Production and characterization of rhamnolipids by Pseudomonas aeruginosa isolated in the Amazon region, and potential antiviral, antitumor, and antimicrobial activity

Biosurfactants encompass structurally and chemically diverse molecules with surface active properties, and a broad industrial deployment, including pharmaceuticals. The interest is growing mainly for the low toxicity, biodegradability, and production from renewable sources. In this work, the optimized biosurfactant production by Pseudomonas aeruginosa BM02, isolated from the soil of a mining area in the Brazilian Amazon region was assessed, in addition to its antiviral, antitumor, and antimicrobial activities. The optimal conditions for biosurfactant production were determined using a factorial design, which showed the best yield (2.28 mg/mL) at 25 °C, pH 5, and 1% glycerol. The biosurfactant obtained was characterized as a mixture of rhamnolipids with virucidal properties against Herpes Simplex Virus, Coronavirus, and Respiratory Syncytial Virus, in addition to antimicrobial properties against Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecium), at 50 µg/mL. The antitumor activity of BS (12.5 µg/mL) was also demonstrated, with potential selectivity in reducing the proliferation of breast tumor cells, after 1 min of exposure. These results demonstrate the importance of studying the interconnection between cultivation conditions and properties of industrially important compounds, such as rhamnolipid-type biosurfactant from P. aeruginosa BM02, a promising and sustainable alternative in the development of new antiviral, antitumor, and antimicrobial prototypes.

www.nature.com/scientificreports/according to the chemical composition and microbial origin, into glycolipids, lipopeptides, lipoproteins, fatty acids, neutral lipids, phospholipids, polymeric, and particulate surfactants 2 .Among them, glycolipids have been widely studied due to their excellent emulsification properties, especially rhamnolipids (RL) produced by the bacteria Pseudomonas aeruginosa 3 , one of the most studied bacterial producers of biosurfactants.This ubiquitous bacterium can be found in different environments and can also be advantageously used in biotechnological applications 4 .
BS has been an environmentally acceptable alternative for numerous applications, as it is produced from renewable resources, has low toxicity, and presents an attractive yield under extreme conditions of pH, temperature, and salinity.Furthermore, the value global BS (rhamnolipid) market was US$ 8.45 billion in 2022, with a projection growth of 5% (US$ 14.3 billion) by 2032 5 .However, the optimization of BS production can be a time-consuming process when individual factors are assessed separately 4 .To make BS production economically feasible, studies have been carried out to optimize the factors that influence its production, ensuring optimum conditions for microbial growth and BS yield 5 .Among the main concerns, the use of bacterial strains notoriously efficient in BS production and the characteristics of microbial cultivation, such as the composition of the culture medium, supply of carbon and nitrogen sources, balance of pH, and temperature, stand out 6 .Considering factorial design in BS production also ensures efficient performance, since factorial design is a statistical tool capable of establishing and quantitatively assessing the factors that are more relevant in the response of interest 7 .
Biosurfactant produced by P. aeruginosa have excellent physicochemical characteristics, in addition to presenting low toxicity, and high biodegradability and yield, compared with other bacterial BSs 8 .Several biological activities are described for BS produced by P. aeruginosa, including antiviral, antitumor, and antimicrobial activities 2,9 , suggesting its indication as potential prophylactic and/or therapeutic adjuvant.The discovery of new, safe, and effective broad-spectrum antitumor and antimicrobial/antiviral agents is essential.In this context, biosurfactants are promising alternatives in developing these agents as prophylactic and/or therapeutic.The present work assessed the optimized production of biosurfactant obtained by bacteria isolated from the soil of a mining area in the Amazon region (Brazil), in addition to investigating its antiviral, antitumor, and antimicrobial activities.Brazil is one of the largest ore producers in the world, mainly iron and bauxite, with the Eastern Amazon and specifically the state of Pará being home to the largest deposits of these ores 10 .Therefore, the discovery and characterization of a new molecule from a mining area is very promising due to the influence of the environment on the microorganism physiology, in addition to expanding the literature with relevant pharmacological compounds, considering the limitations in available therapies.

Cell growth and emulsification production
During the incubation period, the bacterial growth was spectrophotometrically monitored (Bel UV-M51; Ab 600 nm ).Aliquots were taken at time zero and every 24 h of incubation, and centrifuged (4000 rpm, 4 °C, 20 min) to obtain a cell-free broth.The emulsification index (IE24) was performed in tubes containing 2 mL of cell-free broth and 2 mL of mineral oil (Nujol).This mixture was vortexed at a high speed for two minutes and kept at rest for 24 h at 25 °C.The IE24 was calculated using the following formula: IE24 (%) = x/y × 100, where x and y represent the www.nature.com/scientificreports/heights of the emulsified layer and the total layer of mixture, respectively 15 .The negative control was made with uninoculated MSM, and the positive control, with 1% sodium dodecyl sulfate (SDS).

BS quantification
After the incubation period, the microbial culture was centrifuged (4000 rpm, 4 °C, 20 min) and the cell-free broth was acidified to pH 2.0 using hydrochloric acid solution (6 N) and then kept at 4 °C overnight.The precipitate was centrifuged (4000 rpm, 4 °C, 10 min), resuspended with chloroform-ethanol (2:1, v/v) solution, vigorously mixed, and then kept at rest (24 h) in a phase separation flask.This procedure was repeated twice more.Afterward, the organic phase was evaporated in a rotary evaporator (Quimis, Brazil) to obtain crude BS, followed by drying in a circulated air oven to constant weight 16 .

Stability analysis
Crude BS was dissolved in ultra-pure water (1 g/L) and submitted to analysis under the following conditions: temperature (10-100 °C), pH (3-11), and NaCl concentration (2-10%).The surface tension was assessed using a tensiometer (DataPhysics, Oca15 plus, Germany), by the hanging drop method, using an automatic video imaging system and program Oca 10/Oca 20.

Structural determination
The chemical structure of BS was determined by Fourier-Transform Infrared Spectroscopy (FT-IR), Nuclear Magnetic Resonance (NMR), and Electrospray Ionization Mass Spectrometry (ESI-MS).The FT-IR (Bruker FT-IR spectrometer, model Vertex 70, Germany) spectra was recorded within the region 4000-400 cm −1 and analyzed using the OriginPro 9.0 program.One-dimensional NMR analysis was performed on a Bruker Spectrometer (Model Avance III 400 MHz, Germany), operating at 400 MHz for 1 H with a 5 mm probe at 25 °C.The experiment was carried out using the standard pulse programs of the NMR equipment and the spectral analysis was performed using the program MestreNova-6.0.The sample for NMR analysis was prepared from the crude BS at 12.5 mg/mL in CDCl 3 and the chemical shifts were reported in ppm, with the solvent peak as a standard.ESI-MS analysis was performed on a QTOF spectrometer: quadrupole and TOF system for ultra-high resolution and mass precision (Bruker Daltonics, Model Compact, Germany) with direct injection.The mass spectrum was obtained in positive ion mode and the full scan data ranged from 50 to 1500 m/z.The equipment conditions were: capillary voltage of 3.8 kV, gaseous phase of nitrogen, gas flow of 4.0 L/min, gas temperature of 200 °C, and ion energy of 5.0 eV.The sample for ESI-MS analysis was prepared from the crude BS, dissolved in MS grade methanol at 0.01 mg/mL.
Vol:.( 1234567890 www.nature.com/scientificreports/Clonogenic assay (CL) MCF-7 cells were seeded in a 24-well plate (5 × 10 4 cells/well).After 24 h, treatments with BS (1-10 µg/mL), NC (phosphate buffered saline), and PC (DT50, 50 µM) were added to the cultures for 72 h.Then, the cells were trypsinized and counted, and 250 viable cells were seeded in 6-well plates, kept immobile for 14 days.For staining, the plates were washed with NC and fixative solution (ethanol/acetone, 1:2, v/v) for 10 min, followed by staining with 5% Giemsa (Dinamica, Brazil).Then, the plates were washed with distilled water and photographed, and the colonies were counted using the software Zen 2.3 (Zeiss, Germany).The survival fraction of the treated cells was obtained in relation to the NC (100% survival fraction) 23 .

Antiviral activity of BS
The BS effect was assessed against enveloped (Herpes Simplex Virus Type 1: HSV-1, KOS strain; Murine Coronavirus Type 3: MHV-3; Respiratory Syncytial Virus A: RSV, strain Long), and non-enveloped (Poliovirus: PV-1, strain Sabin) viruses, kept in a biobank at − 80 °C, in the Virology Laboratory of State University of Londrina, Londrina-PR, Brazil.Previously, the viral stocks were obtained by inoculating Vero (HSV-1 and PV-1), L-929 (MHV-3), or HEp-2 (RSV) cells, grown in culture flasks 25 cm 2 .The cells were monitored daily for 5 to 10 days to observe the cytopathic effect (CPE).Viral titration was carried out using the TCDI 50 technique 24 .Then, the viruses were incubated (1, 15, 30 and 60 min) with BS (500-6.25 µg/mL).Afterward, an aliquot of each suspension was diluted 10 or 100 times for inoculation in the respective cells (MOI 0.1), grown in 96-well microplates (10 4 cells/mL), and at the non-cytotoxic dose.The plates were incubated (37 °C, 5% CO 2 ) with daily observation of the presence or absence of CPE, for 72 h.The virus control (VC; infected and untreated cells) was performed simultaneously and under the same conditions, but without BS.The antiviral activity was determined by the reduction in viral titer concerning VC 25 .

Statistical analysis
The

Results
Alignment test analyses by Clustal Omega indicate 100% similarity of the BM02 sequence with other 16S rRNA sequences from Pseudomonas spp., including P. aeruginosa (Table S1).These analyses also suggest that all 16S rRNA sequences recovered from NCBI are close, indicating very recent divergence, with the BM02 showing high similarity (> 97%) with other Pseudomonas sequences.According to the phylogenetic analyzes, the BM02 sequence is closer to the P. aeruginosa DSM 50,071 and NBRC 12,689 sequences (Fig. S1).Pseudomonas aeruginosa NBRC 12,689 was determined for the rooting of the phylogenetic tree due to the close proximity of the BM02 obtained in the test of alignment by Clustal Omega (Fig. S1).
Factorial design analyses reveal variation in the BS production concerning the variables temperature (p = 0.0317) and glycerol content (p = 0.0408); only the pH variable was not significant (p = 0.0651) (Table S2).As described in Table 1, the factorial combinations of test 1 (pH 5.0, 25 °C, and 1.0% glycerol) promoted the highest yield in BS production (2.28 mg/mL), although the central point replicates (tests 9 and 10) also stands out in the BS production, with the average of the central point equivalent to 82% of the highest obtained value.
Pareto chart (Fig. 1D) analyzes reveal that the pH variation in the range studied did not influence the BS production.In turn, the temperature influenced the BS production (Fig. 1A, C), being the 25 °C was the most favored for BS production.The pH was not a significant variable alone, but the interactions of pH 5.0 and temperature of 25 °C (Fig. 1A), and pH 9.0 and 3.0% glycerol (Fig. 1B) contributed to the higher BS yield.
For purposes of follow-up of BS production by P. aeruginosa BM02, the IE24 and the optical density of tests (entry) 1 and 9 (tests with higher yield based on factorial design) were monitored.The IE24 resulted within the range of 54% to 60% (Fig. 2), SDS was 72% and uninoculated MSM did not form emulsified.The optical density (OD) varied from 0.70 to 2.0 in the exponential growth phase, between 24 and 48 h (Fig. 2).
The BS demonstrated stability in all tested conditions (Table 2), with the minimum value of surface tension (~ 27 mN/m) at pH 3.0, 2% NaCl and 80 °C.The assay control (uninoculated MSM) presented a surface tension  The 1 H NMR spectrometry (Fig. S3) analysis revealed signals with chemical shifts (δH) between 0.86 and 0.89 ppm, which are characteristic of the methyl groups in both the rhamnose moiety and the fatty acid.The signals between δH 1.36 to 2.63 ppm refer to the fatty acid chain hydrogens, and the rhamnose moiety hydrogens appear between δH 3.29 and 4.88 ppm.In addition, the three signals in the range of δH 4.85 to 5.42 ppm stand out, which show two rhamnose units in the structure and two bonded fatty acids, since the shift at 5.42 ppm is characteristic of CH that connects the fatty acids.There was no clear evidence of the presence of unsaturated fatty acids, as the spectrum did not show relevant signals with δH > 8.0 ppm.Thus, based on the attributions made by the FT-IR and NMR analyses, it can be stated that the BS consists of RL.The low resolution of the NMR spectrum can be an indication that the BS contemplates a mixture of RL congeners.
BS was submitted to mass spectrometry analysis to identify its lipid side chains and the structural homologs (Fig. S4).Table 3 shows the putative composition of the RL congeners of BS, consisting of a mixture of mono-RL (one rhamnose unit) and di-RL (two rhamnose units).Due to the analysis in positive mode, one RL was detected   in the H + form, while the others were mainly detected with the addition of Na + counter-ions.The obtained BS might be a complex mixture of these RL, emphasizing the mono-and di-RL containing two saturated C 10 fatty acids.
The cytotoxicity of BS on Vero, L-929 and HEp-2 cells were dose-dependent, with a reduction in viability proportional to the increase in concentrations, after 72 h of exposure (Fig. 3A), with CC 50 of 25 µg/mL, 27.25 µg/mL, and 20.16 µg/mL, respectively.Due to their well-established use in viral replication studies, Vero cells were used in determination trials of the antiviral and antitumor potential of BS.The BS concentration of 12.5 µg/mL kept the cells viable (> 88.8%) at all assessed times (Fig. 3B).A similar result was obtained only for the two shortest times (1 and 15 min) at 25 µg/mL, reducing the viability to 54.9% and 50% at 30 and 60 min of exposure, respectively.At 50 µg/mL, Vero cells maintained 63.2% cell viability in 1 min, reducing to less than 50% in the other assessed times.However, lower viability (CC 50 = 5.71 µg/mL) was found for MCF-7 cells after 72 h of exposure, and the LDH release assay was performed to assess whether BS alters plasma membrane integrity (Fig. 3C).All tested BS concentrations decreased MCF-7 cell viability (CC 50 = 4.43 µg/mL) to levels close to the standard chemotherapy (DT50).The SI of BS found for MCF-7 was 4.4, considered potentially selective for this tumor cell lineage.The antitumor activity of BS in MCF-7 was assessed (Fig. 3D), and after 72 h of treatment, the number of viable cells reduced by 37%, 29%, and 6%, at BS concentrations of 1.0, 5.0, and 10 µg/mL, respectively.Furthermore, BS induced cell death observed by the increase of cells in initial apoptosis, 35% and 37% at concentrations of 1.0 and 5.0 µg/mL, respectively; similar results were observed to the positive control of the assay (36.5%; data not shown).At the highest BS concentration (10 µg/mL), most cells (87%) were in terminal apoptosis.BS appears not to induce necrosis in MCF-7 cells, or weakly stimulate this cellular event, under assay conditions.
Analyses of the virucidal effects of BS highlight its ability to inhibit 96.4% of the CPE of all enveloped viruses (HSV-1, MHV-3 and RSV) evaluated, in 1 min contact, at 250 µg/mL (Fig. 5).At 50 µg/mL and same contact time, the absence of CPE (100%) was observed only for HSV-1, with a reduction to 71.6% and 77.8% for MHV-3 and RSV, respectively.In the other contact times (15, 30, and 60 min), the inhibition occurred for all enveloped viruses, from the BS concentration of 50 µg/mL.Based on BS virucidal effect results, the anti-HSV-1 activity of BS was also investigated, showing total CPE inhibition after 1 min of exposure, up to 12.5 µg/mL, without cytotoxicity.At BS concentration of 6.25 µg/mL, the absence of CPE depended on the exposure time, with 85% inhibition in 30 and 60 min, reduction to 68% in 15 min, and no inactivation when exposed to 1 min of contact.However, BS did not inactivate the Poliovirus (PV-1), a non-enveloped virus, at all tested conditions.
The antimicrobial potential of BS was also investigated, and the analyses suggest that Gram-positive bacteria (S. aureus and E. faecium) were more susceptible to BS.In contrast, Gram-negative bacteria (S. choleraesuis, E. coli and P. aeruginosa) and yeast (C.albicans) showed weak antimicrobial activity (Table 4).

Discussion
Pseudomonas aeruginosa is a common, Gram-negative environmental organism, known to exhibiting adaptable metabolism and ability to colonize a range of niches 26 .An important characteristic of P. aeruginosa, reported in the literature, is its ability to produce BS.However, its function for bacterial cells is still not fully understood 27 ; some physiological applications have been suggested, such as solubilization of insoluble substrates, adhesion to interfaces in natural ecosystems, and metal complexation, as a form of adaptation of microorganisms in metalrich environments, to reduce or inhibit cellular toxicity 28 .Bonds between metal and BS micelles can occur by attraction between anionic BS and non-ionic metallic forms, or by the formation of chelates on the surface of micelles 1 .In this work, the BM02 lineage was isolated from the soil of the bauxite mining industry, an aluminumrich environment that may have influenced the BS production.
Factorial design analyses revealed the production of BS by P. aeruginosa is influenced by pH and glycerol concentration, corroborating preliminary studies 29,30 , namely: moderately acidic pH (~ 6.0), and glycerol concentration between 0.5% and 3.0% are considered optimal.However, unlike our findings, Silva et al. 30 did not record differences in BS production by P. aeruginosa at temperatures up to 37 °C.These authors provided no information on the biological origin of the bacterial strain.In our study, the BM02 strain was isolated from the acid soil of a Brazilian municipality with temperatures around 28 ± 6 °C31 , a temperature range close to that considered optimal for BS production, according to factorial design analyses.These conditions may have favored the best BS yield, under the tested culture conditions.The carbon source in the culture medium is an essential element in the BS production 7 .However, it is essential to establish sustainable production routes, employing environmentally friendly bioprocesses and reagents, and glycerol is a highly available and low-cost option applied to green chemistry 32 .Glycerol was used as the only carbon source in this work, an alternative substrate commonly used in the production of microbial BS.The use of factorial design, allied to the use of industrial process wastes, is an important tool to optimize BS production conditions from independent variables, enabling the prediction of adequate parameters to achieve the best BS yield and correct disposal of glycerol residues 33 .However, the yield of BS produced by P. aeruginosa BM02 was considered low when compared to other studies, making it necessary to evaluate the production capacity of the bacteria species against different growth and synthesis factors.
The obtained BS showed the highest IE24 (60%) using 2% glycerol.The emulsified production remaining constant throughout the monitored period (120 h).Emulsion production is a widely used technique to determine the presence of BS in cell-free broth 34 , considering a satisfactory emulsifying agent when forming 50% emulsion, maintained for 24 h 35   applicability of BS in industry (e.g., medicine, cosmetic, bioremediation) 4 , and the presented results strengthen the potential of the obtained BS for industrial application.
The application of BS depends on its ability to remain stable under different conditions, considering variations in pH, temperature, and salinity 36 .Joshi et al. 37 reported that the BS was stable at a temperature of 80 °C, and variations in pH and NaCl between 6 and 12, and 1-7%, respectively; however, it increased its surface tension at NaCl concentrations greater than 10%, and at acidic pH, with precipitate formation.Gudiña et al. 36 observed that the surface activity of BS remained stable at temperatures from 25 to 60 °C, however, there was lower surface tension at pH 7, and the highest at pH below 6, showing turbidity and partial precipitation of BS.Giri et al. 38 showed minimum values of surface tension at pH 7 and 20 °C, with reduction of BS activity at temperatures higher than 100 °C.In this work, BS showed stability in all tested variables; there was no formation of precipitate or reduction of tensioactive activity.Some studies characterize the BS as primary metabolites associated with  www.nature.com/scientificreports/exponential bacterial growth 39 .In this work, it was not possible to determine in which phase of bacterial growth the BS was produced, enabling the performance of studies in this theme.
According to FT-IR and 1 H NMR analyses, the found signals are consistent with the functional groups of the RL described in the literature 3,27 .In this study, the putative composition of the RL congeners of BS was described as consisting of a mixture of mono-and di-RL.The most abundant P. aeruginosa RL reported in the literature contains fatty acids from C 8 to C 14 , considering that C 10 is the most common 40 .Furthermore, based on the Table 3 data, it can be observed that the peaks at m/z 499, 553, and 647 provide the possibility of RL congeners with different dispositions of the fatty acid chains.It should be pointed out that there was no quantification of each congener; therefore, some of them, especially the less common ones, may be present in minor quantities.This may explain the fact that the presence of unsaturated chain congeners was not evidenced by NMR analysis, despite the very weak signals in the 7.0-8.2ppm region, but was detected in mass spectrometry.Finally, the information provided by mass spectrometry revealed that there may be both mono-and di-rhamnose moieties, as well as mono-and di-lipid congeners.In addition, high diversity was observed in the length and the nature (saturated or unsaturated) of the lipid chains in relation to the most common chemical structures for the species.
The cytotoxic activity of BS was determined by the MTT assay, which is widely used to assess cellular metabolic activity 41 , evidencing selective toxicity for MCF-7 cells (CC 50 = 5.71 µg/mL), a breast cancer cell line, corroborating with studies on antitumor/anticancer potential of BS vary according to cell lines and exposure time 42,43 .Similar results were obtained by RL by P. aeruginosa BN10, with CC 50 = 8.6 µg/mL for MCF-7 44 .RL by P. aeruginosa B189 showed CC 50 = 6.25 µg/mL for MCF-7, but was not cytotoxic (CC 50 > 50 µg/mL) for Vero cells, and lung (NCI-H187), and oral epidermoid (KB) cell carcinomas 45 .The LDH release assay analyses suggest the cytotoxic safety of the obtained BS; all tested BS concentrations decreased MCF-7 cell viability (SI = 4.4) to levels close to the standard chemotherapy (DT50), similar to results reported by Tambone et al. 46 for RL by P. aeruginosa.The antitumor activity of RL-type BS was demonstrated for different cells 47,48 , strengthening the feasibility of indicating the obtained BS as an alternative to conventional chemotherapeutic agents.
In addition to compromising the membrane integrity, BS induced cell death by apoptosis and decreased the clonogenic capacity of MCF-7 cells, probably due to the amphiphilic nature of the molecule, similar to that reported in other studies 49 .Cytotoxic or cytostatic activity of BS seems to depend on interaction/penetration into the lipid bilayer influenced by the chain size and the micelle formation capacity 50 .However, the greater cytotoxicity found for MCF-7 cells does not rule out the hypothesis that our BS may interfere with other mechanisms, resulting in cancer cell apoptosis.The potentially selective and effective inhibition of MCF-7 cell proliferation, at low BS concentrations, are promising for anticancer therapies, considering the limited efficacy of most available therapeutic drugs.
BS showed virucidal effects against enveloped viruses HSV-1, MHV-3 and RSV after 1 min contact.The anti-HSV effect of BS has been widely investigated 51 ; however, the activity for RSV virus is still little reported.The inactivation of RSV shows promise in the investigation of BS antiviral properties in general therapy for respiratory viruses, where the use of chemical surfactants is indicated 52 .The virucidal effect of BS is attributed mainly to its physicochemical characteristics that favor interaction with the viral envelope lipid membrane.Many viruses of epidemiological importance have this structure, which is fundamental for the recognition of the host cell and the onset of the infectious cycle 51 .Molecules containing fatty acids are strongly suggested as inactivating enveloped viruses 53,54 , especially HSV-1, the most susceptible viral strain to BS.In the present study, BS presented RL containing units of C 10 saturated fatty acids in its composition, capable of interact physiochemically with the viruses, which may explain the viral inactivation.Additionally, several BS were assessed against coronaviruses, using SARS-CoV-2 or similar species 55 .In this study, the murine coronavirus (MHV-3) was used as a model for assessment the virucidal effect of BS, as it belongs to the same genus of SARS-CoV-2 56 .The inactivation of MHV-3 after 1 min of contact makes BS a promise in the coronaviruses' management, contributing to world public health.However, BS did not inactivate the Poliovirus (PV-1), a non-enveloped virus, at all tested conditions.
The BS also has antimicrobial activity against pathogenic bacteria and yeast.Reports on the antimicrobial activity of BS by P. aeruginosa against Gram-positive and Gram-negative bacteria and yeast investigated in this research, as well as other microorganisms of industrial and medical importance, have already been described in the literature 57,58 .Gram-positive bacteria usually are more susceptible to BS 59 , whose inhibition mechanism Table 4. Minimum inhibitory concentration (MIC, µg/mL) of the biosurfactant produced by P. aeruginosa BM02 against pathogenic microbial strains.Tetracycline and fluconazole were used as positive controls for bacterial and fungal strains, respectively.The tested compounds were added at different concentrations (50,000-25 µg/mL) to the microbial strains for 24 h for bacteria and 48 h for yeast.MIC was determined as the lowest concentration, which did not show any color.NI: There was no inhibition up to the highest tested concentration (50,000 µg/mL).NT: Not tested.www.nature.com/scientificreports/occurs through the insertion of fatty acid components (short acyl tails) into the bacterial cell membrane, causing a break between the cytoskeletal elements and the plasma membrane 57,58 .However, Gram-negative bacteria are normally more resistant to BS, possibly due to the presence of lipopolysaccharide and extracellular polymers in the outer membrane, which is little permeable to hydrophobic and amphipathic molecules 59 .The weak antifungal activity can be attributed to the BS composition, as suggested by Rodrigues et al. 60 ; compared to di-RL, mono-RL congeners interact weakly with the fungal phospholipid bilayer, and in our heterogeneous mixture of rhamnolipids, the ratio of mono-RL is higher.Surfactant activity due to the ability of their amphiphilic regions to interact with membrane structures, channel formation and significant conformational changes, has been proposed for biosurfactants 61 .In this study, we suggest that this non-specific mechanism is mainly responsible for the found biological activities.However, other mechanisms by which biosurfactants affect membrane integrity such as the property of adhesion to cell surfaces, ability to internalize the plasma membrane, rupture of the cytoskeleton, accumulation of intramembranous particles, increase in electrical conductance of the membrane, and induction of intracellular signaling pathways infections should not be ruled out 62,63 .New studies will contribute to elucidating the structure-activity relationships of this BS.

Conclusions
The possibilities of enhancing BS production by P. aeruginosa BM02 using media composition design and resulting BS properties were studied.The BM02 lineage was isolated in the Brazilian Amazon region, under the influence of the bauxite extraction, and identified as P. aeruginosa, a BS-producing bacterium RL-type under cultivation conditions using a temperature of 25 °C, pH 5 and 1.0% glycerol.The applicability of prediction was verified for media composition optimization with significant BS yield above 2.0 mg/mL and stability of surfactant properties for up to 120 h.The applications of BS include strong virucidal effect against enveloped viruses and selective antitumor activity for breast tumor cells, as well as inhibition of the growth of Gram-positive bacteria.
Our results indicate RL-type BS from P. aeruginosa BM02 as a promising compound for the development of new antitumor and antimicrobial prototypes, as a management strategy for important public health concerns.The Amazonian mining environment, particularly that of bauxite, is still little known in microbiological studies, but it may present itself as an important microbial reservoir with specific biological functions, as presented in this work.This study also enables discussions on the use of contaminated soil and mining waste, especially from the Brazilian Amazon region, the largest rainforest in the world, for the development of low-cost and ecologically friendly processes.
https://doi.org/10.1038/s41598-024-54828-wranging from 54.2 to 67.8 mN/m, according to the glycerol concentration (1% to 3%), and ultrapure water was of 72.45 mN/m.According to FT-IR analyses, the important absorption bands observed in BS were located at 3318, 2923, 2855, 1736, and 1036 cm −1 (Fig.S2).The broad absorption band at 3318 cm −1 is characteristic of hydroxyl group stretching vibrations.The bands at 2923 and 2855 cm −1 refer to the asymmetric and symmetric vibrations of the stretching of C-H bonds of sp 3 carbon, respectively.These signals may come from the long aliphatic chains of the RL.The peak with maximum absorption (1736 cm −1 ) is characteristic of the stretching vibration of the ester C=O bond, superimposed with the stretching band of the carboxylic acid carbonyl group (1707 cm −1 ).The absorption bands of 1300-1000 cm −1 are characteristic of stretching vibrations of C-O bonds, indicating the

Figure 3 .
Figure 3. Percentage of cell viability (A) after 72 h of exposure in Vero, L-929 and Hep-2 cells and (B) after exposure at different times (1-60 min) in Vero cells; and antitumor activity by (C) lactate dehydrogenase (LDH) release and (D) assessment of cell morphology of biosurfactant and antitumor activity by (C) lactate dehydrogenase (LDH) release and (D) assessment of cell morphology (MCF-7) of biosurfactant (BS) from Pseudomonas aeruginosa BM02.

Table 2 .
Stability of the biosurfactant by Pseudomonas aeruginosa BM02.Results of thre replicates are expressed as mean ± SD.

Table 3 .
Putative chemical composition of biosurfactant by Pseudomonas aeruginosa BM02.Rha, rhamnose; Fa, fatty acid; C8:x to C14:x: length of the fatty acids and presence of unsaturations.