Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa.

The opportunistic human pathogen Pseudomonas aeruginosa produces a variety of virulence factors, including exotoxin A, elastase, alkaline protease, alginate, phospholipases, and extracellular rhamnolipids. The previously characterized rhlABR gene cluster encodes a regulatory protein (RhlR) and a rhamnosyltransferase (RhlAB), both of which are required for rhamnolipid synthesis. Another gene, rhII, has now been identified downstream of the rhlABR gene cluster. The putative RhlI protein shares significant sequence similarity with bacterial autoinducer synthetases of the LuxI type. A P. aeruginosa rhlI mutant strain carrying a disrupted rhlI gene was unable to produce rhamnolipids and lacked rhamnosyltransferase activity. Rhamnolipid synthesis was restored by introducing a wild-type rhlI gene into such strains or, alternatively, by adding either the cell-free spent supernatant from a P. aeruginosa wild-type strain or synthetic N-acylhomoserine lactones. Half-maximal induction of rhamnolipid synthesis in the rhlI mutant strain required 0.5 microM N-butyrylhomoserine lactone or 10 microM N-(3-oxohexanoyl)homoserine lactone. The P. aeruginosa rhlA promoter was active in the heterologous host Pseudomonas putida when both the rhlR and rhlI genes were present or when the rhlR gene alone was supplied together with synthetic N-acylhomoserine lactones. The RhlR-RhlI regulatory system was found to be essential for the production of elastase as well, and cross-communication between the RhlR-RhlI rhamnolipid regulatory system and the LasR-LasI elastase regulatory system was demonstrated.

Electrokinetic and Rheological Properties of Nano Zirconia in the Presence of Rhamnolipid Biosurfactant

In this paper, the effect of rhamnolipid biosurfactant on the electrokinetic and rheological behavior of nanozirconia particles is reported. The effect of pH, concentration of biosurfactant, and solids loading on ζ-potential and rheological behavior was investigated. ζ-potential measurements showed that adsorption of biosurfactant shifted the iso-electric point of zirconia with increasing biosurfactant concentration. The surface of zirconia became more electronegative in the presence of biosurfactant indicating a strong interaction. Maximum charge was obtained in the presence of about 230 ppm of biosurfactant. Rheological tests at pH 7 revealed that the zirconia suspension is viscous at high solids loading and addition of biosurfactant decreased the viscosity substantially especially at high solids loading (>50 wt%). Sedimentation tests confirmed that the biosurfactant is a good dispersant for zirconia particles at pH values of 7 and above.

Biosurfactant synthesis by Pseudomonas aeruginosa LBI isolated from a hydrocarbon-contaminated site

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Oil wastes as unconventional substrates for rhamnolipid biosurfactant production by Pseudomonas aeruginosa LBI

Oil wastes were evaluated as alternative low-cost substrates for the production of rhamnolipids by Pseudomonas aeruginosa LBI strain. Wastes obtained from soybean, cottonseed, babassu, palm, and corn oil refinery were tested. The soybean soapstock waste was the best substrate, generating 11.7 g/L of rhamnolipids with a surface tension of 26.9 mN/m, a critical micelle concentration of 51.5 mg/L, and a production yield of 75%. The monorhamnolipid RhaC10C10 predominates when P. aeruginosa LBI was cultivated on hydrophobic substrates, whereas hydrophilic carbon sources form the dirhamnolipid Rha2C10C10 predominantly. © 2005 American Chemical Society and American Institute of Chemical Engineers.

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

The las and rhl quorum sensing (QS) systems regulate the expression of several genes in response to cell density changes in Pseudomonas aeruginosa. Many of these genes encode surface-associated or secreted virulence factors. Proteins from stationary phase culture supernatants were collected from wild-type and P. aeruginosa PAO1 mutants deficient in one or more of the lasRI, rhIRI and vfr genes and analysed using two-dimensional gel electrophoresis. All mutants released significantly lower amounts of protein than the wild-type. Protein spot patterns from each strain were compared using image analysis and visible spot differences were identified using mass spectrometry. Several previously unknown OS-regulated proteins were characterized, including an aminopeptidase (PA2939), an endoproteinase (PrpL) and a unique 'hypothetical' protein (PA0572), which could not be detected in the culture supernatants of Delta/as mutants, although they were unaffected in Deltarhl mutants. Chitin-binding protein (CbpD) and a hypothetical protein (PA4944) with similarity to host factor I (HF-1) could not be detected when any of the lasRI or rhIRI genes were disrupted. Fourteen proteins were present at significantly greater levels in the culture supernatants of OS mutants, suggesting that QS may also negatively control the expression of some genes. Increased levels of two-partner secretion exoproteins (PA0041 and PA4625) were observed and may be linked to increased stability of their cognate transporters in a CS-defective background. Known QS-regulated extracellular proteins, including elastase (lasB), LasA protease (lasA) and alkaline metalloproteinase (aprA) were also detected.

Sub-inhibitory concentrations of ceftazidime and tobramycin reduce the quorum sensing signals of Pseudomonas aeruginosa

Aim: Concentrations of antimicrobials below minimum inhibitory concentration (subMIC) may reduce the production by Pseudomonas aeruginosa of virulence factors such as elastase. We sought to determine whether the reduction in elastase production may be mediated by a reduction in acyl-homoserine lactones. Methods: Pseudomonas aeruginosa in broth was exposed to three conditions for ceftazidime and tobramycin: control, 6% MIC and 25% MIC. Elastase was assayed using elastin congo red. N-(3-Oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl-homoserine lactone (C4-HSL) were assayed using biosensor Escherichia coli. Results: Elastase was unchanged with ceftazidime. Elastase was reduced by 16% at 6% MIC tobramycin and reduced by 70% at 25% MIC tobramycin (P

Rhamnolipids as biosurfactants from renewable resources: Concepts for next-generation rhamnolipid production

Several microorganisms are known to produce a wide variety of surface-active substances, which are referred to as biosurfactants. Interesting examples for biosurfactants are rhamnolipids, glycolipids mainly known from Pseudomonas aeruginosa produced during cultivation on different substrates like vegetable oils, sugars, glycerol or hydrocarbons. However, besides costs for downstream processing of rhamnolipids, relatively high raw-material prices and low productivities currently inhibit potential economical production of rhamnolipids on an industrial scale. This review focuses on cost-effective and sustainable production of rhamnolipids by introducing new possibilities and strategies regarding renewable substrates. Additionally, past and recent production strategies using alternative substrates such as agro-industrial byproducts or wastes are summarized. Requirements and concepts for next-generation rhamnolipid producing strains are discussed and potential targets for strain-engineering are presented. The discussion of potential new strategies is supported by an analysis of the metabolism of different Pseudomonas species. According to calculations of theoretical substrate-to-product conversion yields and current world-market price analysis, different renewable substrates are compared and discussed from an economical point of view. A next-generation rhamnolipid producing strain, as proposed within this review, may be engineered towards reduced formation of byproducts, increased metabolic spectrum, broadened substrate spectrum and controlled regulation for the induction of rhamnolipid synthesis. (C) 2012 Elsevier Ltd. All rights reserved.

Dispersibility of Barium Titanate Suspension in the Presence of Polyelectrolytes: A Review

Dispersibility of colloidal barium titanate suspensions is reviewed with an emphasis on the use of various polyelectrolytes as dispersants. The fundamentals of colloidal stability are discussed followed by the colloidal properties of barium titanate powder. Dispersion behavior of BaTiO3 in both nonaqueous and aqueous media has been reviewed. Several studies on the stabilization of micron and nano-sized barium titanate using various polymeric dispersants and a rhamnolipid biosurfactant are presented and discussed. The article attempts to provide a comprehensive review of the current state-of-the-art in the area of colloidal processing of barium titanate.

Microbial Biosurfactants – Review

Biosurfactants are surface active compounds released by microorganisms. They are biodegradable non-toxic and eco-friendly materials. In this review we have updated the information about different microbial surfactants. The biosurfactant production depends on the fermentation conditions, environmental factors and nutrient availability. The extraction of the biosurfactants from the cell-free supernatant using the solvent extraction procedure and the qualitative and quantitative analysis has been discussed with appropriate equipment details. The application of the biosurfactant includes biomedical, cosmetic and bioremediation. The type of microbial biosurfactants include trehalose lipids, rhamnolipids, sophorolipids, glycolipids, cellobiose lipids, polyol lipids, diglycosyl diglycerides, lipoloysaccharides, arthrofactin, lichensyn A and B, surfactin, viscosin, phospholipids, sulphonyl lipids and fatty acids. Rhamnolipid biosurfactants produced by Pseudomonas aeruginosa DS10-129 showed significant applications in the bioremediation of hydrocarbons in gasoline spilled soil and petroleum oily sludge. Rhamnolipid biosurfactant enhanced the bioremediation process by releasing the weathered oil from the soil matrices and enhanced the bioavailability of hydrocarbons for microbial degradation. It is having potential applications in the remediation of hydrocarbon contaminated sites. Biosurfactants from marine microorganisms also offer great potential in bioremediation of oil contaminated oceanic environments

Investigação dos efeitos tóxicos do biossurfactante ramnolipídio e suas implicações quando usado na biorremediação de águas contaminadas por petróleo

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

Bioremediation of PAHs - Limitations and soultions

Introduction 1.1 Occurrence of polycyclic aromatic hydrocarbons (PAH) in the environment Worldwide industrial and agricultural developments have released a large number of natural and synthetic hazardous compounds into the environment due to careless waste disposal, illegal waste dumping and accidental spills. As a result, there are numerous sites in the world that require cleanup of soils and groundwater. Polycyclic aromatic hydrocarbons (PAHs) are one of the major groups of these contaminants (Da Silva et al., 2003). PAHs constitute a diverse class of organic compounds consisting of two or more aromatic rings with various structural configurations (Prabhu and Phale, 2003). Being a derivative of benzene, PAHs are thermodynamically stable. In addition, these chemicals tend to adhere to particle surfaces, such as soils, because of their low water solubility and strong hydrophobicity, and this results in greater persistence under natural conditions. This persistence coupled with their potential carcinogenicity makes PAHs problematic environmental contaminants (Cerniglia, 1992; Sutherland, 1992). PAHs are widely found in high concentrations at many industrial sites, particularly those associated with petroleum, gas production and wood preserving industries (Wilson and Jones, 1993). 1.2 Remediation technologies Conventional techniques used for the remediation of soil polluted with organic contaminants include excavation of the contaminated soil and disposal to a landfill or capping - containment - of the contaminated areas of a site. These methods have some drawbacks. The first method simply moves the contamination elsewhere and may create significant risks in the excavation, handling and transport of hazardous material. Additionally, it is very difficult and increasingly expensive to find new landfill sites for the final disposal of the material. The cap and containment method is only an interim solution since the contamination remains on site, requiring monitoring and maintenance of the isolation barriers long into the future, with all the associated costs and potential liability. A better approach than these traditional methods is to completely destroy the pollutants, if possible, or transform them into harmless substances. Some technologies that have been used are high-temperature incineration and various types of chemical decomposition (for example, base-catalyzed dechlorination, UV oxidation). However, these methods have significant disadvantages, principally their technological complexity, high cost , and the lack of public acceptance. Bioremediation, on the contrast, is a promising option for the complete removal and destruction of contaminants. 1.3 Bioremediation of PAH contaminated soil & groundwater Bioremediation is the use of living organisms, primarily microorganisms, to degrade or detoxify hazardous wastes into harmless substances such as carbon dioxide, water and cell biomass Most PAHs are biodegradable unter natural conditions (Da Silva et al., 2003; Meysami and Baheri, 2003) and bioremediation for cleanup of PAH wastes has been extensively studied at both laboratory and commercial levels- It has been implemented at a number of contaminated sites, including the cleanup of the Exxon Valdez oil spill in Prince William Sound, Alaska in 1989, the Mega Borg spill off the Texas coast in 1990 and the Burgan Oil Field, Kuwait in 1994 (Purwaningsih, 2002). Different strategies for PAH bioremediation, such as in situ , ex situ or on site bioremediation were developed in recent years. In situ bioremediation is a technique that is applied to soil and groundwater at the site without removing the contaminated soil or groundwater, based on the provision of optimum conditions for microbiological contaminant breakdown.. Ex situ bioremediation of PAHs, on the other hand, is a technique applied to soil and groundwater which has been removed from the site via excavation (soil) or pumping (water). Hazardous contaminants are converted in controlled bioreactors into harmless compounds in an efficient manner. 1.4 Bioavailability of PAH in the subsurface Frequently, PAH contamination in the environment is occurs as contaminants that are sorbed onto soilparticles rather than in phase (NAPL, non aqueous phase liquids). It is known that the biodegradation rate of most PAHs sorbed onto soil is far lower than rates measured in solution cultures of microorganisms with pure solid pollutants (Alexander and Scow, 1989; Hamaker, 1972). It is generally believed that only that fraction of PAHs dissolved in the solution can be metabolized by microorganisms in soil. The amount of contaminant that can be readily taken up and degraded by microorganisms is defined as bioavailability (Bosma et al., 1997; Maier, 2000). Two phenomena have been suggested to cause the low bioavailability of PAHs in soil (Danielsson, 2000). The first one is strong adsorption of the contaminants to the soil constituents which then leads to very slow release rates of contaminants to the aqueous phase. Sorption is often well correlated with soil organic matter content (Means, 1980) and significantly reduces biodegradation (Manilal and Alexander, 1991). The second phenomenon is slow mass transfer of pollutants, such as pore diffusion in the soil aggregates or diffusion in the organic matter in the soil. The complex set of these physical, chemical and biological processes is schematically illustrated in Figure 1. As shown in Figure 1, biodegradation processes are taking place in the soil solution while diffusion processes occur in the narrow pores in and between soil aggregates (Danielsson, 2000). Seemingly contradictory studies can be found in the literature that indicate the rate and final extent of metabolism may be either lower or higher for sorbed PAHs by soil than those for pure PAHs (Van Loosdrecht et al., 1990). These contrasting results demonstrate that the bioavailability of organic contaminants sorbed onto soil is far from being well understood. Besides bioavailability, there are several other factors influencing the rate and extent of biodegradation of PAHs in soil including microbial population characteristics, physical and chemical properties of PAHs and environmental factors (temperature, moisture, pH, degree of contamination). Figure 1: Schematic diagram showing possible rate-limiting processes during bioremediation of hydrophobic organic contaminants in a contaminated soil-water system (not to scale) (Danielsson, 2000). 1.5 Increasing the bioavailability of PAH in soil Attempts to improve the biodegradation of PAHs in soil by increasing their bioavailability include the use of surfactants , solvents or solubility enhancers.. However, introduction of synthetic surfactant may result in the addition of one more pollutant. (Wang and Brusseau, 1993).A study conducted by Mulder et al. showed that the introduction of hydropropyl-ß-cyclodextrin (HPCD), a well-known PAH solubility enhancer, significantly increased the solubilization of PAHs although it did not improve the biodegradation rate of PAHs (Mulder et al., 1998), indicating that further research is required in order to develop a feasible and efficient remediation method. Enhancing the extent of PAHs mass transfer from the soil phase to the liquid might prove an efficient and environmentally low-risk alternative way of addressing the problem of slow PAH biodegradation in soil.

Production of biosurfactant “Biosur-Pm” by Pseudomonas maltophila CSV89: characterization and role in hydrocarbon uptake

Pseudomonas maltophilia CSV89, a soil bacterium, produces an extracellular biosurfactant, ''Biosur-Pm''. The partially purified product is nondialyzable and chemically composed of 50% protein and 12-15% sugar, which indicates the complex nature of Biosur-Pm. It reduces the surface tension of water from 73 to 53 x 10(-3) N m(-1) and has a critical micellar concentration of 80 mg/l. Compared to aliphatic hydrocarbons, Biosur-Pm shows good activity against aromatic hydrocarbons. The emulsion formed is stable and does not require any metal ions for emulsification. The kinetics of Biosur-Pm production suggest that its synthesis isa growth-associated and pH-dependent process. At pH 7.0, cells produced more Biosur-Pm with less cell surface hydrophobicity. At pH 8.0, however, the cells produced less Biosur-Pm with more cell surface hydrophobicity and showed a twofold higher affinity for aromatic hydrocarbons compared to the cells grown at pH 7.0. The Biosur-Pm showed a pH-dependent release, stimulated growth of the producer strain on mineral salts medium with 1-naphthoic acid when added externally, and facilitated the conversion of salicylate to catechol. All these results suggest that Biosur-Pm is probably a cell-wall component and helps in hydrocarbon assimilation/uptake.

Structure and Applications of a Rhamnolipid Surfactant Produced in Soybean Oil Waste

Soybean oil soapstock was utilized as an alternative carbon source for the production of rhamnolipids by Pseudomonas aeruginosa LBI strain. The chemical composition and properties of the rhamnolipid mixture obtained were determined to define its potential applications. The chemical characterization of the rhamnolipid has revealed the presence of ten different homologues. The monorhamnolipid RhaC(10)C(10) and the dirhamnolipid Rha(2)C(10)C(10) were the main components of the mixture that showed predominance of 44% and 29%, respectively, after 144-h of cultivation. The biosurfactant was able to form stable emulsions with several hydrocarbons and showed excellent emulsification for soybean oil and chicken fat (100%). The rhamnolipid removed 67% of crude oil present in sand samples and presented antimicrobial activity against Bacillus cereus and Mucor miehei at 64 mu g/mL and inhibition of Neurospora crassa, Staphylococcus aureus, and Micrococcus luteus at 256 mu g/mL. The results demonstrated that the rhamnolipid produced in soybean oil soapstock can be useful in environmental and food industry applications.

Rhamnolipid produced from agroindustrial wastes enhances hydrocarbon biodegradation in contaminated soil

A crude biosurfactant solution was produced by Pseudomonas aeruginosa growing on agroindustrial wastes as the substrate and used to study its effect on hydrocarbon biodegradation by the indigenous soil microflora under laboratory conditions. Two concentrations were studied at first and 1 mg of biosurfactant/g of soil showed to be the most efficient for the total petroleum hydrocarbon reduction, which reached 85% at the first 20 days in soil microcosms. Respirometric and microbial analyses showed that the biosurfactant added did not have toxic effects over the microbial population. The use of a biosurfactant for bioremediation has been limited because of its high cost production. Biosurfactants produced from cost-free by-products combines waste minimization with economic potential bioremediation process.