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.

High-solids enzymatic hydrolysis is the premise of obtaining high concentration ethanol by fermentation. In this study, corn stover was first pretreated with formic acid under mild conditions, and more than 70 % of xylan and lignin were removed within the first hour. 173.0 g/L glucose was achieved from total 30 % solid of the pretreated corn stover via fed-batch mode. Moreover, the glucose concentration rose to 194.5 g/L and the hydrolysis time was significantly reduced by 42.9 % with the addition of di-rhamnolipid. On this basis, 89.1 g/L ethanol was obtained by fermentation, and the presence of di-rhamnolipid had no negative effect on fermentation. The effective conversion of corn stover to high titer ethanol provides support for the conversion of stover to ethanol in industrial production.

OBJECTIVE: Bacillus subtilis subsp. subtilis (VCRC B471) and Pseudomonas fluorescens (B426) produce mosquitocidal biosurfactant, surfactin and di-rhamnolipid. The objective of the study was to carry out a small-scale field evaluation of the two biosurfactants to determine the efficacy, application dosage, residual activity and frequency of application against Anopheles stephensi immatures in selected sites in Goa, India.
METHODS: Surfactin (VCRC B471) and di-rhamnolipid (VCRC B426) were formulated as aqueous suspensions (5% AS), and were applied at the dosages of 34, 51 and 68 mL/m2 and 27, 41 and 54 mL/m2 respectively. Two experiments were carried out with the two formulations.
RESULTS: Surfactin (VCRC B471) formulation was effective at all the dosages and there was sustained reduction (>80%) in immature density in the treated sites up to 18 days in experiment 1 and up to 15 days in experiment 2. No pupae were found in the treated sites throughout the study. Di-rhamnolipid (VCRC B426) formulation was also found to reduce the immature density in the treated sites up to 14 days in experiment 1 and up to 15 days in experiment 2.
CONCLUSIONS: For VCRC B471, the optimum application dosage determined was 51 mL/m2 and for VCRC B426, 27mL/m2. The formulations are to be applied fortnightly for effective control of Anopheles. The application dosage determined in the present study can be used for large scale field evaluation to assess their suitability for use in public health programmes for the control of Anopheles mosquitoes vectoring malaria.

The objective of this study was to compare the potential of mono-rhamnolipids (mono-RML) and di-rhamnolipids (di-RML) against biofilm formation on carbon steel coupons submitted to oil produced water for 14 days. The antibiofilm effect of the RML on the coupons was analyzed by scanning electron microscopy (SEM) and by epifluorescence microscopy, and the contact angle was measured using a goniometer. SEM analysis results showed that all RML congeners had effective antibiofilm action, as well as preliminary anticorrosion evaluation confirmed that all RML congeners prevented the metal deterioration. In more detail, epifluorescence microscopy showed that mono-RML were the most efficient congeners in preventing microorganism\'s adherence on the carbon steel metal. Image analyses indicate the presence of 15.9%, 3.4%, and <0.1% of viable particles in di-RML, mono/di-RML and mono-RML pretreatments, respectively, in comparison to control samples. Contact angle results showed that the crude carbon steel coupon presented hydrophobic character favoring hydrophobic molecules adhesion. We calculated the theoretical polarity of the RML congeners and verified that mono-RML (log P = 3.63) presented the most hydrophobic character. This had perfect correspondence to contact angle results, since mono-RML conditioning (58.2°) more significantly changed the contact angle compared with the conditioning with one of the most common surfactants used on oil industry (29.4°). Based on the results, it was concluded that rhamnolipids are efficient molecules to be used to avoid biofilm on carbon steel metal when submitted to oil produced water and that a higher proportion of mono-rhamnolipids is more indicated for this application.

Surfactants, both synthetic and natural, are used in a wide range of industrial applications, including the degradation of petroleum hydrocarbons. Organisms from extreme environments are well-adapted to the harsh conditions and represent an exciting avenue of discovery of naturally occurring biosurfactants, yet microorganisms from cold environments have been largely overlooked for their biotechnological potential as biosurfactant producers. In this study, four cold-adapted bacterial isolates from Antarctica are investigated for their ability to produce biosurfactants. Here we report on the physical properties and chemical structure of biosurfactants from the genera Janthinobacterium, Psychrobacter, and Serratia. These organisms were able to grow on diesel, motor oil, and crude oil at 4 °C. Putative identification showed the presence of sophorolipids and rhamnolipids. Emulsion index test (E24) activity ranged from 36.4-66.7%. Oil displacement tests were comparable to 0.1-1.0% sodium dodecyl sulfate (SDS) solutions. Data presented herein are the first report of organisms of the genus Janthinobacterium to produce biosurfactants and their metabolic capabilities to degrade diverse petroleum hydrocarbons. The organisms\' ability to produce biosurfactants and grow on different hydrocarbons as their sole carbon and energy source at low temperatures (4 °C) makes them suitable candidates for the exploration of hydrocarbon bioremediation in low-temperature environments.

Rhamnolipids produced by P. aeruginosa MR01 were fractionated into mono- and di-rhamnolipids, and their dominant congeners, Rha-C10-C10 and Rha-Rha-C10-C10, were shown by mass spectrometry. Minimum surface tensions and critical micelle concentrations (CMC) were determined as \"≃34 mN/m; ≃26.17 mg/l;\" and \"≃29 mN/m; ≃29.63 mg/l\" for mono- and di-rhamnolipids, respectively. Spectrophotometry measurements provided a close approximation of CMC. Contact angle and diameter of wet area were determined for rhamnolipid-containing drops on hydrophobic paper to display their capability for alteration of surface wettability. Wet area measurement is a simple, reliable method not requiring a Drop Shape Analyzer. Cell viabilities determined by MTT assay showed a decline in a dose-dependent manner and estimated IC50 values were 25.87 μg/ml and 31.00 μg/ml for mono- and di-rhamnolipids treating MCF-7 cells for 48 h. Morphological observations using the inverted phase-contrast microscopy and fluorescence microscopy via Hoechst staining revealed the apoptotic characteristics in treated MCF-7 cells. The semi-quantitative RT-PCR method demonstrated that expression of the p53 gene in mRNA levels significantly (P < 0.05) increased when treated with 30 μg/ml of each rhamnolipid compound for 12 h. It can be concluded that rhamnolipids derived from MR01 show significant anticancer potential against MCF-7 cell line and should be further investigated as natural, therapeutic anti-tumor agents.

Rhamnolipids are a mixture of the homologs species due to variations in the rhamnose units and β-hydroxy fatty acid moieties, mainly including Rha-C10-C10, Rha-Rha-C10-C10, and Rha-C10. In this study, strain P. aeruginosa YM4 was selected for its capacity to efficiently produce di-rhamnolipid (Rha-Rha-C10-C10) as the predominant component with soybean oil and glycerol as carbon source, accounting for 64.8% and 85.7% of total products, respectively. The critical micelle concentration (CMC) of rhamnolipid products varies with the content of di-rhamnolipid, whereby lower CMC values corresponding to higher di-rhamnolipid contents. The rhamnolipids containing 85.7% di-rhamnolipid had the lowest CMC value of 50 mg/L. Accordingly the viscosity-reducing efficiency and oil-washing efficiency of rhamnolipids increased with higher di-rhamnolipid component. At a concentration of 500 mg/L, the rhamnolipids containing 85.7% di-rhamnolipid worked best and showed 82.5% oil-washing efficiency, which offered great promise for applications in enhanced oil recovery. The results showed the variation of structure and composition of rhamnolipids had a significant effect on their application.

Pseudomonas fluorescens Migula (VCRC B426) produces a secondary metabolite, which was found to be active against pupae of vector mosquitoes namely Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti. The mosquito pupicidal metabolite from P. fluoescens was mass produced and separated by ethyl acetate extraction and purified further by silica gel column chromatography, FPLC, HPLC and TLC. The purified metabolite was characterized by NMR, FT-IR, LC-MS and MALDI-TOF. The FT-IR, (1)H and (13)C NMR results showed that it is a rhamnolipid (di-rhamnolipid). The matrix assisted laser desorption and ionization-time-of-flight spectrum of the sample showed predominant pupicidal component produced by P. fluorescens was the molecule mass of 673.40 Da. Owing to its high toxicity to mosquito pupae, especially Anopheles sp., and Aedes sp., the di-rhamnolipd has potential in the control of the vectors of dengue, chikungunya, yellow fever and malaria. This is the first report of mosquito pupicidal di-rhamnolipid from P. fluorescens.

Recent investigations of extreme environments have revealed numerous bioactive natural products. However, biosurfactant-producing strains from deep sea extreme environment are largely unknown. Here, we show that Dietzia maris As-13-3 isolated from deep sea hydrothermal field could produce di-rhamnolipid as biosurfactant. The critical micelle concentration (CMC) of the purified di-rhamnolipid was determined to be 120 mgL(-1), and it lowered the surface tension of water from 74 ± 0.2 to 38 ± 0.2 mN m(-1). Further, the alkane metabolic pathway-related genes and di-rhamnolipid biosynthesis-related genes were also analyzed by the sequencing genome of D. maris As-13-3 and quantitative real-time PCR (Q-PCR), respectively. Q-PCR analysis showed that all these genes were induced by n-Tetradecane, n-Hexadecane, and pristane. To the best of our knowledge, this is first report about the complete pathway of the di-rhamnolipid synthesis process in the genus Dietzia. Thus, our study provided the insights into Dietzia in respects of oil degradation and biosurfactant production, and will help to evaluate the potential of Dietzia in marine oil removal.