Lubes em Foco Magazine – issue 95
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Authors:
Chemical Engineer Emerson Leite – Universal Ind. e Com Laboratories Ltd.,
Chemical Engineer Leonard Giordanni Batista – Universal Ind. Com Laboratories Ltd.,
Dr. Tiago Palladino Delforno – ISI – Biotechnology,
Dr. Isabel Teresa Santos Correa – ISI – Biotechnology,
Dr. Angie Alejandra Calderon Fajardo – ISI – Biotechnology,
Dr. Erica Janaina Rodrigues de Almeida – ISI – Biotechnology,
B.Sc. Carolina Dos Santos Silva – ISI – Biotechnology
Introduction
In recent decades, climate change has generated concern about fossil fuels, particularly diesel. Therefore, the development of renewable alternatives is encouraged. Among the most widely used matrices, methyl-based biodiesel has been the most widely used due to its cost-productivity trade-off.
Biodiesel consists of monoalkyl esters of long-chain fatty acids, formed in a reaction between alcohols and vegetable oils or animal fats. The biodiesel production process produces byproducts that must be purified, but are not completely eliminated by current processes. Therefore, contaminants such as sulfur, metals, and the moisture present in diesel and storage tanks lead to the formation of resin, gum, and sludge that impact combustion and engine performance, leading to maintenance and downtime costs.
Since 2010, the addition of 5% biodiesel to the Brazilian energy matrix has already suffered from sludge formation. The increase in its share of the diesel market to 15% today has been accompanied by a nearly proportional increase in the problem, leading manufacturers of diesel vehicles, machinery, and equipment to question possible solutions.
Laboratórios Universal, a specialty chemicals company with nearly 50 years of experience, has been monitoring market needs and, through its researchers in partnership with leading universities and research centers, sought a solution that would mitigate or prevent the resulting problems. In this case, the partnership with the Senai Institute of Innovation and Biotechnology sought to evaluate compounds that would inhibit the formation of biological sludge from diesel and biodiesel.
Methodology
For the tests, samples were collected from the storage tanks of the Vibra Energia Fuel Distributor in São José dos Campos, São Paulo, and from the tank of a Volvo truck (model TD 102FS) with six cylinders, identified as black grease (Figure 1). During the research, several compounds were developed and carefully evaluated, leading to the discovery of BX-15, which best inhibited biological activity and oxidative reactions in a mixture of up to 15% diesel. BX-15 is a molecule produced by the organic synthesis of p-hydroxybenzoic acid.
To evaluate its effectiveness, a minimum inhibitory concentration (MIC) test was performed, which consists of serially diluting the compounds in the solvent dimethyl sulfoxide (DMSO) and incubating each dilution, as well as the undiluted compound, with microorganisms. Cultures of the following microorganisms were used for this purpose:
A) Escherichia coli 25922;
B) Pseudomonas aeruginosa AT27853;
C) Bacillus megaterium.
Furthermore, the microorganisms present in the microbial consortia samples were also evaluated:
D) Sludge sample collected from the tank of a Volvo TD 102FS 6-cylinder truck.
E) Indigenous biodiesel sample (composed of 50% biodiesel and 50% conventional Luria-Bertani (LB) culture medium);
F) Allochthonous biodiesel sample (composed of 50% biodiesel (74.5 mL), 50% LB medium (74.5 mL), and 1 mL of methanogenic inoculum from a poultry slaughterhouse).
BX-15 was tested in serial dilutions in a base-2 fashion, at concentrations ranging from 8% to 0.0037%. To ensure assay quality, samples A-D were tested with a positive control of 20% DMSO and a negative control of 0.1% DMSO. For samples E and F, the positive control was performed with 75% DMSO, and the negative control with a suspension of 50% microbial inoculum and 50% culture medium. Additionally, a toxicity control was performed for all tests using 0.5% DMSO. Each well then consisted of Nutrient Broth (or LB medium for biodiesel assays), microbial inoculum at a concentration of 1 106 CFU/mL, and BX-15 at its respective concentration. Assays were performed in triplicate for the samples and in duplicate for the controls. Preparation details for each assay are compared in Table 1.
After 24-hour incubation, resazurin dye was added. This dye indicates microorganism viability by changing color to pink, while blue indicates the absence of microbial growth, representing the compound’s antimicrobial activity. Quantitative analyses were performed using spectrophotometry, measuring the optical density (O.D.) at 600 nm before dye addition. O.D. is directly related to the microbial density resulting from growth even under the action of the compounds.
Results and Discussions
A summary of the results generated by the minimum inhibitory concentration (MIC) tests is described in Table 2, which resulted from qualitative analyses by visual analysis and quantitative analyses by optical density analysis at 600nm.
The results for samples A, B, and C are shown in Figures 3, 4, and 5, respectively. For the Escherichia coli ATCC 25922 strain, significant activity was observed against compound BX-15 at concentrations ranging from 0.5% to 0.0156%. However, bacterial inhibition was observed at concentrations above 0.25%. It can be seen that, starting at a concentration of 0.125%, there was a significant increase in cell density, confirming the compound’s minimum inhibitory concentration in E. coli of 0.25%.
For Bacillus megaterium, activity was also observed against compound BX-15 at concentrations ranging from 0.5% to 0.0156%. In this case, significant bacterial inhibition values were found starting at 0.125%. Quantitatively analyzing the Bacillus optical density, the compound’s minimum inhibitory concentration is confirmed at 0.125%, since more pronounced cell growth occurs above 0.0625%.
In tests performed with the Pseudomonas aeruginosa ATCC 27853 strain, a profile similar to E. coli was observed, despite its more pronounced growth. Visual analysis also showed activity against the BX-15 compound tested at concentrations ranging from 0.125% to 0.0156%. Under the conditions studied and at the standardized cell concentration, Pseudomonas aeruginosa exhibited a significantly higher growth rate than the other strains. However, quantitatively analyzing the optical density, the compound’s minimum inhibitory concentration is observed at 0.25%.
For samples containing mixed microbial communities, the sludge collected from the tank of a Volvo TD 102FS truck (Sample D) had its growth inhibited with BX-15 at a minimum concentration of 0.125%, as shown in Figure 6. Similar results were seen for plating, as shown in Figure 7.
Regarding the tests performed with biodiesel samples, sample E, containing the autochthonous microbial population—i.e., microorganisms native to biodiesel—was effectively inhibited by BX-15 at concentrations of 0.125%, 0.25%, and 0.5%. However, concentrations below 0.125% were not sufficient to inhibit microbial growth, making this the concentration equivalent to the MIC in this sample. The results for sample E can be seen in Figure 8, which shows the MIC test plate.
Finally, in sample F of allochthonous microbiota, with biodiesel inoculated with poultry slaughterhouse sludge, compound BX-15 was shown to be effective at concentrations starting at 0.25%, as can be seen in Figure 9.
Conclusions
BX-15 is a lipophilic, hydrophobic product, perfectly miscible with biodiesel, diesel, and their blends up to 15% biodiesel. The results generated by the study indicate that the effectiveness of BX-15 varies depending on the composition and characteristics of the substrate to be applied. Minimum inhibitory concentration (MIC) tests revealed the following results for pure cultures:
• 0.25% for Escherichia coli ATCC 25922
• 0.25% for Pseudomonas aeruginosa ATCC 27853
• 0.125% for Bacillus megaterium
In complex microbial consortia—which best represent real-world contamination conditions in fuel systems—the product demonstrated significant effectiveness, with MICs ranging from 0.125% to 1%. This performance was proven in both biodiesel samples and truck tank sludge, confirming its inhibitory action even under the most challenging conditions of mixed microbial contamination.
