Optimization and characterization of biosurfactant produced by indigenous Brevibacillus borstelensis isolated from a low permeability reservoir for application in MEOR.

Biosurfactants are expected to be a key factor for microbial enhanced oil recovery (MEOR). In this study, we described the novel biosurfactant-producing strain Brevibacillus borstelensis YZ-2 isolated from a low permeability oil reservoir. We purified and characterized the biosurfactants produced by this YZ-2 strain via thin-layer chromatography and MALDI-TOF-MS, revealing them to be fengycins. We additionally used a Box-Behnken design approach to optimize fermentation conditions in order to maximize the biosurfactants production. Core flooding experiments showed that biosurfactants produced by YZ-2 can significantly enhance crude oil recovery. Micro-model tests showed that emulsification and IFT reduction was the main EOR mechanism of the YZ biosurfactant in the oil wet model. In summary, these findings highlight the potential of Brevibacillus borstelensis YZ-2 and its metabolites for MEOR.

Surfactants: physicochemical interactions with biological macromolecules.

Macromolecules are essential cellular components in biological systems responsible for performing a large number of functions that are necessary for growth and perseverance of living organisms. Proteins, lipids and carbohydrates are three major classes of biological macromolecules. To predict the structure, function, and behaviour of any cluster of macromolecules, it is necessary to understand the interaction between them and other components through basic principles of chemistry and physics. An important number of macromolecules are present in mixtures with surfactants, where a combination of hydrophobic and electrostatic interactions is responsible for the specific properties of any solution. It has been demonstrated that surfactants can help the formation of helices in some proteins thereby promoting protein structure formation. On the other hand, there is extensive research towards the use of surfactants to solubilize drugs and pharmaceuticals; therefore, it is evident that the interaction between surfactants with macromolecules is important for many applications which includes environmental processes and the pharmaceutical industry. In this review, we describe the properties of different types of surfactants that are relevant for their physicochemical interactions with biological macromolecules, from macromolecules-surfactant complexes to hydrophobic and electrostatic interactions.

The effect of sophorolipids against microbial biofilms on medical-grade silicone.

Recent medical strategies rely on the search for effective antimicrobials as surface coatings to prevent and treat infections in humans and animals. Biosurfactants have recently been shown to have properties as antiadhesive and antibiofilm agents. Sophorolipids in particular are biosurfactant molecules known to act as therapeutic agents. This study aimed to evaluate antimicrobial properties of sophorolipids in medical-grade silicone discs using strains of clinical relevance. Sophorolipids were produced under fed batch conditions, ESI-MS analyses were carried out to confirm the congeners present in each formulation. Three different products were obtained SLA (acidic congeners), SL18 (lactonic congeners) and SLV (mixture of acidic and lactonic congeners) and were tested against Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 10145 and Candida albicans IHEM 2894. All three congener mixtures showed a biofilms disruption effect (> 0.1 % w/v) of 70 %, 75 % and 80 % for S. aureus, P. aeruginosa and C. albicans, respectively. On pre-coated silicone discs, biofilm formation of S. aureus was reduced by 75 % using SLA 0.8 % w/v. After 1.5 h the inhibition of C. albicans attachment was between 45-56 % whilst after 24 h incubation the percentage of inhibition for the cell attachment increased to 68-70 % when using SLA 0.8 % w/v. Finally, in co-incubation experiments SLA 0.05 % w/v significantly reduced the ability of S. aureus and C. albicans to form biofilms and to adhere to surfaces by 90-95 % at concentrations between 0.025-0.1 % w/v. In conclusion sophorolipids significantly reduced the cell attachment of both tested strains which suggests that these molecules could have a potential role as coating agents on medical grade silicone devices for the preventions of Gram positive bacteria and yeast infections.

Microbial biosurfactants: current trends and applications in agricultural and biomedical industries.

Synthetic surfactants are becoming increasingly unpopular in many applications due to previously disregarded effects on biological systems and this has led to a new focus on replacing such products with biosurfactants that are biodegradable and produced from renewal resources. Microbially derived biosurfactants have been investigated in numerous studies in areas including: increasing feed digestibility in an agricultural context, improving seed protection and fertility, plant pathogen control, antimicrobial activity, antibiofilm activity, wound healing and dermatological care, improved oral cavity care, drug delivery systems and anticancer treatments. The development of the potential of biosurfactants has been hindered somewhat by the myriad of approaches taken in their investigations, the focus on pathogens as source species and the costs associated with large-scale production. Here, we focus on various microbial sources of biosurfactants and the current trends in terms of agricultural and biomedical applications.

The performance of surfactant mixtures at low temperatures.

Optimising detergency at lower temperatures is of increasing interest due to environmental and economic factors, and requires a greater understanding of the effects of temperature on the adsorption of surfactant mixtures at interfaces. The adsorption properties of surfactant mixtures and biosurfactant/surfactant mixtures have been studied at room temperatures and at temperatures below ambient using surface tension and neutron reflectivity measurements. For the ternary surfactant mixture of octaethylene monododecyl ether, C12E8, sodium dodecyl 6-benzene sulfonate, LAS, and sodium dioxyethylene glycol monododecyl sulfate, SLES, the surface tension at the air-water interface increases with decreasing temperature. In contrast, there is a notable reduction in the increase in the surface tension with a decrease in temperature from 25 °C to 10 °C for the 5 component rhamnolipid/surfactant mixture of the mono-rhamnose, R1, and di-rhamnose, R2, with C12E8/LAS/SLES. The associated neutron reflectivity data for the ternary C12E8/LAS/SLES mixture and the significant observation is that the 3, 4, and 5-component mixtures containing rhamnolipids in conjunction with the other surfactants show changes in composition and adsorbed amounts of the individual components which are close to the experimental error. However the significant observation is that the neutron reflectivity data indicate that the improved surface tension tolerance at lower temperatures is associated with the dominance of the rhamnolipid adsorption in such mixtures. Hence the introduction of the rhamnolipids provides a tolerance to the adverse effects associated with reduced temperatures, and a potential for improved detergency at relatively low temperatures.

Identification and characterisation of short chain rhamnolipid production in a previously uninvestigated, non-pathogenic marine pseudomonad.

This study aimed to identify and characterise biosurfactant compounds produced by bacteria associated with a marine eukaryotic phytoplankton bloom. One strain, designated MCTG214(3b1), was isolated by enrichment with polycyclic aromatic hydrocarbons and based on 16S rDNA, and gyrB sequencing was found to belong to the genus Pseudomonas, however not related to P. aeruginosa. Cell-free supernatant samples of strain MCTG214(3b1) at stationary phase showed significant reductions in surface tension. HPLC-MS and NMR analysis of these samples indicated the presence of five different rhamnolipid (RL) congeners. Di-rhamnolipids accounted for 87% relative abundance and all congeners possessed fatty acid moieties consisting of 8-12 carbons. PCR screening of strain MCTG214(3b1) DNA revealed homologues to the P. aeruginosa RL synthesis genes rhlA and rhlB; however, no rhlC homologue was identified. Using the Galleria mellonella larvae model, strain MCTG214(3b1) was demonstrated to be far less pathogenic than P. aeruginosa. This study identifies for the first time a significantly high level of synthesis of short chain di-rhamnolipids by a non-pathogenic marine Pseudomonas species. We postulate that RL synthesis in Pseudomonas sp. MCTG214(3b1) is carried out by enzymes expressed from rhlA/B homologues similar to those of P. aeruginosa; however, a lack of rhlC potentially indicates the presence of a second novel rhamnosyltransferase responsible for the di-rhamnolipid congeners identified by HPLC-MS.

Biosurfactant-facilitated leaching of metals from spent hydrodesulphurization catalyst.

AIMS: To investigate the capabilities of different types of biosurfactants (rhamnolipids, lipopeptides, sophorolipids) to remove metals and carbon from the hazardous spent hydrodesulphurization (HDS) catalyst generated by petroleum refineries. METHODS AND RESULTS: Biosurfactants were prepared and used to treat spent HDS catalyst. Metal and carbon contents were analysed and compared with those from no-biosurfactant control treatments. All biosurfactant treatments increased carbon loss percentage from the spent HDS catalyst. The lipopeptide treatment LI, containing 17·34 mg ml-1 of crude biosurfactants, caused the highest carbon loss percentage (44·5%). Rhamnolipids were, in general, better than sophorolipids and lipopeptides as metal-removing agents. The metal content decreased as the concentration of rhamnolipids decreased. The R5 treatment, which contained 0·4 mg l-1 of crude rhamnolipids, caused the highest reduction in metal content. Molybdenum, nickle and vanadium contents were reduced by 90, 30 and 70% respectively. CONCLUSIONS: Biosurfactants might have potential application for metals and coke removal from spent HDS catalysts. The bioleaching capability depends on the type and concentration of the biosurfactant. SIGNIFICANCE AND IMPACT OF THE STUDY: This study, after further in-depth investigations, might lead to the development of an eco-friendly and economic technology to treat or even regenerate the environmentally hazardous spent HDS catalysts, which are generated in huge amounts by the petroleum refineries.

Rhamnolipids and lactonic sophorolipids: natural antimicrobial surfactants for oral hygiene.

AIMS: To assess the efficacy of rhamnolipid (mixture of monorhamnolipid and dirhamnolipid congeners), purified monorhamnolipid, dirhamnolipid and lactonic sophorolipid biosurfactants against pathogens important for oral hygiene. METHODS AND RESULTS: Acquired and produced biosurfactants were fully characterized to allow the antimicrobial activity to be assigned to the biosurfactant congeners. Antimicrobial activity was assessed using the resazurin-aided microdilution method. Mixed rhamnolipid JBR425 (MR) and lactonic sophorolipids (LSLs) demonstrated the lowest minimum inhibitory concentration (MIC) which ranged between 100 and 400 µg ml-1 against Streptococcus mutans, Streptococcus oralis, Actinomyces naeslundii, Neisseria mucosa and Streptococcus sanguinis. Combining these biosurfactants with standard antimicrobial agents namely chlorhexidine, sodium lauryl sulphate, tetracycline HCl and ciprofloxacin showed a dramatic drop in the MIC values. In addition, in vitro studies demonstrated the biosurfactants' ability to prevent and disrupt oral pathogens biofilms. The increased permeability of microorganisms treated with biosurfactant, as shown using bisbenzimide dye, in part explains the inhibition effect. CONCLUSION: The results demonstrate that rhamnolipids and LSLs have the ability to inhibit oral pathogens both in planktonic and oral biofilm states. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings indicate the potential value of biosurfactants for both oral hygiene and the pharmaceutical industries since there is a serious need to reduce the reliance on synthetic antimicrobials and antibiotics.

Accelerated in vivo wound healing evaluation of microbial glycolipid containing ointment as a transdermal substitute.

A potent biosurfactant (BS) producing Bacillus licheniformis SV1 (NCBI GenBank Accession No. KX130852) was isolated from oil contaminated soil sample. Physicochemical investigations (TLC, HPLC, FTIR, GC-MS and NMR) revealed it to be glycolipid in nature. Fibroblast culture assay showed cytocompatibility and increased cell proliferation of 3T3/NIH fibroblast cells treated with this biosurfactant when checked using MTT assay and DAPI fluorescent staining. To evaluate the wound healing potential, BS ointment was formulated and checked for its spreadability and viscosity consistency. In vivo wound healing examination of full thickness skin excision wound rat model demonstrated the prompt re-epithelialization and fibroblast cell proliferation in the early phase while quicker collagen deposition in later phases of wound healing when BS ointment was used. These results validated the potential usage of BS ointment as a transdermal substitute for faster healing of impaired skin wound. Biochemical evaluation also substantiated the highest concentration of hydroxyproline (32.18±0.46, p<0.001) in the BS ointment treated animal tissue samples compared to the control. Hematoxylin-Eosin (H&E) and Masson's Trichrome staining validated the presence of increased amount of collagen fibers and blood vessels in the test animals treated with BS ointment.

Self-assembly in dilute mixtures of non-ionic and anionic surfactants and rhamnolipd biosurfactants.

The self-assembly of dilute aqueous solutions of a ternary surfactant mixture and rhamnolipid biosurfactant/surfactant mixtures has been studied by small angle neutron scattering. In the ternary surfactant mixture of octaethylene glycol monododecyl ether, C12E8, sodium dodecyl 6-benzene sulfonate, LAS, and sodium dioxyethylene monododecyl sulfate, SLES, small globular interacting micelles are observed over the entire composition and concentration range studied. The modelling of the scattering data strongly supports the assumption that the micelle compositions are close to the solution compositions. In the 5-component rhamnolipid/surfactant mixture of the mono-rhamnose, R1, di-rhamnose, R2, rhamnolipids with C12E8/LAS/SLES, globular micelles are observed over much of the concentration and composition range studied. However, for solutions relatively rich in rhamnolipid and LAS, lamellar/micellar coexistence is observed. The transition from globular to more planar structures arises from a synergistic packing in the 5 component mixture. It is not observed in the individual components nor in the ternary C12E8/LAS/SLES mixture at these relatively low concentrations. The results provide an insight into how synergistic packing effects can occur in the solution self-assembly of complex multi-component surfactant mixtures, and give rise to an unexpected evolution in the phase behaviour.

Effect of biosurfactants on Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a BioFlux channel.

Recent studies have indicated that biosurfactants play a role both in maintaining channels between multicellular structures in biofilms and in dispersal of cells from biofilms. A combination of caprylic acid (0.01 % v/v) together with rhamnolipids (0.04 % v/v) was applied to biofilms of Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 9144 and a mixed culture under BioFlux flowthrough conditions and caused disruption of the biofilms. The biofilms were also treated with a combination of rhamnolipids (0.04 % v/v) and sophorolipids (0.01 %). Control treatments with PBS 1× had no apparent effect on biofilm disruption. The Gram-positive bacterium (S. aureus ATCC 9144) was more sensitive than P. aeruginosa ATCC 15442 in terms of disruption and viability as shown by Live/Dead staining. Disruption of biofilms of P. aeruginosa ATCC 15442 was minimal. Oxygen consumption by biofilms, after different treatments with biosurfactants, confirms that sophorolipid on its own is unable to kill/inhibit cells of P. aeruginosa ATCC 15442, and even when used in combination with rhamnolipids, under static conditions, no decrease in the cell viability was observed. Cells in biofilms exposed to mono-rhamnolipids (0.04 % v/v) showed behaviour typical of exposure to bacteriostatic compounds, but when exposed to di-rhamnolipids (0.04 % v/v), they displayed a pattern characteristic of bactericidal compounds.

Pseudomonas aeruginosa biofilm disruption using microbial surfactants.

AIMS: 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). METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: The disruption of biofilms has major significance in many industrial and domestic cleaning applications and in medical situations.

A study of anti-cancer effects of Funalia trogii in vitro and in vivo.

Extracts of natural products have been used for many years for health benefits. We report on an in vitro and in vivo study into the anti-tumour efficacy of an aqueous extract of the mycelial form of basidiomycete, Funalia trogii. A variety of biological assays were used to show that a 4h exposure of HT29, LNCaP, PC3, MCF-7 and MDA-MB-231 tumour cells to extract (0.5-5.0 mg/ml) resulted in significant cytotoxicity. In a clonogenic assay, IC50 values were found to range from 0.4-0.72 mg/ml; exposing fibroblast cells to the extract resulted in no cell kill. The extract resulted in significant cell kill in proliferating endothelial cells but had no toxicity to quiescent cells, this is useful in targeting tumour tissue since endothelial cells in tumours proliferate more rapidly that those found in other parts of the body. When tumours grown in immune compromised mice were injected intratumourally with extract (5 mg/ml twice a week for two weeks), a 9 day tumour growth delay was observed. The results indicate that the mycelial extract of F. trogii has a promising anti-tumour property.

Mixing behavior of the biosurfactant, rhamnolipid, with a conventional anionic surfactant, sodium dodecyl benzene sulfonate.

The use of small angle neutron scattering, SANS, neutron reflectivity, NR, and surface tension to study the mixing properties of the biosurfactant rhamnolipid with a conventional anionic surfactant, sodium dodecyl 6-benzene sulfonate, LAS, is reported. The monorhamnose rhamnolipid, R1, mixes close to ideally with LAS at the air-water interface, whereas for mixtures of LAS with the dirhamnose rhamnolipid, R2, the LAS strongly partitions to the air-water interface relative to R2, probably because of the steric hindrance of the larger R2 headgroup. These trends in the binary mixtures are also reflected in the ternary R1/R2/LAS mixtures. However, for these ternary mixtures, there is also a pronounced synergy in the total adsorption, which reaches a maximum for a LAS/rhamnolipid mole ratio of about 0.6 and a R1/R2 mol ratio of about 0.5, an effect which is not observed in the binary mixtures. In solution, the R1/LAS mixtures form relatively small globular micelles, L(1), at low surfactant concentrations (<20 mM), more planar structures (lamellar, L(α), unilamellar/multilamellar vesicles, ulv/mlv) are formed at higher surfactant concentrations for R1 and LAS rich compositions, and a large mixed phase (L(α)/L(1) and L(1)/L(α)) region forms at intermediate surfactant compositions. In contrast, for the R2/LAS mixtures, the higher preferred curvature of R2 dominates the phase behavior. The predominant microstructure is in the form of small globular micelles, except for solution compositions rich in LAS (>80 mol % LAS) where more planar structures are formed. For the ternary mixtures, there is an evolution in the resulting phase behavior from one dominated by L(1) (R2 rich) to one dominated by planar structures, L(α), (R1, LAS rich), and which strongly depends upon the LAS/rhamnolipid and R1/R2 mole ratio.

Solution self-assembly and adsorption at the air-water interface of the monorhamnose and dirhamnose rhamnolipids and their mixtures.

The self-assembly in solution and adsorption at the air-water interface, measured by small-angle neutron scattering, SANS, and neutron reflectivity, NR, of the monorhamnose and dirhamnose rhamnolipids (R1, R2) and their mixtures, are discussed. The production of the deuterium-labeled rhamnolipids (required for the NR studies) from a Pseudomonas aeruginosa culture and their separation into the pure R1 and R2 components is described. At the air-water interface, R1 and R2 exhibit Langmuir-like adsorption isotherms, with saturated area/molecule values of about 60 and 75 Å(2), respectively. In R1/R2 mixtures, there is a strong partitioning of R1 to the surface and R2 competes less favorably because of the steric or packing constraints of the larger R2 dirhamnose headgroup. In dilute solution (<20 mM), R1 and R2 form small globular micelles, L(1), with aggregation numbers of about 50 and 30, respectively. At higher solution concentrations, R1 has a predominantly planar structure, L(α) (unilamellar, ULV, or bilamellar, BLV, vesicles) whereas R2 remains globular, with an aggregation number that increases with increasing surfactant concentration. For R1/R2 mixtures, solutions rich in R2 are predominantly micellar whereas solutions rich in R1 have a more planar structure. At an intermediate composition (60 to 80 mol % R1), there are mixed L(α)/L(1) and L(1)/L(α) regions. However, the higher preferred curvature associated with R2 tends to dominate the mixed R1/R2 microstructure and its associated phase behavior.

Bioemulsifier production by a halothermophilic Bacillus strain with potential applications in microbially enhanced oil recovery.

A halothermotolerant Gram-positive spore-forming bacterium was isolated from petroleum reservoirs in Iran and identified as Bacillus licheniformis sp. strain ACO1 by phenotypic characterization and 16S rRNA analysis. It showed a high capacity for bioemulsifier production and grew up to 60 degrees C with NaCl at 180 g l(-1). The optimum NaCl concentration, pH and temperature for bioemulsifier production were 4% (w/v), 8.0, and 45 degrees C, respectively. Although ACO1 did not utilize hydrocarbons, it had a high emulsifying activity (E (24) = 65 +/- 5%) on different hydrophobic substrates. Emulsification was optimal while growing on yeast extract as the sole carbon source and NaNO(3) as the nitrogen source. The efficiency of the residual oil recovery increased by 22% after in situ growth of B. licheniformis ACO1 in a sand-pack model saturated with liquid paraffin.

Lactic acid production by mixed cultures of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus helveticus.

Lactic acid production using Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) individually or as mixed culture on cheese whey in stirred or static fermentation conditions was evaluated. Lactic acid production, residual sugar and cell biomass were the main features examined. Increased lactic acid production was observed, when mixed cultures were used in comparison to individual ones. The highest lactic acid concentrations were achieved when K. marxianus yeast was combined with L. delbrueckii ssp. bulgaricus, and when all the strains were used revealing possible synergistic effects between the yeast and the two lactic acid bacteria. The same synergistic effects were further observed and verified when the mixed cultures were applied in sourdough fermentations, proving that the above microbiological system could be applied in the food fermentations where high lactic acid production is sought.

Whey-cheese production using freeze-dried kefir culture as a starter.

AIMS: The aim of the present study was to evaluate the use of a freeze-dried kefir culture in the production of a novel type of whey-cheese similar to traditional Greek Myzithra-cheese, to achieve improvement of the quality characteristics of the final product and the extension of shelf-life. METHODS AND RESULTS: The use of kefir culture as a starter led to increased lactic acid concentrations and decreased pH values in the final product compared with whey-cheese without starter culture. The effect of the starter culture on production of aroma-related compounds responsible for cheese flavour was also studied using the solid phase microextraction gas chromatography/mass spectrometry technique. Spoilage in unsalted kefir-whey-cheese was observed on the thirteenth and the twentieth day of preservation at 10 and 5 degrees C, respectively, while the corresponding times for unsalted whey-cheese preservation were 11 and 14 days. CONCLUSIONS: The cheeses produced were characterized as high-quality products during the preliminary sensory evaluation. An indication of increased preservation time was attributed to the freeze-dried kefir culture, which also seemed to suppress growth of pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggested the use of kefir culture as a means to extend the shelf-life of dairy products with reduced or no salt content.

Use of Saccharomyces cerevisiae cells immobilized on orange peel as biocatalyst for alcoholic fermentation.

A biocatalyst was prepared by immobilizing a commercial Saccharomyces cerevisiae strain (baker's yeast) on orange peel pieces for use in alcoholic fermentation and for fermented food applications. Cell immobilization was shown by electron microscopy and by the efficiency of the immobilized biocatalyst for alcoholic fermentation of various carbohydrate substrates (glucose, molasses, raisin extracts) and at various temperatures (30-15 degrees C). Fermentation times in all cases were low (5-15 h) and ethanol productivities were high (av. 150.6 g/ld) showing good operational stability of the biocatalyst and suitability for commercial applications. Reasonable amounts of volatile by-products were produced at all the temperatures studied, revealing potential application of the proposed biocatalyst in fermented food applications, to improve productivities and quality.

Interference in adhesion of bacteria and yeasts isolated from explanted voice prostheses to silicone rubber by rhamnolipid biosurfactants.

AIMS: The effects and extent of adhesion of four different bacterial and two yeast strains isolated from explanted voice prostheses to silicone rubber with and without an adsorbed rhamnolipid biosurfactant layer obtained from Pseudomonasaeruginosa DS10-129 was studied. METHODS AND RESULTS: The ability of rhamnolipid biosurfactant to inhibit adhesion of micro-organisms to silicone rubber was investigated in a parallel-plate flow chamber. The anti-adhesive activity of the biosurfactant at different concentrations was significant against all the strains and depended on the micro-organism tested. The results showed an effective reduction in the initial deposition rates, and the number of bacterial cells adhering after 4 h, for all micro-organisms tested at the 4 g l(-1) undiluted rhamnolipid solution. Maximum initial reduction of adhesion rate (an average of 66%) occurred for Streptococcus salivarius GB 24/9 and Candida tropicalis GB 9/9. The number of cells adhering after 4 h on silicone rubber conditioned with biosurfactant was reduced to 48% for Staphylococcus epidermidis GB 9/6, Strep. salivarius GB 24/9, Staphylococcus aureus GB 2/1 and C. tropicalis GB 9/9 in comparison to controls. Perfusing the flow chamber with biosurfactant containing solution followed by the passage of a liquid-air interface, to investigate detachment of micro-organisms adhering to silicone rubber, produced high detachment (96%) of adhered cells for all micro-organisms studied, except for Staph. aureus GB 2/1 (67%). SIGNIFICANCE AND IMPACT OF THE STUDY: It is concluded that biosurfactant represent suitable compounds that should be considered in developing future strategies to prevent the microbial colonization of silicone rubber voice prostheses.