Agro-Industrial Wastes as Potential Substrates for Rhamnolipid Production by Pseudomonas aeruginosa USM-AR2.

Rhamnolipid has gained much attention in various fields owing to its distinctive functional properties compared to conventional chemical surfactants, which are mostly derived from petroleum feedstock. Production cost is one of the main challenges in rhamnolipid production, particularly when using refined substrates. One possible solution is to use agro-industrial wastes as substrates for rhamnolipid production. This is a promising strategy due to their abundance and commercially low value, while simultaneously alleviating an agro-industrial waste management problem in the environment. This study aims to evaluate agro-industrial wastes from local crops as possible low-cost alternative substrates for rhamnolipid production by a local isolate, Pseudomonas aeruginosa USM-AR2. Various liquid wastes, namely sugarcane molasses, rice washing water, overly mature coconut (OMC) water, empty fruit bunch (EFB) steam effluent, palm sludge oil (PSO) and palm oil mill effluent (POME) were screened as the main carbon source supplementing mineral salt medium (MSM) in the fermentation of P. aeruginosa USM-AR2. Batch fermentation was carried out in a shake flask system, agitated at 200 rpm and incubated at room temperature, 27 ± 2°C for 120 h. Among the substrates tested, PSO exhibited the highest biomass at 20.78 g/L and rhamnolipid production at 1.07 g/L. This study has shown the potential of agro-industrial wastes in Malaysia as an alternative resource for rhamnolipid production, transforming them into value added products, while reducing the amount of wastes discharged into the environment.

Novel Insights into Cr(VI)-Induced Rhamnolipid Production and Gene Expression in Pseudomonas aeruginosa RW9 for Potential Bioremediation.

Rhamnolipid (RL) is renowned for its efficacy in bioremediating several types of organic and metal contaminants. Nevertheless, there has been a scarcity of studies specifically examining the relationship between this substance and metals, especially in terms of their impact on RL formation and the underlying interaction processes. This study addresses this gap by investigating the RL mechanism in Cr (VI) remediation and evaluating its effect on RL production in Pseudomonas aeruginosa RW9. In this study, P. aeruginosa RW9 was grown in the presence of 10 mg l-1 Cr (VI). We monitored RL yield, congeners distribution, and their ratios, as well as the transcriptional expression of the RL-encoded genes: rhlA, rhlB, and rhlC. Our results revealed that RL effectively reduced Cr (VI) to Cr (III), with RL yield increasing threefold, although with a slight delay in synthesis compared to control cells. Furthermore, Cr (VI) exposure induced the transcriptional expression of the targeted genes, leading to a significant increase in di-RL production. The findings confirm that Cr (VI) significantly impacts RL production, altering its structural compositions and enhancing the transcriptional expression of RL-encoded genes in P. aeruginosa RW9. This study represents a novel exploration of Cr (VI)'s influence on RL production, providing valuable insights into the biochemical pathways involved and supporting the potential of RL in Cr (VI) bioremediation.

Investigation of Photodynamic and Rhamnolipid Inhibition on the Dermatophyte Biofilm.

The failure to successfully treat dermatophyte-related diseases is often due to the formation of biofilms, which makes dermatophytes resistant to antifungals. Here, an attempt has been made to assess inhibition of dermatophyte biofilm production using photodynamic therapy and rhamnolipid biosurfactant. Two methods were used to inhibit biofilm formation by dermophytes Trichophyton mentagrophytes, Trichophyton rubrum and Trichophyton verrucosum, Microsporum canis and Microsporum gypseum. The first method was the use of rhamnolipid with concentrations of 39 to 1000 ppm and the second was the use of photodynamic method with concentrations of 8, 16 and 32 µg/ml of methylene blue. In addition, these two methods were evaluated simultaneously. The biofilm formation was evaluated using spectrophotometry and scanning electron microscopy. Biosurfactant has been shown to have an improved ability to inhibit the formation of biofilm by the strains. Although photodynamic therapy has not been successful, but in combination with biosurfactant, it may have a synergistic effect. By investigating the effect of rhamnolipid on the formation of biofilm, it was found that Microsporum species has a relatively stronger attachment to the surfaces of the wells compared to trichophyton species. The biofilms were evaluated with electron microscope in the simultaneous treatment of rhamnolipid and photodynamics. The results showed that after the treatment, the biofilms became discrete and their structural integrity was reduced. Even in Microsporum species, which were among the most resistant dermatophytes, the changes in the fungal biofilm after treatment were significant.

Effects of rhamnolipid replacement of chlortetracycline on growth performance, slaughtering traits, meat quality and antioxidant function in broilers.

BACKGROUND: Rhamnolipids (RLS) are surfactants that improve the growth performance of poultry by improving the absorption of nutrients. This study aims to investigate the effects of RLS replacement of chlortetracycline (CTC) on growth performance, slaughtering traits, meat quality, antioxidant function and nuclear-factor-E2-related factor 2 (Nrf2) signaling pathway in broilers. A total of 600 one-day-old Arbor Acres chicks were randomly assigned to five groups with eight replicates in each group, raised for 42 days. Broilers were respectively fed a basal diet with no CTC or RLS, 75 mg kg-1 CTC, and 250, 500, 1000 mg kg-1 RLS. RESULTS: Dietary supplementation with RLS linearly increased the average daily gain, average daily feed intake, carcass yield, eviscerated yield, ether extract, enhanced total superoxide and glutathione peroxidase (GPx) activities, overexpressed the relative expressions of Nrf2, heme oxygenase 1, Copper/zinc superoxide dismutase, Manganese superoxide dismutase, GPx and catalase and decreased the lightness value at 24 h, drip loss and malondialdehyde contents of broilers (P < 0.05). Compared with the control group, broilers fed 1000 mg kg-1 RLS reduced the drip loss and broilers fed 500 mg kg-1 RLS increased muscle crude fat content (P < 0.05). Compared with the CTC group, dietary supplementation with 1000 mg kg-1 RLS increased eviscerated yield (P < 0.05). CONCLUSION: RLS could improve growth performance, crude fat content, meat quality and antioxidant capacity and activate relative expression of genes in the Nrf2 signaling pathway in broilers. It could be used as an antibiotic substitute in diets and the recommended supplemental dose of RLS in feed of broilers is 1000 mg kg-1. © 2024 Society of Chemical Industry.

Molecular identification of rhamnolipids produced by Pseudomonas oryzihabitans during biodegradation of crude oil.

Introduction: The ability to produce biosurfactants plays a meaningful role in the bioavailability of crude oil hydrocarbons and the bioremediation efficiency of crude oil-degrading bacteria. This study aimed to characterize the produced biosurfactants by Pseudomonas oryzihabitans during the biodegradation of crude oil hydrocarbons. Methods: The biosurfactants were isolated and then characterized by Fourier transform infrared (FTIR), liquid chromatography-mass-spectrometry (LC-MS), and nuclear magnetic resonance spectroscopy (NMR) analyses. Results: The FTIR results revealed the existence of hydroxyl, carboxyl, and methoxyl groups in the isolated biosurfactants. Also, the LC-MS analysis demonstrated a main di-rhamnolipid (l-rhamnopyranosyll-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoate, Rha-Rha-C10-C10) along with a mono-rhamnolipid (l-rhamnopyranosyl-b-hydroxydecanoylb-hydroxydecanoate, Rha-C10-C10). In agreement with these findings, the NMR analysis confirmed the aromatic, carboxylic, methyl, sulfate moieties, and hexose sugar in the biosurfactants. The emulsion capacity of the biosurfactants decreased the surface tension of the aqueous system from 73.4 mN m-1 to around 33 mN m-1 at 200 mg L-1 as the critical micelle concentration. The emulsification capacity of the biosurfactants in the formation of a stable microemulsion for the diesel-water system at a wide range of pH (2-12), temperature (0-80°C), and salinity (2-20 g L-1 of NaCl) showed their potential use in oil recovery and bioremediation through the use of microbial enhancement. Discussion: This work showed the ability of Pseudomonas oryzihabitans NC392 cells to produce rhamnolipid molecules during the biodegradation process of crude oil hydrocarbons. These biosurfactants have potential in bioremediation studies as eco-friendly and biodegradable products, and their stability makes them optimal for areas with extreme conditions.

Adapting nature's own solution: The effect of rhamnolipid and lytic bacteriophage cocktail on enteric pathogens that proliferate in mucilage.

The mucilage event witnessed in the Sea of Marmara in 2021 has emerged as a prominent environmental concern, capturing public attention due to its detrimental effects on ecological, economic, and aesthetic dimensions. Addressing the multifaceted impacts of mucilage demands a nature-centric scientific approach, given its global ramifications spanning economy, public health, international relations, and tourism. Consequently, this study sought to explore alternative approaches for the removal of pathogenic enteric bacteria associated with mucilage occurrences, diverging from conventional methodologies. Specifically, the primary objective was to assess the efficacy of rhamnolipid and a bacteriophage cocktail in mitigating the proliferation of enteric pathogens within mucilaginous environments. During the study, 91 phage isolations were obtained from 45 water samples taken and 10 phages were selected for the broad host range and because of the efficacy tests, a phage cocktail was created with 5 phages. It was found that the mixture of rhamnolipid, phage cocktail and rhamnolipid-phage cocktail reduced bacterial load by 7-9 log10, 9-12 log10 and 9-11 log10 respectively under laboratory conditions. When the study was carried out in seawater, reductions of 4-5 log10, 3 log10 and 4 log10 were achieved. This study has shown that the combined use of rhamnolipid, phage cocktail and rhamnolipid-phage cocktail can be considered as the most effective natural solution proposal for reducing bacterial load, both in laboratory conditions and in sea surface water.

Biomimetic mineralization of hydrated magnesium carbonate for hydrogel reinforcement and heavy metal adsorption.

With excessive Mn(Ⅱ) and Cu(Ⅱ) pollution in aquatic environments posing potential health risks to inhabitants, the emergence of carbon capture, utilization and storage (CCUS) technology has promoted the improvement of heavy metal remediation technologies. Using hydrothermal sediment as a crystal seed, rhamnolipid was used to mediate biomimetic mineralization to prepare hydrated magnesium carbonate (HMC) composites to enhance the Mn(Ⅱ)/Cu(Ⅱ) adsorption performance of alginate hydrogels. Hydrothermal sediment is beneficial for accelerating biomimetic mineralization, while rhamnolipid can induce a crystalline phase transformation from dypingite to nesquehonite. The addition of sediment significantly enhanced the compressive mechanical properties and thermal stability of the hydrogels. The adsorption performances of the nesquehonite and dypingite hydrogels were better for Mn(II) and Cu(II), respectively. An increase in the amount of sediment improved the adsorption of Cu(II) by the hydrogels appropriately, resulting in stronger selectivity for Cu(II). The adsorption of Mn(II) and Cu(II) on the hydrogel beads was thermodynamically spontaneous. The inhibitory effects of sodium dodecyl benzene sulfonate (SDBS), fulvic acid (FA) and alginate on Cu(II) adsorption were more obvious than those of bovine serum albumin (BSA). Both the complexation of functional groups on alginate and mineralization by HMC participated in the adsorption of Mn(II) and Cu(II).

Antibiofilm and wound healing efficacy of rhamnolipid biosurfactant against pathogenic bacterium Staphylococcus aureus.

The present study evaluates the in-vitro antibiofilm activity against the biofilm formed by Staphylococcus aureus, and the wound-healing efficacy of two different types of rhamnolipids produced by Pseudomonas aeruginosa strain JS29 in S.aureus infected wounds. The biosurfactant production was carried out in a mineral salt medium supplemented with 2 % Glucose and 2 % Glycerol individually and thus were designated as RL-Glu and RL-Gly respectively. 0.5 mg/ml of RL-Glu and RL-Gly demonstrated 90 % growth inhibition of S. aureus while exhibiting bactericidal activity at 4 mg/ml of RL-Glu and 1 mg/ml of RL-Gly. Both types of rhamnolipid cause changes in membrane permeability leading to pathogens' non-viability. 90 % inhibition of biofilm formation by S. aureus was observed at 2 mg/ml of RL-Glu and 0.5 mg/ml of RL-Gly, while 0.5 mg/ml of both rhamnolipid disrupted 90 % of the preformed biofilm. 0.5 mg/ml of RL-Glu and RL-Gly decreases the production of exopolysaccharides and also causes structural alteration. 0.5 mg/ml of RL-Glu and RL-Gly were found to exhibit effective wound healing efficacy in S. aureus infected wounds within 7 days of treatment. Histopathological studies of wound sites revealed efficient wound management by both the rhamnolipid. LCMS and GCMS characterization of the biosurfactant revealed that JS29 produces different rhamnolipid congeners when grown on different carbon sources, thereby influencing the antimicrobial, antibiofilm, and wound healing efficacy of rhamnolipid.

Draft genome sequence of rhamnolipid-producing bacteria Thermoanaerobacter thermocopriae strain CM-CNRG TB177 isolated from an oil reservoir in Mexico.

The draft genome of Thermoanaerobacter thermocopriae CM-CNRG TB177 isolated from an oil reservoir in Mexico was determined and annotated. The organism is a thermophilic and strict anaerobe bacterium that produces rhamnolipids, using glucose as a carbon source. The predicted genome size is 2,496,169 bp and 2,550 genes.

Lipid lysination by MprF contributes to hemolytic pigment retention in group B Streptococcus.

Group B Streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis. A major virulence factor is a pigmented beta-haemolytic/cyto-lysin (ß-h/c) toxin with an ornithine rhamnolipid structure. We initially observed that absence of MprF enzyme altered pigmentation and haemolytic activity in GBS. Next, we showed that MprF-dependent lipid lysination contributes to the retention of the ornithine rhamnolipid within GBS membrane. Furthermore, cationic lipidation by MprF altered membrane properties contributing to resistance to the cyclic lipopeptide daptomycin and to acidic pH. This study highlights the importance of cationic lipids in cell envelope homeostasis and in modulating ß-h/c activity.

An integrated preventive and therapeutic magnetic nanoparticle loaded with rhamnolipid and vancomycin for combating subgingival biofilms.

OBJECTIVES: Mechanical debridement supplemented with antibacterial agents effectively eradicates subgingival biofilms formed in the periodontal pockets of severe periodontitis patients. However, the available antimicrobial agents have limited penetrating ability to kill the bacteria encased in the deep layers of biofilms. This study aimed to fabricate a novel magnetic nanoparticle (MNP) loaded with rhamnolipid (RL) and vancomycin (Vanc, Vanc/RL-Ag@Fe3O4) to combat subgingival biofilms. METHODS: The multispecies subgingival biofilm was formed by periodontal pathogens, including Streptococcus oralis (S. oralis), Streptococcus sanguinis (S. sanguinis), Actinomyces naeslundii (A. naeslundii), Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). Scanning electron microscope (SEM), confocal laser scanning microscopy (CLSM), and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the anti-biofilm efficacy of Vanc/RL-Ag@Fe3O4 with or without a magnetic field on multispecies subgingival biofilms. RESULTS: The minimal inhibitory concentration (MIC) values of Vanc/RL-Ag@Fe3O4 on S. oralis, S. sanguinis, A. naeslundii, P. gingivalis, and F. nucleatum were 25, 50, 100, 50, and 25 µg/mL, respectively. Vanc/RL-Ag@Fe3O4 (200 µg/mL) reduced the 7-d biofilm thickness from 22 to 13 µm by degrading extracellular polymeric substance (EPS) and killing most bacteria except for tolerant F. nucleatum. A magnetic field enhanced the anti-biofilm effect of Vanc/RL-Ag@Fe3O4 by facilitating its penetration into the bottom layers of biofilms and killing tolerant F. nucleatum. SIGNIFICANCE: Vanc/RL-Ag@Fe3O4 MNPs can release RL, Vanc, and Ag and eradicate subgingival biofilms by disrupting EPS and killing bacteria. Vanc/RL-Ag@Fe3O4 combined with a magnetic force is a promising approach for combating periodontal infection.

Optimizing Systems for Robust Heterologous Production of Biosurfactants Rhamnolipid and Lyso-Ornithine Lipid in Pseudomonas putida KT2440.

Surfactants are amphiphilic molecules that are capable of mixing water and oil. Biosurfactants are eco-friendly, low-toxicity, and stable to a variety of environmental factors. Optimizing conditions for microorganisms to produce biosurfactants can lead to improved production suitable for scaling up. In this study, we compared heterologous expression levels of the luminescence system luxCDABE operon controlled by regulatable promoters araC-PBAD and its strong version araC-PBAD-SD in Escherichia coli K12, Pseudomonas aeruginosa PAO1, and P. putida KT2440. Real-time monitoring of luminescence levels in the three strains indicated that luxCDABE controlled by araC-PBAD-SD promoter with 0.2% arabinose supplementation in P. putida produced the highest level of luminescence. By using the araC-PBAD-SD promoter-controlled rhlAB expression in P. putida, we were able to produce mono-rhamnolipid at a level of 1.5 g L-1 when 0.02% arabinose was supplemented. With the same system to express olsB, lyso-ornithine lipid was produced at a level of 10 mg L-1 when 0.2% arabinose was supplemented. To our knowledge, this is the first report about optimizing conditions for lyso-ornithine lipid production at a level up to 10 mg L-1. Taken together, our results demonstrate that regulatable araC-PBAD-SD promoter in P. putida KT2440 is a useful system for heterologous production of biosurfactants.

Study on the gelling properties of egg white/surfactant system by different heating intensities.

The aim of this study was to elucidate the different effects and difference mechanism of gelling properties among egg white (EW) treated with different heating intensities and the composite addition of rhamnolipid and soybean lecithin. Particle size analyzer, potentiometric analyzer, surface hydrophobicity method, and Fourier transform infrared spectroscopy techniques were used to determine the physicochemical properties and molecular structure, respectively. Low-field nuclear magnetic resonance, magnetic resonance imaging, texture profile analysis, and scanning electron microscopy techniques were used to analyze the gelling properties and gel structure, respectively. And we illuminate the different mechanisms in the gelling properties of the EW with various treatments and key internal factors that play important roles in improving gelling properties by establishing the link between the gelling properties and relevant characteristics by mixed effects model and visual network analysis. The results indicate raising the content of rhamnolipid decreased the migration of immobilized water in the EW gel and the free water content. At the heating intensities of 55 °C/3.5, 65 °C/2.5, and 67 °C/1.5 min, with an increase in rhamnolipid, the gel's cohesiveness, gumminess, and chewiness gradually increased. The mixed effects model indicated that heating intensities and composite ratios have a 2-way interaction on zeta potential, the relaxation time of bound water (T21), the content of bound water (P21), the content of immobilized water (P22), and fractal dimension (df) attributes (P < 0.05). The visual network analysis showed that the protein solubility, the relaxation time of immobilized water (T22), surface hydrophobicity, zeta potential, average particle size (d43) and the relaxation time of free water (T23) are critical contributors to the different gelling properties of EW subjected to various treatments and the improvement of gelling properties. This study will provide theoretical guidance for the development of egg white products and the expansion of egg white's application scope in the egg product processing industry.

Self-assembling properties of mono and di-rhamnolipids characterized using small-angle X-ray scattering.

Rhamnolipids are glycolipid surfactants composed by a hydrophilic head of either one (mono-RL) or two (di-RL) rhamnose moieties coupled to hydroxyaliphatic chains that can be of different lengths. In spite of their importance in different fields of applications, as bioremediation processes for instance, self-aggregation physico-chemical properties of RLs are not unique. This because a variety of aggregates morphologies (shape and size) can either exist or coexist in aqueous dispersion due to mono-RL:di-RL molar ratio, hydrophobic tails length, pH and the presence of co-surfactants and additives. Recently, a theorethical approach reported the self-assembling morphologies of either pure mono or di-RL in aqueous environment, predicting the formation of spherical to ellipsoidal micelles to worm-like and disk-like aggregates depending on RL concentration and fatty acid chain length. In order to add new information to those previously available, the present work investigated the self-assembling properties of mono-RL-C10-C10 and di-RL-C10-C10 separately in aqueous dispersion by small angle X-Ray scattering (SAXS). A novel approach was applied to the data analysis coupling the scattering length density profiles of the RLs chemical groups and Monte Carlo simulations. Such an approach allowed us to infer about the preferred mono-RL and di-RL conformations that fit better in the self-assembling morphologies. In this way, we show that mono-RL-C10-C10 self-assembles into lamella-like aggregates coexisting with 30 % of multi-lamella aggregates (circa of 5 closed stacked lamella) from a concentration ranging from 10 to 50 mM, with hydrophobic thickness of about 12 Å, a hydrated polar head thickness of 10 Å, and an area per glycolipid of 76 Å2. On the other hand, di-RL prefers to self-associate into flexible cylinder-like aggregates, from 70 mM to 110 mM concentration, with hydrophobic radius on the order of 7.5 Å, a hydrated polar shell of 21.5 Å, with hydropobic/polar interface of 110 Å2 per glycolipid. Interestingly, the parameters obtained from the best fitting to the experimental data associated to the volume fraction distribution of the chemical groups within the aggregates revealed that the hydrophobic chains are more disordered in mono-RL planar aggregates than in di-RL worm-like aggregates, as well as the hydration properties. Further, the addition of 100 mM NaCl in di-RL aqueous dispersion leads to the formation of longer worm-like aggregates. Taking together, this work opens a new avenue regarding characterization of biosurfactants self-assembling properties by using SAXS, also contributing to prepare more efficient biosurfactant dispersions depending on the desired applications in industrial sectors and bioremediation.

Production and characterization of rhamnolipid biosurfactant from thermophilic Geobacillus stearothermophilus bacterium isolated from Uhud mountain.

Introduction: Biosurfactants have been given considerable attention as they are potential candidates for several biotechnological applications. Materials and methods: In this study, a promising thermophilic biosurfactant-producing HA-2 was isolated from the volcanic and arid region of Uhud mountain, Madinah, Saudi Arabia. It was identified using 16S rRNA gene sequence analysis. The biosurfactant production ability was screened using different methods such as the drop collapse test, oil spreading test, hemolytic activity test, CTAB test, and emulsification index. The ability of rhamnolipid production by the tested strain was confirmed by the polymerase chain reaction (PCR) of rhlAB. The affinity of thermophilic HA-2 to hydrophobic substrates was also investigated. Optimization of biosurfactant production was conducted. The biological activities of produced surfactant were investigated. Results and discussion: The isolated HA-1 was identified as Geobacillus stearothermophilus strain OR911984. It could utilize waste sunflower frying oil (WSFF) oil as a low-cost carbon source. It showed high emulsification activity (52 ± 0.0%) and positive results toward other biosurfactant screening tests. The strain showed high cell adhesion to hexane with 41.2% cell surface hydrophobicity. Fourier-transform infrared (FTIR) spectra indicated the presence of hydrophobic chains that comprise lipids, sugars, and hydrophilic glycolipid components. The optimization results showed the optimal factors included potato peel as a carbon source with 68.8% emulsification activity, yeast extract as a nitrogen source with 60% emulsification activity, a pH of 9 (56.6%), and a temperature of 50° (72%). The kinetics showed that optimum biosurfactant production (572.4 mg/L) was recorded at 5 days of incubation. The produced rhamnolipid biosurfactant showed high antimicrobial activity against some human and plant pathogenic bacterial and fungal isolates and high antioxidant activity (90.4%). In addition, it enhanced wheat (Triticum aestivum) growth, with the greatest enhancement obtained with the 5% concentration. Therefore, thermophilic G. stearothermophilus is a promising rhamnolipid biosurfactant producer that utilizes many organic wastes. The produced biosurfactant could be applied as a promising emulsifier, antimicrobial, antioxidant, and plant growth promoter.

The efficiency of micellar solubilization of naphthalene from aqueous solutions using rhamnolipid as a biological surfactant according to NMR diffusometry.

The micellar solubilization of naphthalene from its saturated aqueous solutions using the biosurfactant rhamnolipid was studied. Using the NMR diffusion method, selective measurements of the self-diffusion coefficients of molecules of all components of the solution-naphthalene, rhamnolipid, and water-were carried out at various rhamnolipid concentrations from 0.06 to 100 g/L. Based on the results of diffusometry, the distribution of naphthalene molecules between the states free in solution and states bound by micelles was found. With an increase in the concentration of rhamnolipids, the proportion of bound naphthalene molecules increases from 50% at CRL = 2 g/L to 100% at CRL ≥ 50 g/L. The micelle-water partition coefficient Km and the molar solubilization ratio MSR were calculated.

A Rare Mono-Rhamnolipid Congener Efficiently Produced by Recombinant Pseudomonas aeruginosa YM4 via the Expression of Global Transcriptional Regulator irrE.

Rhamnolipids (RLs) are widely used biosurfactants produced mainly by Pseudomonas aeruginosa and Burkholderia spp. in the form of mixtures of diverse congeners. The global transcriptional regulator gene irrE from radiation-tolerant extremophiles has been widely used as a stress-resistant element to construct robust producer strains and improve their production performance. A PrhlA-irrE cassette was constructed to express irrE genes in the Pseudomonas aeruginosa YM4 of the rhamnolipids producer strain. We found that the expression of irrE of Deinococcus radiodurans in the YM4 strain not only enhanced rhamnolipid production and the strain's tolerance to environmental stresses, but also changed the composition of the rhamnolipid products. The synthesized rhamnolipids reached a maximum titer of 26 g/L, about 17.9% higher than the original, at 48 h. The rhamnolipid production of the recombinant strain was determined to be mono-rhamnolipids congener Rha-C10-C12, accounting for 94.1% of total products. The critical micelle concentration (CMC) value of the Rha-C10-C12 products was 62.5 mg/L and the air-water surface tension decreased to 25.5 mN/m. The Rha-C10-C12 products showed better emulsifying activity on diesel oil than the original products. This is the first report on the efficient production of the rare mono-rhamnolipids congener Rha-C10-C12 and the first report that the global regulator irrE can change the components of rhamnolipid products in Pseudomonas aeruginosa.

Transport of nanoscale zero-valent iron in the presence of rhamnolipid.

Nanoscale zero-valent iron (nZVI) particles have gained widespread use for in-situ treatment of various chlorinated hydrocarbons. Their non-toxic nature, affordability, and minimal maintenance requirements have made them a favored material for nanoremediation. The treatment typically involves the injection of nZVI particles into contaminated sites using direct-push well injection systems. However, their small size leads to high surface energy, causing aggregation that alters their physiochemical properties, reactivity, and transport behavior. To counteract aggregation, nZVI suspension can be stabilized with different surfactants, reducing the surface energy during subsurface soil transport. This study investigates the impact of rhamnolipid, a biosurfactant produced by Pseudomonas aeruginosa during the late growth phase, on the aggregation and mobility of nZVI particles. The retardation factor of nZVI in the model media of zeolite, ZK406H, decreased from 1.66 in the absence of rhamnolipid to 1.03, 0.98, 0.93, and 0.87, corresponding to the presence of rhamnolipid at concentrations of 20, 50, 80, and 100 mg/L. The deposition coefficient also decreased from 2.39 in the absence of rhamnolipid to 0.459, 0.279, 0.217, and 0.0966, corresponding to the presence of rhamnolipid at concentrations of 20, 50, 80, and 100 mg/L. The transport parameters of nZVI in ZK406H were linked to the interactions of nZVI particles with ZK406H by the DLVO theory.

Microbe cultivation guidelines to optimize rhamnolipid applications.

In the growing landscape of interest in natural surfactants, selecting the appropriate one for specific applications remains challenging. The extensive, yet often unsystematized, knowledge of microbial surfactants, predominantly represented by rhamnolipids (RLs), typically does not translate beyond the conditions presented in scientific publications. This limitation stems from the numerous variables and their interdependencies that characterize microbial surfactant production. We hypothesized that a computational recipe for biosynthesizing RLs with targeted applicational properties could be developed from existing literature and experimental data. We amassed literature data on RL biosynthesis and micellar solubilization and augmented it with our experimental results on the solubilization of triglycerides (TGs), a topic underrepresented in current literature. Utilizing this data, we constructed mathematical models that can predict RL characteristics and solubilization efficiency, represented as logPRL = f(carbon and nitrogen source, parameters of biosynthesis) and logMSR = f(solubilizate, rhamnolipid (e.g. logPRL), parameters of solubilization), respectively. The models, characterized by robust R2 values of respectively 0.581-0.997 and 0.804, enabled the ranking of descriptors based on their significance and impact-positive or negative-on the predicted values. These models have been translated into ready-to-use calculators, tools designed to streamline the selection process for identifying a biosurfactant optimally suited for intended applications.

Valorization of waste engine oil to mono- and di-rhamnolipid in a sustainable approach to circular bioeconomy.

This study aims to valorize waste engine oil (WEO) for synthesizing economically viable biosurfactants (rhamnolipids) to strengthen the circular bioeconomy concept. It specifically focuses on investigating the influence of key bioprocess parameters, viz. agitation and aeration rates, on enhancing rhamnolipid yield in a fed-batch fermentation mode. The methodology involves conducting experiments in a stirred tank bioreactor (3 L) using Pseudomonas aeruginosa gi |KP 163922| as the test organism. Central composite design and response surface methodology (CCD-RSM) are employed to design the experiments and analyze the effects of agitation and aeration rates on various parameters, including dry cell biomass (DCBM), surface tension, tensoactivity, and rhamnolipid yield. It is also essential to determine the mechanistic pathway of biosurfactant production followed by the strain using complex hydrophobic substrates such as WEO. The study reveals that optimal agitation and aeration rates of 200 rpm and 1 Lpm result in the highest biosurfactant yield of 29.76 g/L with minimal surface tension (28 mN/m). Biosurfactant characterization using FTIR, 1H NMR, and UPLC-MS/MS confirm the presence of dominant molecular ion peaks m/z 543.9 and 675.1. This suggests that the biosurfactant is a mixture of mono- and di-rhamnolipids (RhaC10C10, RhaRhaC10C12:1, RhaRhaC12:1C10). The findings present a sustainable approach for biosurfactant production in a fed-batch bioreactor. This research opens the possibility of exploring the use of pilot or large-scale bioreactors for biosurfactant production in future investigations.