formation potential by determining the percentage of esters using Equation (1) [28] .

$\%\text{of esters}=100*\frac{\text{IS}-\text{IA}}{\text{IS}}$ (1)

Considering the criteria established by the National Oil and Gas Agency for biodiesel [41] , the material shows potential for biodiesel production, as the theoretical ester content (96.65%) is close to the minimum requirement of 96.5%. However, given the high free fatty acid (FFA) content (~98%), the raw material does not meet this specification without further processing. Therefore, the implementation of acid-catalyzed pre-esterification is essential to convert FFAs into esters and ensure compliance with ANP standards

Comparing these results to the study by Canesin [28] , which reported a saponification index of 196 mg NaOHg⁻¹ of sample for soybean oil and 182.5 for residual poultry slaughterhouse oil, it was concluded that the material under analysis has, respectively, 42.7% and 38.5% less capacity for conversion into biofuel compared to conventional sources. Even with a lower value, the source of the material has its advantage, since it is a residue with no other practical uses.

Furthermore, when compared to other non-conventional lipid sources, landfill leachate shows competitive or even superior performance. For instance, Zhu et al. [31] reported lipid yields ranging from 2.5% to 10.3% (dry basis) from municipal sewage sludge, while hydrothermal pretreatment at 200˚C increased yields up to 14.01% as studied by Chen et al. [46] . Similarly, grease trap waste has been studied as a feedstock with high free fatty acid (FFA) content, requiring pre-esterification to reduce FFA to acceptable levels before transesterification. Tran et al. [47] demonstrated a reduction of FFA to 0.84% after treatment with 3 wt% sulfuric acid at 75˚C for 3 hours. These findings reinforce the potential of landfill leachate as a promising alternative for biodiesel production, especially when optimized pretreatment methods are employed