Rhamnolipid Biosurfactant Research Articles

Obtaining transgenic alfalfa plants for improved phytoremediation of petroleum-contaminated soils

The possibility of using transgenic plants and their complexes with microorganisms to clean up soil from pollution is a topical research area. In our work the transgenic alfalfa plants with the rhlA gene, responsible for the biosynthesis of biosurfactant–rhamnolipids, were obtained. Rhamnolipids help reduce the surface tension of the hydrocarbon oil and its desorption from soil particles, thereby facilitating its recycling by microorganism. Cultivation of the control and the transgenic alfalfa plants in soil polluted with 4% oil showed the advantage of plants emitting rhamnolipids: oil utilization was 20% higher than with the control plants. When used together, the transgenic plants and Candida maltosa microorganism increased the degree of degradation of the oil to 86%. The results suggest bright prospects for the application of transgenic plants and the complex of transgenic plants and microorganisms to increase the efficiency of bioremediation.

Sub-CMC solubilization of dodecane by rhamnolipid in saturated porous media.

Experiments were conducted with a two-dimensional flow cell to examine the effect of monorhamnolipid surfactant at sub-CMC concentrations on solubilization of dodecane in porous media under dynamic flow conditions. Quartz sand was used as the porous medium and artificial groundwater was used as the background solution. The effectiveness of the monorhamnolipid was compared to that of SDBS, Triton X-100, and ethanol. The results demonstrated the enhancement of dodecane solubility by monorhamnolipid surfactant at concentrations lower than CMC. The concentrations (50–210 μM) are sufficiently low that they do not cause mobilization of the dodecane. Retention of rhamnolipid in the porous medium and detection of nano-size aggregates in the effluent show that the solubilization is based on a sub-CMC aggregate-formation mechanism, which is significantly stronger than the solubilization caused by the co-solvent effect. The rhamnolipid biosurfactant is more efficient for the solubilization compared to the synthetic surfactants. These results indicate a strategy of employing low concentrations of rhamnolipid for surfactant-enhanced aquifer remediation (SEAR), which may overcome the drawbacks of using surfactants at hyper-CMC concentrations.

Rhamnolipid mediated enhanced degradation of chlorpyrifos by bacterial consortium in soil-water system

The study was conducted with the aim to develop an environmentally compatible bio-based system which may rapidly detoxify soil and water polluted by inordinate use of organophosphate (OP) pesticides. Chlorpyrifos was used as model pesticide as it degrade slowly due to its low aqueous phase solubility (2ppm) and formation of antibacterial intermediate 3,5,6, trichloropyridinol (TCP). Five potential bacteria used in this study belonging to genus Pseudomonas, Klebsiella, Stenotrophomonas, Ochrobactrum and Bacillus and their mixed culture system efficiently degraded chlorpyrifos and its toxic intermediates TCP and diethylthiophosphate (DETP) in aqueous medium. However, degradation rate in soil-water based slurry system was slow as it took 10 days to degrade 82% of added chlorpyrifos (50mg/kg) by a potential mixed culture CS2 comprised of isolates F-3 and CH-y. This might be due to strong sorption affinity of chlorpyrifos to soil components which limits its bioavailability. Hence, a crude rhamnolipid biosurfactant produced by ChlD was used which improved the aqueous phase solubility of chlorpyrifos by 2–15 folds. This supported CS2 to attain 30% higher degradation within short period of 6 days as compared to biotic control without surfactant. Thus, this combination of mixed bacterial population with biosurfactant significantly improved the rate of chlorpyrifos degradation in soil without accumulation of toxic intermediates. This environmentally benign biosurfactant may be produced “in situ” and can replace commonly used toxic synthetic surfactants for bioremediation purposes.

Genetic construction of recombinant Pseudomonas chlororaphis for improved glycerol utilization

Abstract This study is to use genetic engineering to improve the glycerol metabolic capability of Pseudomonas chlororaphis which is capable of producing commercially valuable biodegradable poly(hydroxyalkanoate) (PHA) biopolymers and biosurfactant rhamnolipids (RLs). In the study, the glycerol uptake facilitator or aquaglyceroporin gene ( glpF ) and the glycerol kinase ( glpK ) gene were PCR-cloned from E. coli , inserted into a shuttle vector pBS29P2- gfp , and expressed in P. chlororaphis by a Pseudomonas promoter P2. The P. chlororaphis recombinants were then tested for cell growth and glycerol metabolism in chemically defined medium containing 0.5% and 1.0% (v/v) glycerol. The simultaneous expression of glpF and glpK resulted in a shorter lag time for cell growth and a more immediate glycerol consumption by P. chlororaphis . In conclusion, the recombinant P. chlororaphis that grows more efficiently in glycerol is expected to improve the technoeconomics of PHA and RL production using the surplus bioglycerol byproduct stream from biodiesel production.

EPS solubilization treatment by applying the biosurfactant rhamnolipid to reduce clogging in constructed wetlands

Application of extracellular polymeric substances (EPS) solubilization treatment with biosurfactant rhamnolipid (RL) to reduce clogging in constructed wetlands was first conducted in this study. The results showed significant improvement in the solubilization and dispersion of clogging matter following the treatment. And RL dosage of 0.09–0.15g/L altered microbial group make-up and had an overall positive effect on the growth of microorganisms. Moreover, RL was found to enhance EPS dissolution and dispersion, which was beneficial for the release of enzymes embedded in the EPS, and resulted in enhanced pollutant removal. The treatment had no apparent detrimental effect on wetland plants. Our results indicate that the optimum dosage of RL is 0.12g/L, and that the approach provides a promising and moderate option to reverse wetland clogging through RL-mediated solubilization treatment.

Development of fermentation calorimeter with improved sensitivity for bioprocess monitoring and control applications

In this report, a method for a simultaneous production and separation of a microbially synthesized rhamnolipid biosurfactant is presented. During the aerobic cultivation of flagella-free Pseudomonas putida EM383 in a 3.1 L stirred tank reactor on glucose as a sole carbon source, rhamnolipids are produced and excreted into the fermentation liquid. Here, a strategy for biosurfactant capture from rhamnolipid enriched fermentation foam using hydrophobic–hydrophobic interaction was investigated. Five adsorbents were tested independently for the application of this capture technique and the best performing adsorbent was tested in a fermentation process. Cell-containing foam was allowed to flow out of the fermentor through the off-gas line and an adsorption packed bed. Foam was observed to collapse instantly, while the resultant liquid flow-through, which was largely devoid of the target biosurfactant, eluted towards the outlet channel of the packed bed column and was subsequently pumped back into the fermentor. After 48 h of simultaneous fermentation and ex situ adsorption of rhamnolipids from the foam, 90% out of 5.5 g of total rhamnolipids produced were found in ethanol eluate of the adsorbent material, indicating the suitability of this material for ex situ rhamnolipid capture from fermentation processes.

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.

Foliar penetration enhanced by biosurfactant rhamnolipid

With recent environmental and health concerns, biosurfactants have obtained increasing interest in replacing conventional surfactants for diverse applications. In agriculture, the use of surfactant in stimulating foliar uptake is mainly for wetting leaf surface, resisting deposition/evaporation, enhancing penetration across cuticular membrane (CM) and translocation. This paper aimed to address the improved foliar uptake by rhamnolipid (RL) in comparison with the currently used alkyl polyglucoside (APG). As found, compared with APG at 900mg/L (1×critical micellar concentration, CMC), RL at a much lower concentration of 50mg/L (1×CMC) showed much better wettability and surface activity, indicative of its high effectiveness as surfactants. Its performance on resistance to deposition and evaporation was at least as same as APG. Moreover, RL could significantly improve the penetration of herbicide glyphosate and other two small water-soluble molecules (phenol red and Fe2+) across CM at an equivalent efficiency as APG at 1×CMC. Finally, the greatly enhanced herbicidal actitivity of glyphosate on greenhouse plants confirmed that RL and APG could both enhance the foliar uptake including translocation. Overall, RL should be more applicable than APG in agriculture due to its more promising properties on health/environmental friendliness.

Mechanism Study on the Severe Foaming of Rhamnolipid in Fermentation

AbstractAlthough the biosurfactant rhamnolipid has been previously characterized as having low foam ability, its fermentation is largely impeded by severe foaming. Hence, the investigation of this paradox is critically important for improving the mass production of rhamnolipid. Unexpectedly, the hydrophobic cell, instead of rhamnolipid, has been claimed to explain such severe foaming in rhamnolipid fermentation. This study tried to systematically investigate the severe foaming in fermentation, aiming to propose an effective strategy for foam control. The overflowing foam sustained a super high stability in terms of half‐time for over 30 min. The major product of rhamnolipid largely contributed to the severe foaming in the fermentation process whereas other products like cells elicited much more limited effects. Furthermore, the foam stability of the fermentation broth increased with rhamnolipid concentration and noticeably increased with agitation speed. In the classic Bikerman foam test system without stirring, rhamnolipid showed foam stability as low as Tween 20 which is well known for its poor foam stability. However, in a stirring Bikerman system, rhamnolipid exhibited a foam stability almost as high as sodium dodecyl sulfate (SDS) at 10 g/L and even surpassed SDS at a higher concentration of 20 g/L. Hence, the extraordinarily increased foam stability of rhamnolipid with both agitation and concentration could explain the severe foaming at its late‐stage fermentation when rhamnolipid‐rich solution is mechanically agitated.

Utilization of Paneer Whey Waste for Cost-Effective Production of Rhamnolipid Biosurfactant

The present study aimed at isolating rhamnolipid biosurfactant-producing bacteria that could utilize paneer whey, an abundant waste source as sole medium for the production purpose. Pseudomonas aeruginosa strain, SR17, was isolated from hydrocarbon-contaminated soil that could efficiently utilize paneer whey for rhamnolipid production and reduce surface tension of the medium from 52 to 26.5mN/m. The yield of biosurfactant obtained was 2.7g/l, upgraded to 4.8g/l when supplemented with 2% glucose and mineral salts. Biochemical, FTIR, and LC-MS analysis revealed that extracted biosurfactant is a combination of both mono and di-rhamnolipid congeners. The critical micelle concentration (CMC) was measured to be 110mg/l. Emulsification activity of the biosurfactant against n-hexadecane, olive oil, kerosene, diesel oil, engine oil, and crude oil were found to be 83, 88, 81, 92, 86, and 100%, respectively. The rhamnolipid was detected to be non-toxic against mouse fibroblastic cell line L292.

Comparative study of the production of rhamnolipid biosurfactants by B. thailandensis E264 and P. aeruginosa ATCC 9027 using foam fractionation

Biosurfactants are surface-active agents that are produced by a variety of microorganisms including yeasts, filamentous fungi and bacteria. In this work, we report on the ability of Pseudomonas aeruginosa ATCC 9027 and Burkholderia thailandensis E264 to produce rhamnolipids via a 10-L bioreactor and their recovery through foam fractionation studies in a continuous stripping mode. The recovery of Rha-C10-C10 (mono-rhamnolipids) produced by P. aeruginosa ATCC 9027 increased (from 6% to 96%), whilst the enrichment decreased (from 2.9 to 1.2) with the increasing airflow rate. These results are consistent with foam fractionation of a single surfactant system with stable foam. The recovery and enrichment of Rha-Rha-C14-C14 (di-rhamnolipids) produced by B. thailandensis E264 (and an unknown molecule) in a single-component system were found to display different characteristics. Both recovery and enrichment were found to decrease with the airflow rate. It is postulated that a competitive adsorption process could occur between the smaller molecule identified by electrospray ionisation–mass spectrometry (ESI–MS) and Rha-Rha-C14-C14.

Pseudomonas aeruginosa biofilm disruption using microbial surfactants.

To establish the ability of the rhamnolipids biosurfactants from Pseudomonas aeruginosa, in the presence and absence of caprylic acid and ascorbic acid, to disrupt bacterial biofilms, compared with the anionic alkyl sulphate surfactant Sodium dodecyl sulphate (SDS). Pseudomonas aeruginosa ATCC 15442 biofilms were disrupted by rhamnolipids at concentrations between 0·5 and 0·4 g l(-1) and with SDS at 0·8 g l(-1) . The combination of rhamnolipids 0·4 g l(-1) and caprylic acid at 0·1 g l(-1) showed a remarkable effect on biofilm disruption and cell killing. After 30 min of treatment most of the biofilm was disrupted and cell viability was significantly reduced. Neither caprylic acid nor ascorbic acid has any effect on biofilm disruption at 0·1 g l(-1) . SDS is an effective antimicrobial agent; however, in the presence of caprylic acid its effect was neutralized. The results show that rhamnolipids at low concentration in the presence of caprylic acid are promising molecules for inhibition/disruption of biofilms formed by Ps. aeruginosa ATCC 15442. The disruption of biofilms has major significance in many industrial and domestic cleaning applications and in medical situations.

Biosynthesis of Rhamnolipid Biosurfactant by Newly Isolated Marine Bacterium Methylobacterium mesophilicum MTCC 6839 from Oil Contaminated Sites at Alang Coast, Gujarat, India

Biosynthesis of Rhamnolipid Biosurfactant by Newly Isolated Marine Bacterium Methylobacterium mesophilicum MTCC 6839 from Oil Contaminated Sites at Alang Coast, Gujarat, India

Utilization of Crude Glycerol as a Substrate for the Production of Rhamnolipid by Pseudomonas aeruginosa.

Biosurfactants are produced by bacteria or yeast utilizing different substrates as sugars, glycerol, or oils. They have important applications in the detergent, oil, and pharmaceutical industries. Glycerol is the product of biodiesel industry and the existing glycerol market cannot accommodate the excess amounts generated; consequently, new markets for refined glycerol need to be developed. The aim of present work is to optimize the production of microbial rhamnolipid using waste glycerol. We have developed a process for the production of rhamnolipid biosurfactants using glycerol as the sole carbon source by a local Pseudomonas aeruginosa isolate that was obtained from an extensive screening program. A factorial design was applied with the goal of optimizing the rhamnolipid production. The highest production yield was obtained after 2 days when cells were grown in minimal salt media at pH 6, containing 1% (v/v) glycerol and 2% (w/v) sodium nitrate as nitrogen source, at 37°C and at 180 rpm, and reached 2.164 g/L after 54 hours (0.04 g/L h). Analysis of the produced rhamnolipids by TLC, HPLC, and FTIR confirmed the nature of the biosurfactant as monorhamnolipid. Glycerol can serve as a source for the production of rhamnolipid from microbial isolates providing a cheap and reliable substrate.

Mixed reverse micellar extraction and effect of surfactant chain length on extraction efficiency

Abstract This study investigated the effect of surfactant chain length on mixed reverse micellar extraction of cellulase. The mixed reverse micelle systems were formed by biosurfactant rhamnolipid and a nonionic surfactant tween (tween-20, tween-40, tween-60, or tween-80). Electron spin resonance (ESR) method was used in order to find the suitable chain length of tween and the best ratio of rhamnolipid/tween mixture. The key parameters such as pH and temperature in forward and backward extraction were studied, and the results showed that mixed reverse micelle with different surfactant chain length had different pH sensitivities. Rhamnolipid/tween-80 system was more sensitive to pH than other systems, and rhamnolipid/tween-20 system was the least sensitive to pH among four systems. The experimental results also showed that chain length had limited sensitivity to temperature. Rotation correlation time of cellulase solutions (original and extraction) were investigated, which proved the superiority of longer chain length system (rhamnolipid/tween-80 system).

Rhamnolipid Biosurfactants Produced by Pseudomonas Species

Surfactants are chemical products widely used in our daily life in toothpaste and other personal hygiene and cosmetic products, and in several industries. Biosurfactants are surfactants of biological origin that can be produced by microorganisms and have many advantages, such as low toxicity and high biodegradability, compared to synthetic counterparts. Unfortunately, high production costs limit the use of biosurfactants. Low-cost production is the most important factor for biosurfactants to be able to compete in the global market place. This review presents general information on rhamnolipid biosurfactant produced by Pseudomonas species, as well as on their production and applications. In addition, industrial products and their wastes used for rhamnolipid production are reviewed in detail based on recent studies.

Editorial: Microbiotechnology Based Surfactants and Their Applications.

This editorial is an annotation on the exciting research topic “Microbiotechnology based surfactants and their applications” that covers a compilation of original research articles, reviews and mini-reviews submitted by researchers enthusiastically working in the field of biosurfactants.

Optimization of rhamnolipid biosurfactant production by mangrove sediment bacterium Pseudomonas aeruginosa KVD-HR42 using response surface methodology

Abstract Rhamnolipid biosurfactant production by a novel Pseudomonas aeruginosa strain KVD-HR42 was optimized with statistical approaches. The produced biosurfactant showed surface active properties with stable emulsification activities. Based on the results of Plackett–Burman design, first-order polynomial model was developed and the following significant variables were determined viz., Karanja oil, sodium nitrate and pH. Response surface methodology experimental design was performed by Box–Behnken design to study the concentration of each component. The response plots resulted in the following optimized conditions; Karanja oil (23.85 g/L) sodium nitrate (9.17 g/L) and pH (7.8) which yielded an average biosurfactant production of 5.90±2.1 g/L at 48 h, and 37 °C temperature. The statistical approach resulted in enhanced biosurfactant production. The biosurfactant showed excellent emulsion forming capabilities and could reduce the surface tension to 30.14 mN/m at a CMC value of 100 mg/L. The biosurfactant was found to be stable at extreme conditions of temperature, pH and NaCl concentrations. Additionally surface active nature of the crude biosurfactant was demonstrated using oil displacement assay with a clearance zone of 19.26±0.23 cm 2 . Our results signify that the biosurfactant has a great industrial potential as a cleansing agent at adverse environmental conditions and provide better alternative to synthetic surfactants.

Separation and purification of six biosurfactant rhamnolipids by high-speed countercurrent chromatography utilizing novel solvent selection method

ABSTRACTIn this study, an original solvent selection method was developed for the separation of biosurfactant rhamnolipids from fermentation broth by high-speed countercurrent chromatography. For this method, calculations were based on solubility and functional group parameters that enabled the rapid selection of suitable solvent systems at no more than 10% of average time by traditional method. Based on selection results, a three-phase solvent system composed of n-hexane-methyl acetate-acetonitrile-water (2:2:2:5, v/v) was efficiently used for one-step separation of a mixture of six rhamnolipids. Thereby, RhaC10C10 (0.73 mg), RhaC10 (0.51 mg), Rha2C10 (0.12 mg), RhaC10C12 (1.26 mg), Rha2C10C10 (1.03 mg), and Rha2C10C12 (0.87 mg) were extracted from prepared Pseudomonas aeruginosa (100.0 mg) with purities of 96.37, 95.20, 91.25, 84.41, 89.8, and 90.26%, respectively. The purified compounds were identified by high-performance liquid chromatography/mass spectroscopy, plus 1H and 13C nuclear magnetic resonance studies. This study provides a new perspective on concepts involving efficient solvent system selection.

Effect of rhamnolipid biosurfactant on solubilization of polycyclic aromatic hydrocarbons

Rhamnolipid biosurfactant-producing bacteria, Bacillus Lz-2, was isolated from oil polluted water collected from Dongying Shengli oilfield, China. The factors that influence PAH solubilization such as biosurfactant concentration, pH, ionic strength and temperature were discussed. The results showed that the solubilities of naphthalene, phenanthrene and pyrene increased linearly with the rise of rhamnolipid biosurfactant dose above the biosurfactant critical micelle concentration (CMC). Furthermore, the molar solubilization ratio (MSR) values decreased in the following order: naphthalene>phenanthrene>pyrene. However, the solubility percentage increased and followed the opposite order: pyrene>phenanthrene>naphthalene. The solubilities of PAHs in rhamnolipid biosurfactant solution increased with the rise of pH and ionic strength, and reached the maximum values under the conditions of pH11 and NaCl concentration 8g·L−1. The solubility of phenanthrene and pyrene increased with the rise of temperature.