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High production cost and potential pathogenicity of Pseudomonas aeruginosa , commonly used for rhamnolipid synthesis, have led to extensive research for safer producing strains and cost-effective production methods. This has resulted in numerous research publications claiming new non-pathogenic producing strains and novel production techniques many of which are unfortunately without proper characterisation of product and/or producing strain/s. Genes responsible for rhamnolipid production have only been confirmed in P. aeruginosa , Burkholderia thailandensis and Burkholderia pseudomallei . Comparing yields in different publications is also generally unreliable especially when different methodologies were used for rhamnolipid quantification. After reviewing the literature in this area, we strongly feel that numerous research outputs have insufficient evidence to support claims of rhamnolipid-producing strains and/or yields. We therefore recommend that standards should be set for reporting new rhamnolipid-producing strains and production yields. These should include (1) molecular and bioinformatic tools to fully characterise new microbial isolates and confirm the presence of the rhamnolipid rhl genes for all bacterial strains, (2) using gravimetric methods to quantify crude yields and (3) use of a calibrated method (high-performance liquid chromatography or ultra-performance liquid chromatography) for absolute quantitative yield determination.

In this work, the antifungal activity of rhamnolipids produced by Pseudomonas aeruginosa #112 was evaluated against Aspergillus niger MUM 92.13 and Aspergillus carbonarius MUM 05.18. It was demonstrated that the di-rhamnolipid congeners were responsible for the antifungal activity exhibited by the crude rhamnolipid mixture, whereas mono-rhamnolipids showed a weak inhibitory activity. Furthermore, in the presence of NaCl (from 375 mM to 875 mM), the antifungal activity of the crude rhamnolipid mixture and the purified di-rhamnolipids was considerably increased. Dynamic Light Scattering studies showed that the size of the structures formed by the rhamnolipids increased as the NaCl concentration increased, being this effect more pronounced in the case of di-rhamnolipids. These results were confirmed by Confocal Scanning Laser Microscopy, which revealed the formation of giant vesicle-like structures (in the µm range) by self-assembling of the crude rhamnolipid mixture in the presence of 875 mM NaCl. In the case of the purified mono- and di-rhamnolipids, spherical structures (also in the µm range) were observed at the same conditions. The results herein obtained demonstrated a direct relationship between the rhamnolipids antifungal activity and their aggregation behaviour, opening the possibility to improve their biological activities for application in different fields.

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.

In plants, cell-surface immune receptors sense molecular non–self-signatures. Lipid A of Gram-negative bacterial lipopolysaccharide is considered such a non–self-signature. The receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) mediates plant immune responses to Pseudomonas and Xanthomonas but not enterobacterial lipid A or lipopolysaccharide preparations. Here, we demonstrate that synthetic and bacterial lipopolysaccharide-copurified medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) metabolites elicit LORE-dependent immunity.The mc-3-OH-FAs are sensed in a chain length– and hydroxylation-specific manner, with free (R)-3-hydroxydecanoic acid [(R)-3-OH-C10:0] representing the strongest immuneelicitor. By contrast, bacterial compounds comprising mc-3-OH-acyl building blocks but devoid of free mc-3-OH-FAs—including lipid A or lipopolysaccharide, rhamnolipids, lipopeptides, and acyl-homoserine-lactones—do not trigger LORE-dependent responses. Hence, plants sense low-complexity bacterial metabolites to trigger immune responses.

Burkholderia thailandensis E264 is a rhamnolipid (RL)-producing gram-negative bacterium first isolated from the soils and stagnant waters of central and north-eastern Thailand. Growth of B. thailandensis E264 under two different incubation temperatures (25 and 30 °C) resulted in a significantly higher dry cell biomass production at 30 °C (7.71 g/l) than at 25 °C (4.75 g/l) after 264 h; however, incubation at the lower temperature resulted in consistently higher concentration of RL production throughout the growth period. After 264 h, the concentration of crude RL extract for the 25 °C culture was 2.79 g/l compared to 1.99 g/l for the 30 °C culture. Overall RL production concentration after 264 h was 0.258 g/g dry cell biomass (DCB) for the 30 °C culture compared to 0.587 g/g DCB for the 25 °C culture. Real-time PCR (qPCR) was also used to analyse expression of the RL biosynthesis genes throughout the incubation period at 25 °C showing that the expression of the rhlA , rhlB and rhlC genes is continuous. During the log and early stationary phases of growth, expression levels remain low and are increased upon entry to the late stationary phase. B. thailandensis E264 produces mostly di-RLs and the Di-RL C14-C14 in most abundance (41.88 %). Fermentations were also carried out in small-scale bioreactors (4 l working volume) under controlled conditions, and results showed that RL production was maintained. Our findings show that B. thailandensis E264 has excellent potential for industrial scale RL production.

Biosurfactant rhamnolipids have been claimed to show biological activities of inhibiting the proliferation of cancer cells. In this study, the cytotoxicity of rhamnolipids was examined on four cancer cells (HepG2, Caco-2, Hela, MCF-7 cells) and two normal cells (HK-2 cell, primary hepatocyte). Interestingly, both cancer cells and normal cells exhibited similar sensitivities to the addition of rhamnolipids in culture medium, and the cytotoxicity was largely attenuated by the presence of fetal bovine serum (FBS) in culture medium. In correlation of the mono-/di-rhamnolipid cytotoxicity with the surface tension of culture medium, it was found that rhamnolipids triggered cytotoxicity whenever the surface tension of culture medium decreased below 41 mN/m irrespective of the FBS content in culture medium, cell line, or rhamnolipid congener. Similarly, each chemical surfactant (Tween-80, sodium dodecyl sulfate, and sodium dodecyl benzene sulfonate) could cause cytotoxicity on HepG2 cells whenever its addition made the surface tension under 41 mN/m in culture medium with or without the presence of FBS. It seems that rhamnolipids, like chemical surfactants, exhibited cytotoxicity by reducing the surface tension of culture medium rather than by changing its specific molecular structure, which had no selection on tumor cells. This study could offer helps to correct the misleading biological activity of rhamnolipids and to avoid the possible large wastes of time and expenses on developing the applications in antitumor drugs.

Pseudomonas spp. are the main producers of rhamnolipids. These products have applications in pharmaceuticals, cosmetics, food industry and bioremediation. The biosynthesis of rhamnolipids is influenced by nutrient composition, pH and temperature. In this study, the impact of nutrients on the expression levels of rhamnolipid synthesis genes was evaluated in P. aeruginosa ATCC 15442. Glucose and glycerol were used as carbon sources; while, NaNO3, NH4NO3 and yeast extract/peptone were employed as nitrogen sources. The effect of different concentrations of Fe2+ and Fe3+ on rhamnolipid synthesis genes was also evaluated. Highest biosurfactant production was obtained in minimal medium supplemented with glucose, NaNO3 and Fe2+. Two rhamnolipid synthesis genes, rhlA and rhlB, were amplified with PCR. CapLC ESI–Ion trap-MS/MS detected only mono-rhamnolipid Rha–C10–C10 in the extract. Although similar induction levels were recorded in the presence of 0.05 g/L iron ions, the presence of Fe2+ resulted in higher expression levels than Fe3+ at concentrations equivalent to 0.025 and 0.075 g/L.

Background Rhamnolipids are the most extensively studied biosurfactants and has been successfully used in various areas from bioremediation to industrial fields. Rhamnolipids structural composition decide their physicochemical properties. Different physicochemical properties influence their application potential. Rhamnolipids can be produced at both aerobic conditions and anaerobic conditions by Pseudomonas aeruginosa . This study aims to evaluate the oxygen effects on the rhamnolipids yield, structural composition, physicochemical properties and the rhl -genes expression in P. aeruginosa SG. Results will guide researchers to regulate microbial cells to synthesize rhamnolipids with different activity according to diverse application requirements. Results Quantitative real-time PCR analysis revealed that rhlAB genes were down-regulated under anaerobic conditions. Therefore, strain P. aeruginosa SG anaerobically produced less rhamnolipids (0.68 g/L) than that (11.65 g/L) under aerobic conditions when grown in media containing glycerol and nitrate. HPLC–MS analysis showed that aerobically produced rhamnolipids mainly contained Rha-C 8 -C 10 , Rha–Rha-C 10 -C 12:1 and Rha–Rha-C 8 -C 10 ; anaerobically produced rhamnolipids mainly contained Rha-C 10 -C 12 and Rha-C 10 -C 10 . Anaerobically produced rhamnolipids contained more mono-rhamnolipids (94.7%) than that (54.8%) in aerobically produced rhamnolipids. rhlC gene was also down-regulated under anaerobic conditions, catalyzing less mono-rhamnolipids to form di-rhamnolipids. Aerobically produced rhamnolipids decreased air–water surface tension (ST) from 72.2 to 27.9 mN/m with critical micelle concentration (CMC) of 60 mg/L; anaerobically produced rhamnolipids reduced ST to 33.1 mN/m with CMC of 80 mg/L. Anaerobically produced rhamnolipids emulsified crude oil with EI 24  = 80.3%, and aerobically produced rhamnolipids emulsified crude oil with EI 24  = 62.3%. Both two rhamnolipids products retained surface activity (ST < 35.0 mN/m) and emulsifying activity (EI 24  > 60.0%) under temperatures (4–121 °C), pH values (4–10) and NaCl concentrations less than 90 g/L. Conclusions Oxygen affected the rhl -genes expression in P. aeruginosa , thus altering the rhamnolipids yield, structural composition and physicochemical properties. Rhamnolipids produced at aerobic or anaerobic conditions was structurally distinct. Two rhamnolipids products had different application potential in diverse biotechnologies. Although both rhamnolipids products were thermo-stable and halo-tolerant, aerobically produced rhamnolipids possessed better surface activity, implying its well wetting activity and desorption property; anaerobically produced rhamnolipids exhibited better emulsifying activity, indicating its applicability for enhanced oil recovery and bioremediation of petroleum pollution.

In the present research, magnetic rhamnolipid-Co/Al layered double hydroxide (MR-LDH) was synthesized to uptake methylene blue (MB) and reactive orange 16 (RO16) from aqueous solution. The main parameters, including pH, adsorbent dosage, contact time, and initial analyte concentration, were optimized to achieve the best adsorption efficiency. Accordingly, the elimination of MB on MR-LDH is improved in the basic medium due to the electrostatic interactions between the negative charge of MR-LDH and the positive charge of MB dye. In contrast, the acidic medium (pH = 3) was favored for RO16 adsorption because of hydrogen bonding between the protonated form of azo dye and protonated hydroxyl groups at the surface of MR-LDH. The calculated maximum adsorption capacities for MB and RO16 were 54.01 and 53.04 mg/g at 313 K, respectively. The Langmuir model, which assumes monolayer adsorption on the adsorbent surface, provides the best explanation for the adsorption of both dyes (R 2  = 0.9991 for MB and R 2  = 0.9969 for RO16). Moreover, the pseudo-second-order kinetic model best described the adsorption process for MB (R 2  = 0.9970) and RO16 (R 2  = 0.9941). The proposed adsorbent maintains stable adsorption performance for four consecutive cycles. After each adsorption process, MR-LDH is easily separated by an external magnet. The findings show that MR-LDH was found to be an excellent adsorbent for the removal of both cationic and anionic organic dyes from aqueous solutions.

To construct a derivative of the avirulent Pseudomonas aeruginosa ATCC 9027 that produces high levels of di-rhamnolipid, that has better physico-chemical characteristics for biotechnological applications than mono-rhamnolipid, which is the sole type produced by ATCC 9027. We used plasmids expressing the rhlC gene, which encodes for rhamnosyl transferase II that transforms mono- to di-rhamnolipids under different promoters and in combination with the gene coding for the RhlR quorum sensing regulator, or the mono-rhamnolipid biosynthetic rhlAB operon. The plasmids tested carrying the rhlC gene under the lac promoter were plasmid prhlC and prhlRC, while prhlAB-R–C expressed this gene from the rhlA promoter, forming part of the artificially constructed rhlAB-R–C operon. We measured rhamnolipds concentrations using the orcinol method and determined the proportion of mono-rhamnolipids and di-rhamnolipids by UPLC/MS/MS. We found that the expression of rhlC in P. aeruginosa ATCC 9027 caused the production of di-rhamnolipids and that the derivative carrying plasmid prhlAB-R–C gives the best results considering total rhamnolipids and a higher proportion of di-rhamnolipids. A P. aeruginosa ATCC 9027 derivative with increased di-rhamnolipids production was developed by expressing plasmid prhlAB-R–C, that produces similar rhamnolipids levels as PAO1 type-strain and presented a higher proportion of di-rhamnolipids than this type-strain.