Rhamnolipid Congeners Research Articles

Designer rhamnolipids by reduction of congener diversity: production and characterization

BackgroundRhamnolipids are biosurfactants featuring surface-active properties that render them suitable for a broad range of industrial applications. These properties include their emulsification and foaming capacity, critical micelle concentration, and ability to lower surface tension. Further, aspects like biocompatibility and environmental friendliness are becoming increasingly important. Rhamnolipids are mainly produced by pathogenic bacteria like Pseudomonas aeruginosa. We previously designed and constructed a recombinant Pseudomonas putida KT2440, which synthesizes rhamnolipids by decoupling production from host-intrinsic regulations and cell growth.ResultsHere, the molecular structure of the rhamnolipids, i.e., different congeners produced by engineered P. putida are reported. Natural rhamnolipid producers can synthesize mono- and di-rhamnolipids, containing one or two rhamnose molecules, respectively. Of each type of rhamnolipid four main congeners are produced, deviating in the chain lengths of the β-hydroxy-fatty acids. The resulting eight main rhamnolipid congeners with variable numbers of hydrophobic/hydrophilic residues and their mixtures feature different physico-chemical properties that might lead to diverse applications. We engineered a microbial cell factory to specifically produce three different biosurfactant mixtures: a mixture of di- and mono-rhamnolipids, mono-rhamnolipids only, and hydroxyalkanoyloxy alkanoates, the precursors of rhamnolipid synthesis, consisting only of β-hydroxy-fatty acids. To support the possibility of second generation biosurfactant production with our engineered microbial cell factory, we demonstrate rhamnolipid production from sustainable carbon sources, including glycerol and xylose. A simple purification procedure resulted in biosurfactants with purities of up to 90%. Finally, through determination of properties specific for surface active compounds, we were able to show that the different mixtures indeed feature different physico-chemical characteristics.ConclusionsThe approach demonstrated here is a first step towards the production of designer biosurfactants, tailor-made for specific applications by purposely adjusting the congener composition of the mixtures. Not only were we able to genetically engineer our cell factory to produce specific biosurfactant mixtures, but we also showed that the products are suited for different applications. These designer biosurfactants can be produced as part of a biorefinery from second generation carbon sources such as xylose.

Profiling of Microbial Colonies for High-Throughput Engineering of Multistep Enzymatic Reactions via Optically Guided Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) imaging has been used for rapid phenotyping of enzymatic activities, but is mainly limited to single-step conversions. Herein we report a label-free method for high-throughput engineering of multistep biochemical reactions based on optically guided MALDI-ToF MS analysis of bacterial colonies. The bacterial cells provide containment of multiple enzymes and access to substrates and cofactors via metabolism. Automated MALDI-ToF MS acquisition from randomly distributed colonies simplifies procedures to prepare strain libraries without liquid handling. MALDI-ToF MS profiling was utilized to screen both substrate and enzyme libraries for natural product biosynthesis. Computational algorithms were developed to process and visualize the resulting mass spectral data sets. For analogues of the peptidic antibiotic plantazolicin, multivariate analyses by t-distributed stochastic neighbor embedding were used to group similar spectra for rapid identification of nonisobaric variants. After MALDI-ToF MS screening, follow-up analyses using high-resolution MS and tandem MS were readily performed on the same sample target. Separately, relative ion intensities of rhamnolipid congeners with various lipid moieties were evaluated to engineer enzymatic specificity. The glycolipid profiles of each colony were overlaid with optical images to facilitate the recovery of desirable mutants. For both the antibiotic and rhamnolipid cases, large populations of colonies were rapidly surveyed at the molecular level, providing information-rich insights not easily obtained with traditional screening assays. Utilizing standard microbiological techniques with routine microscopy and MALDI-ToF MS instruments, this simple yet effective workflow is applicable for a wide range of screening campaigns targeting multistep enzymatic reactions.

Characterization of rhamnolipid biosurfactants produced by recombinant Pseudomonas aeruginosa strain DAB with removal of crude oil.

To improve rhamnolipid production and its potential application in removal of crude oil, the recombinant Pseudomonas aeruginosa strain DAB was constructed to enhance yield of rhamnolipids. Strain DAB had a higher yield of 17.3g rhamnolipids l-1 in the removal process with crude oil as the sole carbon source than 10g rhamnolipids l-1 of wild-type strain DN1, where 1% crude oil was degraded more than 95% after 14days cultivation. These rhamnolipids reduced the surface tension of water from 72.92 to 26.15 mN m-1 with CMC of 90mgl-1. The predominant rhamnolipid congeners were Rha-C10-C10 and Rha-Rha-C10-C10 detected by MALDI-TOF MS analysis with approx. 70% relative abundance, although a total of 21 rhamnolipid congeners were accumulated. Increasing the copy number of rhlAB genes efficiently enhanced the production of rhamnolipids by the recombinant P. aeruginosa DAB and thus presents a promising application for the bioremediation process.

Variants of lipopeptides and glycolipids produced by Bacillus amyloliquefaciens and Pseudomonas aeruginosa cultured in different carbon substrates

The quantitative and qualitative effect of water immiscible and miscible carbon-rich substrates on the production of biosurfactants, surfactin and rhamnolipids, by Bacillus amyloliquefaciens ST34 and Pseudomonas aeruginosa ST5, respectively, was analysed. A small-scale high throughput 96 deep-well micro-culture method was utilised to cultivate the two strains in mineral salt medium (MSM) supplemented with the water miscible (glucose, glycerol, fructose and sucrose) and water immiscible carbon sources (diesel, kerosene and sunflower oil) under the same growth conditions. The biosurfactants produced by the two strains were isolated by acid precipitation followed by an organic solvent extraction. Ultra-performance liquid chromatography coupled to electrospray ionisation mass spectrometry was utilised to analyse yields and characterise the biosurfactant variants. For B. amyloliquefaciens ST34, maximum surfactin production was observed in the MSM supplemented with fructose (28 mg L−1). In addition, four surfactin analogues were produced by ST34 using the different substrates, however, the C13–C15 surfactins were dominant in all extracts. For P. aeruginosa ST5, maximum rhamnolipid production was observed in the MSM supplemented with glucose (307 mg L−1). In addition, six rhamnolipid congeners were produced by ST5 using different substrates, however, Rha–Rha–C10–C10 and Rha–C10–C10 were the most abundant in all extracts. This study highlights that the carbon sources utilised influences the yield and analogues/congeners of surfactin and rhamnolipids produced by B. amyloliquefaciens and P. aeruginosa, respectively. Additionally, glucose and fructose were suitable substrates for rhamnolipid and surfactin, produced by P. aeruginosa ST5 and B. amyloliquefaciens ST34, which can be exploited for bioremediation or as antimicrobial agents.

Characterization of Biosurfactant Produced during Degradation of Hydrocarbons Using Crude Oil As Sole Source of Carbon.

Production and spillage of petroleum hydrocarbons which is the most versatile energy resource causes disastrous environmental pollution. Elevated oil degrading performance from microorganisms is demanded for successful microbial remediation of those toxic pollutants. The employment of biosurfactant-producing and hydrocarbon-utilizing microbes enhances the effectiveness of bioremediation as biosurfactant plays a key role by making hydrocarbons bio-available for degradation. The present study aimed the isolation of a potent biosurfactant producing indigenous bacteria which can be employed for crude oil remediation, along with the characterization of the biosurfactant produced during crude oil biodegradation. A potent bacterial strain Pseudomonas aeruginosa PG1 (identified by 16s rDNA sequencing) was isolated from hydrocarbon contaminated soil that could efficiently produce biosurfactant by utilizing crude oil components as the carbon source, thereby leading to the enhanced degradation of the petroleum hydrocarbons. Strain PG1 could degrade 81.8% of total petroleum hydrocarbons (TPH) after 5 weeks of culture when grown in mineral salt media (MSM) supplemented with 2% (v/v) crude oil as the sole carbon source. GCMS analysis of the treated crude oil samples revealed that P. aeruginosa PG1 could potentially degrade various hydrocarbon contents including various PAHs present in the crude oil. Biosurfactant produced by strain PG1 in the course of crude oil degradation, promotes the reduction of surface tension (ST) of the culture medium from 51.8 to 29.6 mN m−1, with the critical micelle concentration (CMC) of 56 mg L−1. FTIR, LC-MS, and SEM-EDS studies revealed that the biosurfactant is a rhamnolipid comprising of both mono and di rhamnolipid congeners. The biosurfactant did not exhibit any cytotoxic effect to mouse L292 fibroblastic cell line, however, strong antibiotic activity against some pathogenic bacteria and fungus was observed.

Isolation of rhamnolipids-producing cultures from faeces: Influence of interspecies communication on the yield of rhamnolipid congeners

The aim of this study was to evaluate the ability of bacterial cultures isolated from cattle, poultry or pig faeces and manure to produce rhamnolipids, as well as to investigate the influence of interspecies communication on possible quantitative differences in the production of rhamnolipid congeners. Initial screening methods (oil spreading, drop collapse, haemolytic activity and emulsification activity) showed that approximately 36% of the 51 isolated cultures exhibited the ability to produce biosurfactants. Subsequent studies using a selected culturable mixed culture (which included Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli) revealed that only P. aeruginosa was able to produce this biosurfactant. HPLC–MS analysis showed that the surface active compounds were rhamnolipids. Further comparative studies confirmed that the total yield of rhamnolipids was notably higher in the bioreactor inoculated with the selected mixed culture (940.58±1.10mg/L) compared to the bioreactor inoculated with the axenic strain of P. aeruginosa (108.47±0.41mg/L). Twelve rhamnolipid congeners were identified during cultivation of the selected mixed culture, whereas six congeners were detected during cultivation of the sole axenic strain of P. aeruginosa. Furthermore, increased production of rhamnolipids was observed when the concentration of autoinducer molecules (AI-2) responsible for interspecies signaling increased, suggesting the influence of quorum-sensing communication on biosynthesis efficiency. This observation may be of importance for large-scale production of this biosurfactant, as it opens new possible solutions based on the use of mixed cultures or external addition of stimulating autoinducers.

Microemulsion synthesis of silver nanoparticles using biosurfactant extracted from Pseudomonas aeruginosa MKVIT3 strain and comparison of their antimicrobial and cytotoxic activities.

In the present study, an efficient biosurfactant producing bacterial strain Pseudomonas aeruginosa MKVIT3 was isolated from an oil logging area in Vellore district of Tamil Nadu, India. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis was performed for the identification of different congeners present in the extracted biosurfactant. The column purified biosurfactant was used to stabilise the formation of silver nanoparticles (NP) using borohydrate reduction in reverse micelles. The silver NP were characterised using UV-vis absorption spectroscopy, Powder-XRD TEM analysis and zeta potential. A comparative study of the antimicrobial activity and cytotoxic efficacy was done for the extracted purified biosurfactant and the silver NP. The LC-MS/MS analysis of the biosurfactant revealed the presence of five rhamnolipid congeners. The synthesised silver NP showed the characteristic absorption peak in UV-vis at 440 nm. Powder-XRD and TEM analysis revealed the average particle size of the NP as 17.89 ± 8.74 nm as well as their cubic structure. Zeta potential value of -30.9 mV suggested that the silver NPs are stable in the suspension. Comparative study of the antimicrobial activity revealed that the silver NP are more potent than the biosurfactant in inhibiting the growth of microbes. Cytotoxic activity revealed that the biosurfactant are more effective than the synthesised silver NP.

High performance liquid chromatography-charged aerosol detection applying an inverse gradient for quantification of rhamnolipid biosurfactants

A method using high performance liquid chromatography coupled to charged-aerosol detection (HPLC-CAD) was developed for the quantification of rhamnolipid biosurfactants. Qualitative sample composition was determined by liquid chromatography coupled to tandem mass spectrometry (LC⿿MS/MS). The relative quantification of different derivatives of rhamnolipids including di-rhamnolipids, mono-rhamnolipids, and their precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) differed for two compared LC⿿MS instruments and revealed instrument dependent responses. Our here reported HPLC-CAD method provides uniform response.An inverse gradient was applied for the absolute quantification of rhamnolipid congeners to account for the detector⿿s dependency on the solvent composition. The CAD produces a uniform response not only for the analytes but also for structurally different (nonvolatile) compounds. It was demonstrated that n-dodecyl-β-d-maltoside or deoxycholic acid can be used as alternative standards. The method of HPLC-ultra violet (UV) detection after a derivatization of rhamnolipids and HAAs to their corresponding phenacyl esters confirmed the obtained results but required additional, laborious sample preparation steps. Sensitivity determined as limit of detection and limit of quantification for four mono-rhamnolipids was in the range of 0.3⿿1.0 and 1.2⿿2.0μg/mL, respectively, for HPLC-CAD and 0.4 and 1.5μg/mL, respectively, for HPLC-UV. Linearity for HPLC-CAD was at least 0.996 (R2) in the calibrated range of about 1⿿200μg/mL. Hence, the here presented HPLC-CAD method allows absolute quantification of rhamnolipids and derivatives.

Valorization of agro-industrial wastes towards the production of rhamnolipids

In this work, oil mill wastewater (OMW), a residue generated during olive oil extraction, was evaluated as an inducer of rhamnolipid production. Using a medium containing as sole ingredients corn steep liquor (10%, v/v), sugarcane molasses (10%, w/v) and OMW (25%, v/v), Pseudomonas aeruginosa #112 produced 4.5 and 5.1g of rhamnolipid per liter in flasks and reactor, respectively, with critical micelle concentrations as low as 13mg/l. Furthermore, in the medium supplemented with OMW, a higher proportion of more hydrophobic rhamnolipid congeners was observed comparing with the same medium without OMW. OMW is a hazardous waste which disposal represents a serious environmental problem; therefore, its valorization as a substrate for the production of added-value compounds such as rhamnolipids is of great interest. This is the first report of rhamnolipid production using a mixture of these three agro-industrial by-products, which can be useful for the sustainable production of rhamnolipids.

Development and validation of an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the quantitative determination of rhamnolipid congeners.

Rhamnolipids (RLs) are synthesised as a complex mixture of congeners comprising either one or two molecules of rhamnose glycosidically linked to a dimer of 3-hydroxy fatty acids varying in chain length and degree of saturation. Currently, HPLC-MS/MS is the most precise and accurate method for RL determination, while accurate quantification is limited. In this study, a rapid ultra pressure liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the rapid and quantification of individual RL congeners. Increased RLs specificity was achieved using tandem mass spectrometry in multiple reaction monitoring (MRM) mode which was used to quantify RL isomer pairs such as Rha-Rha-C8-C10/Rha-Rha-C10-C8 which are difficult to resolve chromatographically. UPLC showed an 18-fold reduction in retention time for Rha-Rha-C10-C10 (1.07 min) and a 17-fold reduction for Rha-C10-C10 (1.36), the major rhamnolipids present, compared to HPLC, with a total run time less than 2.2 min. The results show that the linear range for the main RL congeners (Rha-C10-C10 and Rha-Rha-C10-C10) is 0.1 to 100 μg/mL. The LOD and LOQ for Rha-C10-C10 is 0.05 and 0.1 μg/mL and for Rha-Rha-C10-C10 is 0.1 and 0.5 μg/mL, respectively. The method was validated for linearity, intra- and inter-day precision and accuracy in accordance with FDA guidelines. The method was applied for the quantification of 14 individual RL congeners produced by Pseudomonas aeruginosa ST5 and comparison of RLs composition on four different carbon sources. Quantification of the individual congeners showed a conserved congener distribution irrespective of carbon source with a preferential selection for C10 β-hydroxyacids as the lipid component of RLs. The only statistically significant differences detected were between actual RL yields on the various carbon sources.

Stimulation of rhamnolipid biosurfactants production in Pseudomonas aeruginosa AK6U by organosulfur compounds provided as sulfur sources

A Pseudomonas aeruginosa AK6U strain produced rhamnolipid biosurfactants to variable extents when grown on MgSO4 or organosulfur compounds as sulfur sources and glucose as a carbon source. Organosulfur cultures produced much higher biosurfactants amounts compared to the MgSO4 cultures. The surface tension of the growth medium was reduced from 72mN/m to 54 and 30mN/m in cultures containing MgSO4 and 4,6-dimethyldibenzothiophene (4,6-DM-DBT), respectively. AK6U cultures produced different rhamnolipid congener profiles depending on the provided sulfur source. The dibenzothiophene (DBT) culture produced more diverse and a higher number of rhamnolipid congeners as compared to the DBT-sulfone and MgSO4 cultures. The number of mono-rhamnolipid congeners in the DBT culture was also higher than that detected in the DBT-sulfone and MgSO4 cultures. Di-rhamnolipids dominated the congener profiles in all the analyzed cultures. The sulfur source can have a profound impact on the quality and quantity of the produced biosurfactants.

Rhamnolipid biosurfactant from Pseudomonas aeruginosa: From discovery to application in contemporary technology

The rhamnolipids are most likely the next generation of biosurfactants which will reach the market. They should follow closely after alkyl polyglycosides, already established in the biosurfactants market, and sophorolipids, which can be found in several cleaning agents. However, the greatest number of recent publications and patents among glycolipid biosurfactants has been dedicated to rhamnolipids. Produced mainly by Pseudomonas aeruginosa, rhamnolipids are mixtures of different rhamnolipid congeners, which show physico-chemical properties that differ from those of single congeners, with the most abundant structure in the mixture having the largest impact on the overall characteristics of the total mixture. Characteristics of biodegradability, low toxicity, production from renewable sources and antimicrobial (particularly antifungal) activity together make rhamnolipid biosurfactants particularly promising for broad commercial application. Although to date, bioremediation has been the major topic filed for patents utilizing rhamnolipids, an increasing number of patents for applications in cosmetics, agronomy and food industries, formulation of cleaners and nanotechnology indicates their future implementation in these fields.

Structural and physiochemical characterization of rhamnolipids produced by Acinetobacter calcoaceticus, Enterobacter asburiae and Pseudomonas aeruginosa in single strain and mixed cultures

Rhamnolipids are naturally occurring biosurfactants with a wide range of potential commercial applications. As naturally derived products they present an ecological alternative to synthetic surfactants. The majority of described rhamnolipid productions are single strain Pseudomonas spp. cultivations. Here we report rhamnolipids producing bacteria Acinetobacter calcoaceticus, Enterobacter asburiae and Pseudomonas aeruginosa that were cultivated separately and as mixed populations. The ratio and composition of rhamnolipid congeners was determined by tandem mass spectrometry with negative electrospray ionization. Mono-rhamnolipid and di-rhamnolipid homologues containing one or two saturated or monounsaturated 3-hydroxy fatty acids were found in all strains.Physiochemical characterization of rhamnolipids was evaluated by the critical micelle concentration determination, the emulsification test, oil displacement test and phenanthrene solubilization. Critical micelle concentrations of rhamnolipids produced by both single strain and mixed cultures were found to be very low (10–63mg/l) and to correspond with saturated/unsaturated fatty acid content of rhamnolipid homologues. The rhamnolipids produced by all strains effectively emulsified crude petroleum in comparison with synthetic surfactants Tween 80 and sodium dodecyl sulfate (SDS). Good performance of phenanthrene solubilization was exhibited by rhamnolipids from E. asburiae.The single strain and co-cultures cultivations were proposed as a possible way to produce rhamnolipid mixtures with a specific composition and different physiochemical properties, which could be exploited in bioremediation of various hydrophobic contaminants.

Bioconversion of agro-industrial by-products in rhamnolipids toward applications in enhanced oil recovery and bioremediation

In this work, biosurfactant production by a Pseudomonas aeruginosa strain was optimized using low-cost substrates. The highest biosurfactant production (3.2g/l) was obtained using a culture medium containing corn steep liquor (10% (v/v)) and molasses (10% (w/v)). The biosurfactant reduced the surface tension of water up to 30mN/m, and exhibited a high emulsifying activity (E24=60%), with a critical micelle concentration as low as 50mg/l. The biosurfactant produced in this alternative medium was characterized as a mixture of eight different rhamnolipid congeners, being the most abundant the mono-rhamnolipid Rha-C10-C10. However, using LB medium, nine different rhamnolipid congeners were identified, being the most abundant the di-rhamnolipid Rha-Rha-C10-C10. The rhamnolipid mixture produced in the alternative medium exhibited a better performance in removing oil from contaminated sand when compared with two chemical surfactants, suggesting its potential use as an alternative to traditional chemical surfactants in enhanced oil recovery or bioremediation.

Production of rhamnolipid surfactant and its application in bioscouring of cotton fabric

In the present study, a biosurfactant was synthesized by using a bacterial strain of Pseudomonas aeruginosa in minimal media provided with n-heptadecane as sole carbon source under shake-flask conditions. The biosurfactant was isolated (by acid precipitation, solvent extraction, and rotary evaporation), purified (by column chromatography and TLC), identified (by FAB-MS, FTIR, and 1D-1H NMR), and chemo-physical characterized (by tensiometry). Two principal rhamnolipid congeners were identified as dirhamnolipid RRC10C10 and monorhamnolipid RC10C10 with a CMC of 50mg/L. The biosurfactant, hence produced, was applied in sole and in combination with pectinase in scouring of cotton fabric in contrast to conventional scouring agents of NaOH and anionic surfactant SDS. The scoured cotton fabric was investigated for its weight loss, residual oil and grease, wettability, whiteness, and tensile strength. The results were compared both for conventional and biological approaches. The scouring with biosurfactant plus pectinase was equivalent to or better in efficiency than conventional alkaline scouring. The former process is additionally environmentally friendly and bio-compatible. Scanning electron microscopy of cotton fabric showed that the alkaline scouring deteriorates the fabric texture whereas bioscouring with biosurfactant plus pectinase gently removes hydrophobic impurities from the cotton fabric.

Structural characterization of rhamnolipid produced by Pseudonomas aeruginosa strain FIN2 isolated from oil reservoir water

Biosurfactant-producing microorganisms inhabiting oil reservoirs are of great potential in industrial applications. Yet, till now, the knowledge about the structure and physicochemical property of their metabolites are still limited. The aim of this study was to purify and structurally characterize the biosurfactant from Pseudomonas aeruginosa strain FIN2, a newly isolated strain from an oil reservoir. The purification was conducted by silica gel column chromatography followed by pre-RP HPLC and the structural characterization was carried out by GC-MS combined with MS/MS. The results show that the biosurfactant produced by FIN2 is rhamnolipid in nature and its four main fractions were identified to be Rha-C10-C10(46.1 %), Rha-Rha-C10-C10(20.1 %), Rha-C8-C10 (7.5 %) and Rha-C10-C12:1(5.5 %), respectively. Meanwhile, the rarely reported rhamnolipid congeners containing β-hydroxy fatty acids of C6, C9, C10:1 and C11 were also proved to be present in the rhamnolipid mixture produced. The rhamnolipid mixture exhibited a strong surface activity by lowering the surface tension of distilled water to 28.6 mN/m with a CMC value of 195 mg/l.

Optimization of biosurfactant production using waste from biodiesel industry in a new membrane assisted bioreactor

The main byproduct of biodiesel production is glycerol. Here, crude glycerol – byproduct of biodiesel industry – was evaluated as sole carbon source in rhamnolipids production by Pseudomonas aeruginosa. The optimal concentration of crude glycerol and sodium nitrate was assessed using response surface methodology, resulting in about 40–50mg/L.h of rhamnolipids, which was about four times higher than previously reported in the literature. Fermentation parameters were similar to those observed with commercial glycerol as sole carbon source. The optimized medium was suitable for production using simple (22.9mg/L.h) and fed-batch (32.4mg/L.h) fermentation in oxygen-controlled bioreactor without foaming formation. Composition and relative abundance of rhamnolipid congeners showed that crude glycerol had little effect on metabolic pathways involved in their production. CMC values were approximately 130mg/L and 230–260mg/L for rhamnolipids from crude and commercial glycerol fermentation, respectively, which were about 2–6 times lower than CMC values of synthetic surfactants.

Rhamnolipids are conserved biosurfactants molecules: implications for their biotechnological potential

A range of isolates of Pseudomonas aeruginosa from widely different environmental sources were examined for their ability to synthesise rhamnolipid biosurfactants. No significant differences in the quantity or composition of the rhamnolipid congeners could be produced by manipulating the growth conditions. Sequences for the rhamnolipid genes indicated low levels of strain variation, and the majority of polymorphisms did lead to amino acid sequence changes that had no evident phenotypic effect. Expression of the rhlB and rhlC rhamnosyltransferase genes showed a fixed sequential expression pattern during growth, and no significant up-regulation could be induced by varying producer strains or growth media. The results indicated that rhamnolipids are highly conserved molecules and that their gene expression has a rather stringent control. This leaves little opportunity to manipulate and greatly increase the yield of rhamnolipids from strains of P. aeruginosa for biotechnological applications.

Structural characterization of a rhamnolipid-type biosurfactant produced by Pseudomonas aeruginosa MR01: Enhancement of di-rhamnolipid proportion using gamma irradiation

We previously reported that MR01, an indigenous strain of Pseudomonas aeruginosa, was able to produce a rhamnolipid-type biosurfactant. Here, we attempted to define the structural properties of this natural product. The analysis of the extracted biosurfactant by thin-layer chromatography (TLC) revealed the presence of two compounds corresponding to those of authentic mono- and di-rhamnolipid. The identity of two structurally distinguished rhamnolipids was confirmed by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Liquid chromatography/mass spectrometry (LC/MS) of extracted biosurfactant revealed up to seventeen different rhamnolipid congeners. Further quantification showed di-rhamnolipids as the major compound (77.2%), while monorhamnolipids comprising a smaller proportion (22.8%) of MR01 biosurfactant. Rha-Rha-C 10-C 10 was verified as the major component of the MR01 biosurfactant (35.93%). Cytotoxic activity of MR01 biosurfactant against human cancer Hela cells showed an excellent inhibitory effect of 5 μg/ml. An isolated mutant strain (MR01-C) created by Gamma ray irradiation demonstrated more than one and a half-fold biosurfactant production and activity compared with the parent strain. Analysis of the biosurfactant produced by MR01-C showed the magnitude of di-rhamnolipids in the sample increased up to 88.6% (∼15% higher than control) and the quantity of Rha-Rha-C 10-C 10 increased to 52.08% (∼45% higher than control).

Structure, properties and applications of rhamnolipids produced by Pseudomonas aeruginosa L2-1 from cassava wastewater

The properties and applications of rhamnolipid surfactants produced by Pseudomonas aeruginosa L2-1 from cassava wastewater added with waste cooking oil (CWO) as low-cost substrate, were investigated and compared with the commercial rhamnolipid mixture JBR599 (Jeneil Biosurfactant Co., Saukville, USA). The rhamnolipids produced by strain L2-1 were characterized by high performance liquid chromatography–mass spectrometry. Sixteen different rhamnolipid congeners were detected, with Rha-C 10-C 10 and Rha-Rha-C 10-C 10 being the most abundant. The L2-1 rhamnolipids from CWO showed similar or better tensioactive properties than those from JBR599, with a minimal surface tension of 30 mN/m and a critical micelle concentration (CMC) of 30 mg/l. The L2-1 biosurfactants formed stable emulsions with several hydrocarbons and showed excellent emulsification of soybean oil (100%). These rhamnolipids removed 69% of crude oil present in contaminated sand samples at the CMC and presented antimicrobial activity against Bacillus cereus (32 μg/ml), Micrococcus luteus (32 μg/ml) and Staphylococcus aureus (128 μg/ml). These results demonstrate that the rhamnolipids produced in CWO can be useful for industrial applications, such as the bioremediation of oil spills.