Comparison of Susceptibility to Microbiological Contamination in FAMEs Synthesized from Residual and Refined Lard During Simulated Storage

Abstract

The present research features an experimental comparative design and the objective of this work was to determine the susceptibility to microbiological contamination in fatty acid methyl esters (FAMEs) and the FAME–water interface of residual and refined lard, large volume simulating storage conditions as fuel supply chain, and to identify the microorganisms developed. The plates were seeded according to ASTM E-1259 and the instructions provided by the manufacturer of the Bushnell Haas agar. Microbiological growth was observed at the FAME–water interface of FAME obtained from residual lard. Using the MALDI-TOF mass spectrometry technique, Pseudomonas aeruginosa and Streptomyces violaceoruber bacteria were identified in the residual lard FAMEs, with the latter being previously reported in FAMEs. The implications of microorganism development on the physicochemical quality of FAMEs are significant, as it leads to an increase in the acid index, which may negatively impact metals by inducing corrosion. The refined lard FAMEs did not show any development of microorganisms. The present research concluded that residual lard tends to be more prone to microbiological attack if the conditions of water and temperature affect microbial growth. The findings will contribute to the knowledge base for a safer introduction of FAMEs into the biofuel matrix.

1. Introduction

Fatty acid methyl esters (FAMEs), which are the main components of biodiesel, are fueld with physical properties similar to petroleum-derived diesel [1], whose chemical composition is a mixture of alkyl esters, synthesized from triglycerides present in vegetable oil, animal fat, and their residues generated during food preparation. The traditional process of synthesizing FAME is by alkaline transesterification, which uses a short-chain alcohol such as methanol and an alkaline catalyst such as sodium hydroxide (NaOH), which has a high catalytic activity, low reaction time, and is economical and accessible [2]. The product obtained is a mixture of fatty acid methyl esters and glycerin as a by-product. This procedure has proven to be able to produce good quality FAMEs, depending on the feedstock used [3].

First-generation FAME fuel is obtained from vegetable oils and edible animal fats, for example, refined lard (Figure 1a). Second-generation FAMEs are produced mainly from residual vegetable oils and animal fat resulting from food preparation, such as residual lard from frying bacon (Figure 1b), which have already fulfilled their main purpose. Its advantages include being a FAME fuel of recycled origin, requiring low economic resource management, and contributing to the reduction in fossil fuel emissions [4].

Refined lard has food applications due to its high standards of flavor and quality for human consumption. The use of refined lard as a raw material for processing into FAMEs is limited mainly by its high cost and concerns about its competition with food products [5]. Residual lard is lard generated from the preparation of dishes made from pork, such as bacon, as well as the leftovers of what was used in the preparation of various dishes, in which refined lard was used as a fat bath for frying various foods.

Tert-butyl hydroquinone (TBHQ) is a food additive commonly used as an effective protectant in the food, cosmetic, and pharmaceutical industries [6]. TBHQ has strong antioxidative activity due to the presence of two phenolic hydroxyl groups. It is used as a powerful antioxidant against the oxidation of fats and oils at room temperature and under frying conditions [7].

TBHQ is a patented potential oil soluble antioxidant used an effective additive in various products, and is more efficient than other synthetic antioxidants in vegetable oils and animal fat [8]. The damage caused by TBHQ can lead to the loss of bacterial cell membrane integrity, resulting in the release of membrane components and ions into the bacterial broth [9]. Without proper disposal, residual lard becomes an environmental problem when it is disposed of directly into the household garbage or sewage system [10,11]. One of the main recycling alternatives for residual lard is its reassessment as a raw material for its transformation into FAME synthesis. The advantages of FAME synthesis from this residue lie in its renewable and biodegradable capacity, in addition to the fact that the cost of FAMEs depends largely on the cost of the feedstock used, with residual lard being cheaper than vegetable oil obtained from non-edible oleaginous plants [12]. Due to the organic nature of FAMEs, microbiological activity is present when the environmental conditions allow it, such as the presence of nutrients, water, and an adequate temperature [13]. Microbial activity takes place at the water–FAME interface, resulting in the visible formation of a biofilm, which may consist of bacteria, fungi and/or yeasts [14].

Phenolic-based antioxidants are commonly used in the biodiesel industry. In general, most common antioxidants used in the stability of biodiesel are TBHQ, propy gallate, pyrogallol, butylated hydroxytoluene and butylated hydroxyanisole [15]. TBHQ is more effective in vegetable oil-based FAMEs and is ineffective in FAMEs produced from high free fatty acid oil feedstocks [16].

Therefore, the objective of the present work was to determine the comparative susceptibility of microbiological development in FAME synthesized from refined and residual lard, simulating storage conditions in the fuel supply chain, as well as to identify the microorganisms developed at the FAME and FAME–water interface.

2. Materials and Methods

2.1. Reagents

In this study, 99.9% methanol (Chemika, Monterrey, México), 99.4% purity NaOH (Fagalab, Mocorito, México), Bushnell Haas agar culture media (Himedia, Thane, India), alpha-cyano-4-hydroxycinnamic acid in 50% acetonitrile (Sigma-Aldrich, St. Louis, MO, USA), and 2.5% trichloroacetic acid (Lab Alley, Austin, TX, USA) were used. Distilled water was obtained from a local supplier.

2.2. Materials

The residual lard was obtained as a by-product from domestic bacon preparation and was filtered to separate it from the remaining solid residue using Grade 40 filter paper for 8 μm particle retention, to remove possible food waste. It was then dried to 105 °C for 20 min to avoid moisture. The refined lard was purchased from a local market, and the nutritional label reported that antioxidants and citric acid were added to preserve the product. The samples were stored in a laboratory refrigerator in the dark at 4 °C until further use.

2.3. FAME Synthesis

The synthesized FAME was prepared in duplicate following the alkaline transesterification method reported by Parawira [17]. The fat was completely melted, and the reaction was conducted separately for bacon fat and refined fat in a 600 mL borosilicate beaker. The synthesis involved a single-step basic transesterification carried out at 65 °C, using 1.0 wt% NaOH as the catalyst and a 6:1 molar ratio of methanol to fat, with the reaction lasting 60 min.

2.4. FAME Purification

The FAME purification process is necessary to ensure the purity required to be used as a fuel [18]. The purification began with the removal of sedimented glycerin, followed by the removal of other residues derived from the transesterification reaction, such as unreacted glycerin and methoxide, by washing with deionized water [19] (Figure 2).

The washing process of the FAME samples was conducted in a 1 L flask equipped with a rubber stopper. Equal volumes of water and FAME were combined in the flask with mechanical agitation at 200 RPM for 5 min to ensure a proper interaction between the phases. Subsequently, the resulting mixture of FAME and water was transferred to a 1 L separating funnel and allowed to settle for 5 min under ambient conditions. Following the settling period, the water phase was carefully drained, leaving only the upper phase corresponding to the purified FAME.

The FAME washing was repeated until the water used in the purification process showed no turbidity, this process required at least four washes. Finally, the remaining wash water was removed from the FAME by heating at 105 °C until the FAME turned to a clear color. This color change occurs approximately 15 min after reaching the previously mentioned temperature. The FAME obtained complied with the quality parameters established in ASTM D-6751 Standard Specification for Biodiesel Fuel Blend Stock (B100) for Middle Distillate Fuels [20].

2.5. Samples and Microbiological Isolation

Samples of refined and residual FAME were tested in duplicate to determine their susceptibility to microbiological development. For this purpose, 25 mL of each FAME sample were mixed with an equivalent volume of drinking water and left uncovered at 25 °C for 14 days, simulating a large volume under storage conditions representative of the fuel supply chain. Throughout the incubation period, the samples remained in separate phases. This setup facilitated the emergence of differentiated regions exhibiting microbiological contamination, functioning as laboratory-scale microcosms for FAMEd derived from refined fat (Figure 3a) and residual lard (Figure 3b).

The incorporation of equivalent volumes of water aims to simulate the effect of moisture condensation within the storage container. Due to the density difference between the two liquids, the water settles at the bottom of the biodiesel, replicating the conditions observed in real world storage systems. This experimental approach serves as a starting point for investigating differentiated zones of microbial contamination, as the selected water volume enables a clearer visualization of biofilm formation at the water–FAME interface. Moreover, the simulation provides a reproducible platform for assessing the microbiological degradation risks of the biofuel, especially in the absence of regulations governing the maximum permissible water content in industrial biodiesel storage systems.

To assess the growth of microorganisms in the FAME phase and at the FAME–water interface of refined and residual lard, a Bushnell Haas culture medium was prepared, as recommended for microorganisms in fuels and in accordance with ASTM E-1259, Standard Practice for Evaluation of Antimicrobials in Liquid Fuels Boiling Below 390 °C [21]. The preparation of the culture medium in Petri dishes was carried out following the manufacturer’s instructions: 23.27 g of the medium was suspended in 1000 mL of purified or distilled water. The Bushnell Haas culture medium was heated with mechanical stirring at 200 rpm to ensure complete dissolution, and sterilized by autoclaving at 15 lb of pressure (121 °C) for 15 min. Once cooled to 45–50 °C, the medium was mixed thoroughly and poured into sterile Petri plates. For inoculation, 0.1 mL of each sample, previously homogenized, was used, and seeding was performed directly onto the culture medium. Sample dispersion across the surface of the medium was achieved using a Drigalski spatula, and the procedure was conducted in duplicate. The plates were incubated at 30.6 °C for four days using an ECOSHEL Model 9052 incubator. To express the microbial concentration in terms of Colony-Forming Units per milliliter (CFU/mL), an extrapolation was performed by multiplying the obtained value by a factor of 10, given that 1 mL corresponds to ten times the analyzed volume.

2.6. MALDI-TOF MS Assay

The identification of the microorganisms in the FAME samples was carried out using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) technique. This technique allowed for the identification of microorganisms from colonies developed on culture plates in a short time with a simple and automated methodology, considerably reducing errors in the analysis [22].

For the sample preparation, a small amount of a colony was directly deposited onto the ground steel plate of the mass spectrometer, forming a thin film. Over the film, 1 mL of the matrix solution (a saturated solution of alpha-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% trichloroacetic acid) was applied and left to dry at room temperature. The measurements were conducted using a MALDI-TOF-TOF Autoflex III MS (Bruker Daltonics GmbH, Leipzig, Germany) and the spectrum was obtained automatically within the range of 2–20 kD and operated in positive linear mode at a frequency of 200 Hz. The parameters set for the spectrometer were IS1 at 20 kV, IS2 at 18.6 kV, lens at 6 kV, and PIE at 40 ns. The obtained spectrum was automatically compared using algorithms integrated into the system software with the MALDI Biotyper database. The MALDI Biotyper workflow protocol provides optimal sample acquisition by accumulation of 500 laser shots at different locations on the sample. The spectra were externally calibrated using a standard calibrant mixture to cover a range of 4–17 kD.

Spectra Analysis (Bruker Daltonics GmbH, Leipzig, Germany): For microorganism identification, the spectrum obtained from the target microorganisms was processed using the MALDI Biotyper 1.1 software. The generated peak list was compared with the reference library of the MALDI Biotyper 2.0 using a comparison algorithm integrated into the software. Once the spectrum was imported into the program, the entire process from analysis to identification was performed automatically without any user intervention [22].

3. Results

3.1. FAME Synthesis and Purification

The synthesis of the FAMEs was performed separately through the transesterification of refined (Figure 4a) and residual fat (Figure 4b), using methanol and NaOH as a catalyst.

During the transesterification reaction, the alcohol interacts with the triglycerides, breaking the ester bonds and forming a mixture of methyl esters, which constitute the biodiesel, while glycerin is generated as a byproduct, as you can see in Figure 5.

The color of the FAME is not a regulated parameter and can exhibit a wide range of tonal variations, which primarily depend on the quality of the fat or oil used in its transformation. However, a light-colored biodiesel indicates that the feedstock has not undergone transformation processes due to thermal stress [23], as is the case with refined lard (Figure 4a), and the color can range from pale yellow to light amber. The dark orange color of a FAME (Figure 4b) can be an indicator of feedstock degradation, as well as the presence of impurities that could suggest the microscopic appearance of organic residues from the feedstock, which were transferred to the FAME, as could be the case with FAME produced from residual lard.

After the reaction, the products were cooled at room temperature and left to rest to allow phase separation. Figure 6 illustrates a separation funnel containing the reaction products: the upper layer, comprising the majority volume, corresponds to the FAME; a thin white layer in the middle represents the residue formed as foam; and the bottom layer, in a moderate proportion, consists of glycerin.

The two lower layers, products of the reaction, were removed through drainage using a separation funnel, and the purification of the FAME was subsequently performed to eliminate residual impurities. According to the ASTM D6751 standard [20], which establishes the physicochemical parameters of FAMEs, no specific purification procedure is prescribed. However, washing with water is widely recognized as the most effective and economical method commonly employed to remove residual impurities from FAMEs.

3.2. Microbial Profiles of Collected Samples

After incubation at 30.6 °C for four days, on plates seeded with biofilm grown at the interface of the residual lard FAME + water, a colony of microorganisms developed, as observed in Figure 7a, and it was not possible to quantify the CFU due to colony overlap. In Figure 7c, given the sample level of 0.1 mL in the Petri dish, extrapolation estimates the presence of 10 CFU/mL of microorganisms, originating from the FAME phase of the residual lard. This was not observed in the refined lard FAME (Figure 7b,d).

Figure 7b,d show that the FAME synthesized from refined lard did not exhibit microbiological growth, likely due to the loss of nutrients in the industrial refining process. Additionally, the presence of antioxidants, TBHQ, and synthetic preservatives to extend its shelf life inhibits microbiological development [24].

The FAME synthesized from residual lard was the most susceptible to microbiological attack, likely due to the presence of nutrients such as triglycerides, fatty acids, and minor components including cholesterol, phospholipids, vitamins, aldehydes, ketones, and pigments that remained in the lipid fraction [25,26]. Since no direct purification processes are implemented for their removal in residual pork lard, such as bleaching, degumming, deodorizing, and neutralization [27], these compounds are directly transferred to the FAME produced from this source.

3.3. MALDI-TOF MS Analysis

Prior to the analysis, a detailed visual inspection was carried out on the colonies present on the plate corresponding to the sample ‘FAME from residual lard + water’ (Figure 7a). A well-defined, isolated colony with consistent morphology was selected for subsequent mass spectrometry analysis. The spectrum obtained was automatically compared from algorithms integrated in the system software with the MALDI Biotyper database. The mass spectrum obtained was processed with the MALDI Biotyper 1.1 program. The generated peak list was compared with the MALDI Biotyper 2.0 reference library by applying a comparison algorithm integrated into the software. The spectra obtained are shown in Figure 8.

In the mass spectral profile, the two-dimensional relationship between the mass/charge (m/z) it carries, with respect to the intensity of the peak for a given microorganism, can be seen.

4. Discussion

The duration and conditions of the storage simulation were carefully selected. The decision to conduct a 14-day experiment at a constant temperature was based on establishing a controlled reference point that would allow for the assessment of microbial contamination in FAME without interference from environmental variations. This initial approach enabled us to identify microbiological trends and determine differences in susceptibility between FAMEs derived from residual and a refined lard FAME under stable conditions. We recognize that practical biodiesel storage is often subject to temperature fluctuations, variable humidity, water content, and extended storage periods. However, microbial contamination can develop relatively quickly when conditions favor bacterial growth.

The hygroscopic characteristic of FAMEs results in a rapid water absorption rate in high humidity situations [28]. Consequently, contact with ambient humidity can exceed the maximum allowable water content in FAMEs of 500 ppm, as established by ASTM D 2709 “Standard Test Method for Water and Sediment in Middle Distillate Fuels by Centrifuge” [29]. This may result in significant negative effects on the physicochemical properties, particularly the development of microorganisms, such as the reported growth of microorganisms like Cladosporium, Comamonas, Burkholderia, Klebsiella, Tolumonas, Candida, Aspergillus, Fusarium [30,31].

The profile presented in Figure 8 corresponds to two types of microorganisms. Figure 8A depicts Streptomyces violaceoruber, which developed in both samples: the FAME prepared with residual lard + water (Figure 7a), and the FAME from residual lard alone (Figure 7c). This microorganism is a Gram-positive bacterium naturally found in soil [32], and has been associated with infections such as mycetoma. Species of the genus Streptomyces have also been reported to cause septicemia and pulmonary conditions [33]. The mass spectral profile shown in Figure 8B, identified exclusively in the FAME derived from residual lard + water, corresponds to Pseudomonas aeruginosa, a Gram-negative bacillus of the genus Pseudomonas. These bacteria are commonly present in soil, freshwater, and marine environments. Pseudomonas aeruginosa has received particular attention due to its opportunistic pathogenicity and its ability to cause human diseases [34].

The water used as a contamination simulator under FAME storage conditions was likely contaminated with microorganisms, which were subsequently transferred to the FAME, suggesting that the introduction of microorganisms is primarily due to their presence in the water. Although the presence of water in biodiesel is not a desired parameter, it is frequently introduced through ambient humidity and migrates along the container walls, carrying microorganisms. Once inoculated into FAME via the aqueous phase, these microorganisms utilize the nutrients available in the biodiesel, such as carbon-rich compounds, to support their growth and proliferation. This interaction highlights the importance of controlling water presence in FAME during storage and handling to mitigate microbial contamination and its adverse effects on the physicochemical stability of FAME.

Table 1. Pseudomonas aeruginosa development in FAMEs as reported in the scientific literature.

Microorganism	FAMEs Raw Material	Interaction of Microorganism with FAME	Ref.
Pseudomonas aeruginosa	Residual vegetable oil	The impact of Pseudomonas aeruginosa on FAME is mainly related to its ability to cause biodeterioration. These bacteria can contribute to biofilm formation and degradation of FAME components, which can result in decreased fuel quality and operational problems, such as filter plugging.	[35,36]
	90% soy, 10% tallow	The impact of Pseudomonas aeruginosa on FAME is mainly related to its ability to degrade compounds present in this type of fuel. This bacteria can contribute to the biodegradation of FAMEs, affecting its quality and stability during storage.	[37]
	80% soy, 20% tallow	Pseudomonas is one of the principal fuel-biodeteriorating contaminants, together with Comamonas, Burkholderia, Klebsiella, Tolumonas, Candida, Aspergillus, Fusarium.	[28]
	Palm oil	Palm oil and FAMEs are subjected to aerobic biodegradation by bacteria like Pseudomonas, commonly present in natural open environments.	[38]
	J. curcas A. aculeata	The FAMEs exhibited noticeable deterioration by Pseudomonas during simulated storage conditions, primarily due to microbial contamination. Significant shifts in pH, surface tension, and ester content were observed even over a short 30-day period, with fungal inoculation accelerating the degradation process resulting in up to a 12% reduction in ester concentration.	[39]
	Soy oil (B20)	The addition of FAMEs leads to an increase in the number of microorganisms, resulting in the degradation of the fraction corresponding to FAMEs producing aldehydes and ketones.	[40]
Streptomyces violaceoruber		In the present work, this microorganism was identified in the FAME of residual lard, not previously reported by scientific literature. However, this microorganism is very commonly found in various places such as soil [41], industry [42], and water [43].

shows the reports of the presence of Pseudomonas aeruginosa in FAMEs. Regarding the presence of the Streptomyces violaceoruber identified in the FAME derived from residual lard, this study contributes to broadening the current understanding of the microbial diversity associated with this biofuel.

The biodegradation pathway for FAME molecules is a multistep process that is similar under both aerobic and anaerobic conditions. FAMEs are first de-esterified to form free fatty acids and methanol. The free fatty acids then undergo sequential removal of two- carbon components through a process known as β-oxidation. The methanol released is readily biodegraded under aerobic and anaerobic conditions. Metabolism of glyceride esters starts with de-esterification to form free fatty acids and glycerin by esterase enzymes known as lipases, which have been found in microorganisms as in Pseudomonas consortia [44].

The study confirms that the introduction of FAMEs affects the types and activity of the microorganisms present in the fuel microcosm. It is less likely that microorganisms will develop in the FAME; instead, they need water and nutrients to develop as a biofilm at the water–FAME interface. Although the presence of Pseudomonas aeruginosa in biodiesel derived from various raw material sources had been previously reported, there had been no documented cases of this bacterium in biodiesel synthesized from residual lard. Similarly, the identification of Streptomyces violaceoruber in the present study is particularly relevant, as it may support future proposals for mitigation strategies or its potential use as a microorganism for the bioremediation of biodiesel-contaminated soils.

According to studies conducted by Soriano Ururahy et al. [45] and Schleicher et al. [46], in systems where microbiological growth occurs, there is a tendency for an increase in the acidity index, a phenomenon particularly notable in B100 biodiesel, this increase is attributed to microbial activity, which generates organic acids responsible for lowering pH levels. Although there is no regulation that defines the maximum permissible parameters for the presence of microorganisms in stored biodiesel, the addition of broad-spectrum biocidal active substances, such as 3,3-Methylenebis (5-methyloxazolidine) (MBO) and 5-Chloro-2-methyl-4-isothiazolin-3-one + 2-Methylisothiazol3(2H)-one (MIT/CMIT), is highly recommended. This approach helps mitigate microbial growth in stored biodiesel, ensuring its stability and quality over time [47].

5. Conclusions

The results obtained in this research highlight the importance of the quality of the feedstock and the effect of the presence of water in the storage of FAMEs produced from lard as parameters that can be of great significance by influencing microbial contamination, underlining the potential of FAME as a sustainable alternative to conventional biofuels.

Although there is no evidence of microbiological contamination in lard FAMEs, there are reports of contamination in other sources of fatty acids. Residual lard from food preparation could be used to convert this waste into FAMEs, representing an alternative solution to prevent pollution from improper disposal of waste, while offering the opportunity to generate a value-added biofuel, in line with global goals of environmental sustainability and energy security. However, it is crucial that the FAME does not come into contact with water during the synthesis and storage processes and, as a preventive measure, an antimicrobial agent should be incorporated to guarantee its quality and safety. The research concludes that residual lard is more prone to microbiological attack than refined lard, especially when water and temperature conditions favor microbial growth. This process leads to an increase in the acid index, which may negatively impact metals by inducing corrosion. Control methods for preventing microbial contamination in FAMEs, such as the addition of MBO, MIT/CMIT, or a broad-spectrum biocide, are recommended.

In future research, expanding the experimental design to incorporate climatic variations and long-term monitoring would provide a more accurate representation of real industrial storage conditions. Additionally, directly correlating microbial growth with fuel quality degradation and implementing quantitative methods—such as plate count, ATP luminescence, and qPCR—will enable a more robust assessment of the microbial load in FAMEs and at the interface over time, enhancing the evaluation of contamination severity.