Application of cell-free broth containing rhamnolipids in the remediation of soils contaminated by metallic species

Description

/ 12 “APPLICATION OF CELL-FREE BROTH CONTAINING RHAMNOLIPIDS IN THE REMEDIATION OF SOILS CONTAMINATED BY METALLIC SPECIES” Field of invention

[001] The present invention describes obtaining cell-free broth containing rhamnolipid biosurfactant produced by Pseudomonas aeruginosa, a bacterium isolated from the Amazon region of Brazil, using agro-industrial waste as the sole source of carbon and nitrogen, as well as its application as a bioremediation agent for soils contaminated by metallic species. The innovation of the proposal lies in the bioremediation agent being ecologically friendly and economically viable, in addition to its production positively impacting the circular bioeconomy and solid waste management. This invention is situated in the field of biotechnology with an emphasis on the remediation of soils contaminated by potentially toxic metals. Fundamentals of the invention

[002] Contamination by toxic metals has been one of the most important environmental pollution problems, due to the high reactivity, bioaccumulation, biomagnification, and persistence of these metallic species in nature. Some metals, such as lead, mercury, cadmium, arsenic, and nickel, are frequently released into the environment as a consequence of the indiscriminate use of pesticides and industrial waste, and by environmental accidents associated with mining activity, such as acid mine drainage and tailings dam ruptures, as exemplified by the episodes that occurred in Mariana and Brumadinho in 2015 and 2019, respectively, both located in Minas Gerais, Brazil. Episodes like these, where contamination by toxic metals is the main cause, represent a significant risk not only to biodiversity and the quality of aquatic and terrestrial ecosystems, but also to human health. Petition 870260070577, dated 07 / 16 / 2026, page 4 / 18 2 / 12

[003] The scientific literature discusses numerous strategies for mitigating the impacts caused by heavy metal contamination. Among them, bioremediation has been gaining prominence as a green strategy that uses living organisms or their bioproducts to remove or neutralize pollutants from the environment. One of the most used bioproducts as a bioremediation agent is rhamnolipid, a class of biosurfactant produced mainly by the species Pseudomonas aeruginosa, a Gram-negative bacterium found in different environments.

[004] Rhamnolipids are amphipathic surfactant molecules, typically formed by one or two rhamnose units linked to one or two fatty acid chains (Cs to C14), with C10 being the most prevalent. These molecules are capable of reducing the surface and interfacial tensions of miscible and immiscible liquids, respectively, and producing nono- and macroemulsions. In addition, rhamnolipids are biodegradable, exhibit low toxicity when compared to chemical surfactants, and can be produced from natural resources using alternative substrate sources.

[005] The scientific literature describes that the use of agro-industrial waste as raw material for the production of rhamnolipids has presented numerous benefits, including reduced production costs, wide availability and accessibility, and being a renewable source. The waste generated from the agro-industrial processing of Amazonian fruits, particularly andiroba (Carapa guianensis Aubl.), are promising sources of carbon, nitrogen, and phosphorus for the growth and production of rhamnolipids by P. aeruginosa, being considered strategic in biotechnological processes of microbial fermentation and an environmentally responsible option for solid waste management.

[006] Andiroba, belonging to the Meliaceae family, is endemic to Brazil, occurring in the North and Northeast regions of the country, contributing positively to the national and international cultural, economic, and social landscape. However, the industrialization of the processing stages of andiroba Petition 870260070577, dated 07 / 16 / 2026, page 5 / 18 3 / 12 results in waste that is not utilized in quantities proportional to its generation, causing a loss of economic potential in the production chain of this Amazonian fruit and improper disposal in various ecosystems.

[007] The production of rhamnolipids using residual andiroba biomass is a promising field in biotechnology due to its innovative and sustainable nature. Rhamnolipids are normally produced via fermentative processes under aerobic conditions, where the rhamnolipid-producing bacteria oxidize the carbon source to obtain energy, producing rhamnolipids as byproducts or primary metabolites formed during microbial growth. The first step in extracting rhamnolipids involves centrifugation and / or filtration processes to obtain cell-free broth, a solution rich in rhamnolipids and other molecules.

[008] The surfactant properties of rhamnolipids are described in the scientific literature as essential to numerous industrial applications, including bioremediation. In the bioremediation of heavy metals, rhamnolipids play an important role as a chelating agent, a crucial property in the removal of toxic metals from various surfaces. Furthermore, rhamnolipids have a high affinity for heavy metals, mainly cadmium, lead, and mercury, which makes their use in bioremediation even more promising. Rhamnolipids can bind to toxic metals, forming stable complexes that facilitate their removal from the environment. Unlike synthetic chemical chelating agents, rhamnolipids are biodegradable and can be decomposed by microorganisms in the environment, reducing the risk of environmental impact.

[009] However, the cost of producing rhamnolipids on an industrial scale is still quite high, mainly due to the raw materials used in production and the extraction process, which can represent up to 70% of the final product cost. Therefore, the present invention stands out for reducing the production costs of rhamnolipids by using residual andiroba biomass as raw material. Petition 870260070577, dated 07 / 16 / 2026, page 6 / 18 4 / 12 prima, and by employing cell-free broth containing rhamnolipids (CLCR) as a bioremediation agent for environments contaminated by metallic species. CLCR exhibits physicochemical properties similar to isolated rhamnolipids, but with the advantage of not requiring a purification step, which also positively impacts the cost of the final product and the yield of active compound. Related Technique

[010] Researchers have dedicated efforts to the study and development of technologies for the remediation of heavy metal pollution. These technologies encompass physical, chemical, and biological methods, the latter highlighting bioremediation. Although chemical adsorbents have contributed to the removal of metals, they are often inadequate due to secondary pollution and the high cost of the process. On the other hand, the bioremediation process with biosurfactants has been widely recommended.

[011] The invention BR102022004611A2 relates to a biosurfactant produced by yeast using industrial waste. The biosurfactant was characterized as an anionic and polymeric molecule, composed of carbohydrates, lipids, and proteins. This surfactant is described as having high emulsifying capacity, stability over a wide pH range (2-12), temperature (0, 5, 70, 100, and 120 °C), and salinity (2-12), in addition to being biodegradable, non-toxic, and having the ability to remove heavy metals such as Fe, Zn, and Pb from contaminated soil and effluents under dynamic conditions. Tests in packed columns also confirmed the biosurfactant's ability to remove Fe, Zn, and Pb under static conditions.

[012] Invention CN106077056A describes a method for bioremediating soils contaminated with heavy metals in mining areas. The method comprises the following steps: 1) spreading sufficiently composted manure and calcium superphosphate on the soil surface. Petition 870260070577, dated 07 / 16 / 2026, page 7 / 18 5 / 12 contaminated with heavy metals in mining areas and spraying a rhamnolipid biosurfactant at a mass concentration of 1.5%; 2) plowing, leveling, and irrigating the soil contaminated with heavy metals in the mining areas; 3) coating alfalfa seeds with a microbial inoculum containing Rhizobium meliloti; 4) coating the alfalfa seeds coated with the microbial inoculum; 5) performing low-temperature treatment on Leymus chinensis seeds; 6) mixing the coated alfalfa seeds and the L. chinensis seeds subjected to low-temperature treatment and sowing the mixed seeds in the soil contaminated with heavy metals in the mining areas; and 7) performing field management after seedling emergence. The invention describes that by using the bioremediation method, the soil contaminated with heavy metals in the mining areas could be effectively adsorbed and the residual heavy metals in the soil could be reduced.The method is characterized by high remediation speed, good effectiveness, simplicity of operation, and high treatment capacity.

[013] Invention CN107365585A relates to an environmentally friendly method for treating cadmium-contaminated soil. The eluent preparation method specifically comprises the following steps: preparing 10-30 parts of a natural chelating agent with a mass concentration of 1-5%, 20-40 parts of a biosurfactant with a mass concentration of 0.5-5%, and 0.1-1 part of a pH regulator; and uniformly mixing the solutions. Then, diluting with distilled water to 100 parts is performed, thus obtaining the eluent for treating cadmium-contaminated soil, with a pH value between 6-8. The invention describes that the eluent used for treating cadmium-contaminated soil is simple, low-cost, and the raw materials are non-toxic, easily degradable, and poses no risk of causing secondary soil pollution.

[014] Invention CN109135756A relates to an eluent agent for soil contaminated with heavy metals and methods for eluting and recycling the same. The eluent agent is a mixed aqueous solution of organic acid and alkyl glycoside biosurfactant. The concentration of the organic acid in Petition 870260070577, dated 07 / 16 / 2026, p. 8 / 18 The aqueous mixed solution of 6 / 12 has a concentration of 0.05-0.1 mol / L, and the alkyl glycoside concentration is 0.2-8%. The raw materials used by the eluting agent are low-cost and environmentally friendly. The invention describes that the elution method is simple and easy to perform, the eluted soil can be directly filled, and the eluting agent can be recycled several times.

[015] The present invention differs from those found in the prior art search in that none mentions the use of cell-free broth containing rhamnolipid biosurfactant (CLCR) produced by P. aeruginosa, using residual biomass of andiroba (Carapa guianensis Aubl.) as raw material, to act as a bioremediation agent for soils contaminated by metallic species.

[016] A particularity of the present invention is the fact that CLCR presents financial and environmental advantages, as it is produced from accessible and abundant renewable natural resources - prioritizing the use of those that make up the Brazilian Amazon region (bacteria and andiroba residue) -, and by dispensing with extraction and purification steps. Detailed description of the invention

[017] Although the present invention is described herein, by way of example, using terms such as “including”, “comprising”, “having”, “contemplating”, “containing”, “encompassing” and variations thereof, these are understood to be synonymous for applicable legal purposes, intended to be broad and encompass the subject matter listed below and additional matters not mentioned, without excluding other additives, components and / or steps. Additionally, the term “may” is used in a permissive sense (i.e., meaning to have the potential to), rather than the obligatory sense (i.e., meaning must), unless otherwise indicated.

[018] Any discussion of documents, acts, materials, devices, articles and the like is included in the descriptive report only for the purpose of providing context for the present invention. Petition 870260070577, dated 07 / 16 / 2026, page 9 / 18 7 / 12 It is not suggested or represented that any or all of these matters form part of the basis of prior art or are of general common knowledge in the field relevant to the present invention, unless otherwise indicated.

[019] The present invention utilizes CLCR produced by the bacterium P. aeruginosa, using residual biomass of andiroba (Carapa guianensis Aubl.) as raw material, as a bioremediation agent for soils contaminated by metallic species. In this regard, before explaining in detail the object of the invention, it should be understood that the invention is not limited in its application to the details of the set of rules and arrangements of the various models set forth below.

[020] The rhamnolipid biosurfactant-producing strain of Psuedomonas aeruginosa BM02 was isolated from a soil sample from a mining operation in the state of Pará, in the Amazon region of Brazil. The genetic sequence of the bacterial strain was deposited in the NCBI, under the GenBank registration: OP410927.1. The use of the bacterium was registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen, No. A4DA401). The bacterium is preserved in a glycerol solution at -20 °C in the Bioassays and Bioprocesses Laboratory (L@eio) of the Federal University of Southern and Southeastern Pará (Unifesspa), Marabá-PA, Brazil.

[021] Andiroba residue was chosen as raw material for the production of rhamnolipid biosurfactant due to its oily composition, high safety, low cost, high availability and because it is the result of the commercial processing of a fruit that is part of the scope of traditional uses by Amazonian communities, enabling avenues for the commercial competitiveness of the present invention.

[022] Alternatively or additionally, one or more parts of the plant (organ, tissue, system, among others) or its residues may be used, separately or as a whole. Non-limiting examples include derivatives of these plants, including their exudates or processed products, such as essential oils, plant extracts, fractionated, purified and / or their isolated constituents, separately. Petition 870260070577, dated 07 / 16 / 2026, page 10 / 18 8 / 12 or a combination of both.

[023] The process of obtaining CLCR begins with the reactivation of the bacterial strain, transferring 100 μL of the preserved culture to Petri dishes containing Luria Bertani (LB) medium with 1.5% agar, previously sterilized at 121 °C for 20 minutes, and incubated in an oven at 35 °C for 18 to 24 hours.

[024] The reactivated bacteria proceed to the pre-inoculum stage, transferring 3 to 5 bacterial colonies, using a bacteriological loop, to 125 mL Erlenmeyer flasks containing 25 mL of LB broth previously sterilized at 121 °C for 20 minutes, and incubated in an orbital shaker at 36 °C, 200 rpm, for a period ranging from 12 to 16 hours.

[025] The bacterial inoculum is obtained by transferring 2% of the pre-inoculum to 125 mL Erlenmeyer flasks containing 25 mL of previously sterilized LB broth (121 °C for 20 minutes), and incubating in a shaker at 36 °C, 200 rpm, for 1 to 2 hours, or until reaching an optical density (600 nm) between 0.6-0.8.

[026] The CLCR was produced in 250 mL Erlenmeyer flasks containing 50 mL of vegetable saline medium (VSM), with the following composition in g / L: 0.15 g of MgSO4.7H2U; 0.35 g of (NH4)2SO4; and 2.5% of residual andiroba biomass. Then, 4% of the bacterial inoculum was transferred to the flasks, which were incubated at 36 °C, 180 rpm, for 10 days. After the incubation period, the flasks were centrifuged at 4500 rpm for 15 minutes at 25 °C. The CLCR (phase of interest) was separated from the precipitate (cell biomass, other biological compounds, salts and residue byproducts) and sterilized by filtration (gram weight 85 g / m2) for testing.

[027] The rhamnolipid biosurfactant was characterized using Fourier transform infrared spectroscopy (Fox-transformed infrared spectroscopy, FT-IR), with a Platinum ATR reflectance accessory. The spectrum was analyzed in the 4000 - 400 cm-1 region and processed in the OriginPro 8.0 program; and by mass spectrometry with ionization by Petition 870260070577, dated 07 / 16 / 2026, p. 11 / 18 9 / 12 ultra-high resolution and precision electrospray spectroscopy (ESI-MS). By FT-IR, important absorption bands of rhamnolipids were observed, located at 3355, 2921, 2852, 1710, and 1024 cm⁻¹. The prevalent ion observed at 479 m / z in the ESI-MS analysis indicates that the probable composition of the biosurfactant is formed by a di-rhamnolipid (two rhamnose units) linked to a C10 fatty acid chain.

[028] The structure of the rhamnolipid functional groups present in CLCR was determined by coupled attenuated total reflectance FT-IR spectra. Spectra were also recorded in the 400-4000 cm-1 range. According to the FT-IR analysis, the absorption bands located at 3310, 2928, 2847 and 1089 cm-1 in CLCR are consistent with the rhamnolipid functional groups, corroborating the results of the isolated rhamnolipid biosurfactant.

[029] The concentration of rhamnose, an integral part of the rhamnolipid molecule, present in CLCR was determined by the orcinol method. The standard curve was calculated using L-rhamnose solutions at different concentrations (1 to 200 μg / mL). The results showed that the maximum rhamnose value was 0.37 g / L.

[030] Batch experiments were conducted to compare the ability of CLCR to adsorb Ni2+, Cr2+, and Cd2+ ions from contaminated soil. For the preliminary removal experiments, 10 mL of CLCR were placed in contact with 5 mg of soil contaminated with different salts of the metal ions. The system was kept under agitation at 50 rpm for 180 minutes in a separation column. At the end of the experiment, the exhaust valve of the separation column was opened, the soil was separated from the CLCR and reserved for metal quantification analysis.

[031] After the decontamination process, the soil samples were oven-dried for 72 hours at 60 °C, weighed, and sieved. To quantify the metals in the control and decontaminated soil samples, the soils were subjected to a digestion process with concentrated nitric acid and 30% hydrogen peroxide at 120 °C. The concentrations of heavy metals in Petition 870260070577, dated 07 / 16 / 2026, page 12 / 18 10 / 12 extracts obtained were analyzed using inductively coupled plasma mass spectrometry (ICP-MS). CLCR removed approximately 97% of Cd2+ metal species, 82% of Ni2+, and 68% of Cr2+ through chemical chelation.

[032] To evaluate the metal removal capacity of CLCR, several parameters were examined, including the solid-liquid ratio (1-10 g / mL), initial metal concentration (1-25 mg / kg), and contact time (30-180 minutes). Isothermal and kinetic data were collected simultaneously to elucidate the physicochemical parameters governing the adsorption process. With this methodology, it was observed that the best solid-liquid ratio was 1 g / mL, that is, for every 1 gram of soil, 1 mL of CLCR is needed for washing. It was observed that the best treatment occurred at a concentration of 25 mg / kg of metals and with 120 minutes of washing with CLCR under agitation of 50 rpm, removing 99% of Cd2+, 84% of Ni2+, and 62% of Cr2+.

[033] According to the FT-IR analysis, it was possible to observe, in addition to the composition of CLCR, the process of metal removal from the soil. The absorption bands located at 3310, 2928, 2847 and 1089 cm-1 in CLCR are consistent with the functional groups of rhamnolipids. In CLCR, the broad absorption band at 3310 cm-1 is attributed to hydroxyl groups (OH bond elongation); the bands at 2928 and 2847 cm-1 result from CH elongation of aliphatic groups, such as CH, CH2 or CH3, in the glycosidic structure and fatty acids of CLCR; the absorption bands at 1633 cm-1 are characteristic of C=O ester bond elongation vibrations, possibly overlapping the C=O carboxylic acid bond band. The signal at 1444 cm-1 refers to the CH bending of aliphatic chains; The band at 1089 cm-1 is associated with COC elongation, characteristic of glycosidic structures.After washing the soil contaminated with CLCR, the resulting spectrum showed redshifts in the characteristic rhamnolipid bands, indicating the presence of metallic species in the CLCR and confirming the removal of metals from the soil. Petition 870260070577, dated 07 / 16 / 2026, page 13 / 18 / 12

[034] The present invention positively impacts the innovative and sustainable reuse of solid waste, by considering the use of an Amazonian plant residue as raw material for the production of a product (CLCR) with potential in the bioremediation of soils impacted with metals, contributing strategically to the circular bioeconomy and the conservation of Brazilian natural resources. Furthermore, the process for obtaining CLCR is free of potentially toxic organic solvents, making it an economically and ecologically sustainable product. The present invention and its processes are aligned with the Sustainable Development Goals foreseen in the 2030 Agenda, especially SDG 9 (Industry, innovation and infrastructure), SDG 12 (Responsible consumption and production) and SDG 15 (Life on land). Examples of embodiments of the invention

[035] The P. aeruginosa BM02 strain produced CLCR with a rhamnose concentration above 0.30 g / L (Table 1). The CLCR reduced the surface tensions between air and water and the interfacial tensions between oil and water, in addition to achieving an emulsifying capacity above 50%, remaining stable after 24 hours (Table 1). Furthermore, the properties shown in Table 1 presented an ideal condition on the 10th day, due to the stabilization of the CLCR composition. Table 1. Physicochemical properties and rhamnose dosage of rhamnolipid cell-free broth (CLCR) produced by Pseudomonas aeruginosa BMI2. Tension Index Tension Days / Control Surface Emulsification Interfacial Rhamnose (g / L) (%) (mN / m) (mN / m) Day 3 54.5 ± 0.5 29.12 ± 0.07 4.80 ± 0.06 0.09 ± 0.01 Day 8 63.5 ± 1.0 31.20 ± 0.05 5.15 ± 0.09 0.31 ± 0.03 Petition 870260070577, dated 07 / 16 / 2026, page 14 / 18 12 / 12 10th day 64.0 ± 1.4 36.15 ±0.11 5.20 ± 0.03 0.34 ± 0.04 18th day 64.5 ± 1.8 36.23 ± 0.09 5.25 ±0.01 0.34 ± 0.02 SDS at 65.1 ±0.5 - - - a: 1% sodium dodecyl sulfate was used as a positive control. Results were expressed as mean ± standard deviation of three replicates (n = 3).

[036] The metal removal capacity of RCFB was evaluated based on the following parameters: solid-liquid ratio (1-10 g / mL), initial metal concentration (1-25 mg / kg) and contact time (30-180 minutes). For each new condition tested, removal results were obtained from each optimized condition, and a new condition was tested based on the previous analysis, as shown in Table 2. Table 2. Metal removal by CLCR for each variable of conditions analyzed. Metal Removal under different conditions (%) Ratio (solid / liquid) (g / mL) Metal concentration (mg / kg) Washing time (min.) 1 4 6 8 10 1 5 10 20 25 30 60 90 120 180 Cd 100% 90% 80% 65% 60% 55% 65% 70% 90% 99% 85% 90% 95% 99% 100% Ni 87% 65% 59% 45% 40% 40% 45% 60% 75% 86% 79% 80% 82% 84% 84% Cr 62% 60% 50% 40% 38% 25% 40% 50% 59% 63% 50% 60% 62% 62% 62% Petition 870260070577, dated 07 / 16 / 2026, p. 15 / 18

Claims

1 / 3 CLAIMS 1. APPLICATION OF CELL-FREE BROTH CONTAINING RHAMNOLIPIDS IN THE REMEDIATION OF SOILS CONTAMINATED BY METALLIC SPECIES, characterized by the use of cell-free rhamnolipid broth obtained from natural Amazonian sources as a bioremediation agent for soils contaminated by metallic species.

2. APPLICATION OF CELL-FREE BROTH CONTAINING RHAMNOLIPIDS IN THE REMEDIATION OF SOILS CONTAMINATED BY METALLIC SPECIES, according to claim 1, characterized by including the bacterium of the species Pseudomonas aeruginosa, isolated from mining soil in the Amazon region, which produces a biosurfactant of the glycolipid class, of the rhamnolipid type.

3. APPLICATION OF CELL-FREE BROTH CONTAINING RHAMNOLIPIDS IN THE REMEDIATION OF SOILS CONTAMINATED BY METALLIC SPECIES, according to claim 1, characterized by the fact that it uses residual biomass of andiroba (Carapa guianensis Aubl.) as the sole source of carbon and nitrogen in the cell-free broth production process.

4. APPLICATION OF CELL-FREE BROTH CONTAINING RHAMNOLIPIDS IN THE REMEDIATION OF SOILS CONTAMINATED BY METALLIC SPECIES, according to claim 3, characterized by the use of the plant species selected in the present invention, whether a part (organ, tissue, system, among others) of the plant separately or the whole plant, as well as its combination with other plants, non-limiting examples include Petition 870240101176, dated 11 / 27 / 2024, page 21 / 24 2 / 3 derivatives of these plants or their total or partial processing, such as residues, oils (essential or not), extracts, fractionated, purified and / or their isolated constituents, separately or in combination thereof.

5. APPLICATION OF CELL-FREE BROTH CONTAINING RHAMNOLIPIDS IN THE REMEDIATION OF SOILS CONTAMINATED BY METALLIC SPECIES, according to claim 1, characterized in that the rhamnolipid cell-free broth acts as a chelating agent for metallic species, preferably in soils.

6. APPLICATION OF CELL-FREE BROTH CONTAINING RHAMNOLIPIDS IN THE REMEDIATION OF SOILS CONTAMINATED BY METALLIC SPECIES, according to claim 5, characterized in that the metallic species are cadmium, nickel and chromium, but not limited to this group of metals, encompassing the use of the rhamnolipid cell-free broth for other metallic species.

7. APPLICATION OF CELL-FREE BROTH CONTAINING RHAMNOLIPIDS IN THE REMEDIATION OF SOILS CONTAMINATED BY METALLIC SPECIES, according to claim 6, characterized by the fact that the metals cadmium, nickel and chromium present in the contaminated soils are in concentration ranges varying from 1 mg / kg to 25 mg / kg, the most effective treatment being, but not limited to, the concentration of 25 mg / kg.

8. APPLICATION OF CELL-FREE BROTH CONTAINING RHAMNOLIPIDS IN THE REMEDIATION OF SOILS CONTAMINATED BY METALLIC SPECIES, according to claim 5, characterized by Petition 870240101176, dated 11 / 27 / 2024, page 22 / 24 3 / 3, in the fact that the ratio of cell-free rhamnolipid broth and soil contaminated by metallic species is described as between 1 g:mL and 10 g:mL, with the most effective treatment, but not limited to this, being at a ratio of 1 g:mL.

9. APPLICATION OF CELL-FREE BROTH CONTAINING RHAMNOLIPIDS IN THE REMEDIATION OF SOILS CONTAMINATED BY METALLIC SPECIES, according to claim 8, characterized by the fact that washing the soil contaminated by metallic species with the cell-free rhamnolipid broth occurs in a period of 30 to 180 minutes under agitation at 50 rpm, with the most effective treatment, but not limited to this, occurring in 120 minutes.

10. APPLICATION OF CELL-FREE BROTH CONTAINING RHAMNOLIPIDS IN THE REMEDIATION OF SOILS CONTAMINATED BY METALLIC SPECIES, according to claim 1, characterized by the fact that liquid forms of the ready-to-use formulation and / or its solid or semi-solid forms are preferred for commercialization; non-limiting examples include the administration of the cell-free rhamnolipid broth, free or incorporated into inert substances and adjuvants, in the form of liquids, solids, semi-solids, powders, sprays or the like, as well as incorporated into or integrating parts or all of products, processes, applications or the like, with purposes analogous to or the same as those described in this invention. Petition 870240101176, dated 11 / 27 / 2024, pp. 23 / 24