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The involvement of rhamnolipids in microbial cell adhesion and biofilm development - an approach for control?
Abstract
Biofilms are omnipresent in clinical and industrial settings and most of the times cause detrimental side effects. Finding efficient strategies to control surface-growing communities of microorganisms remains a significant challenge. Rhamnolipids are extracellular secondary metabolites with surface active properties mainly produced by Pseudomonas aeruginosa. There is growing evidence for the implication of this biosurfactant in different stages of biofilm development of this bacterium. Furthermore, rhamnolipids display a significant potential as anti-adhesive and disrupting agents against established biofilms formed by several bacterial and fungal species. Their low toxicity, biodegradability, efficiency and specificity, compared to synthetic surfactants typically used in biofilm control, might compensate for the economic hurdle still linked to their superior production costs and make them promising antifouling agents. This article is protected by copyright. All rights reserved.
... When a microorganism is deposited on a certain surface, a structured biological community or bacterial biofilm is formed [52]. This biofilm is self-regulated by quorum-sensing molecules and grows enveloped in an extracellular matrix which protects it from the environment, prevents the action of antimicrobial agents, and, consequently, greatly hinders their elimination [53]. Recent studies have shown that approximately 80% of chronic and recurrent microbial infections are caused by biofilms [54]. ...
... Rhamnolipids also play an important role in the disruption of biofilms. Similar to that observed in sophorolipids, the suitability of rhamnolipids to prevent microbial colonization of medical-grade instruments has also been investigated [53]. An investigation of the physicochemical and biological properties of rhamnolipids carried out by Ramos Da Silva (2019) found that the antibiofilm activity of these compounds is concentration-dependent. ...
The rapid emergence of multidrug-resistant pathogens worldwide has raised concerns regarding the effectiveness of conventional antibiotics. This can be observed in ESKAPE pathogens, among others, whose multiple resistance mechanisms have led to a reduction in effective treatment options. Innovative strategies aimed at mitigating the incidence of antibiotic-resistant pathogens encompass the potential use of biosurfactants. These surface-active agents comprise a group of unique amphiphilic molecules of microbial origin that are capable of interacting with the lipidic components of microorganisms. Biosurfactant interactions with different surfaces can affect their hydrophobic properties and as a result, their ability to alter microorganisms’ adhesion abilities and consequent biofilm formation. Unlike synthetic surfactants, biosurfactants present low toxicity and high biodegradability and remain stable under temperature and pH extremes, making them potentially suitable for targeted use in medical and pharmaceutical applications. This review discusses the development of biosurfactants in biomedical and therapeutic uses as antimicrobial and antibiofilm agents, in addition to considering the potential synergistic effect of biosurfactants in combination with antibiotics. Furthermore, the anti-cancer and anti-viral potential of biosurfactants in relation to COVID-19 is also discussed.
... Thus, plants can sense bacterial molecules as well as their direct precursors to trigger distinct immune responses. motility and for normal biofilm development (18)(19)(20). These glycolipids are produced from L-rhamnose and 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) precursors (15,21). ...
... In Pseudomonas and Burkholderia species, swarming motility is intimately related to the production of extracellular surfaceactive RLs and HAAs (22,25,(53)(54)(55). In addition, RL production affects bacterial biofilm architecture and increases affinity of cells for initial adherence to surfaces through increasing the cell's surface hydrophobicity (19,56). These exoproducts are therefore at the frontline during host colonization. ...
Significance
Activation of plant innate immunity relies on the perception of microorganisms through elicitors. Rhamnolipids and their precursor HAAs are exoproducts produced by bacteria. They are involved in bacterial surface dissemination and biofilm development. As these compounds are released in the extracellular milieu, they have the potential to be perceived by the plant immune system. Our work shows that both compounds independently activate plant immunity. We demonstrate that HAAs are perceived by the receptor protein kinase LORE. By contrast, rhamnolipids are not sensed by LORE but activate a noncanonical immune response influenced by the sphingolipid composition of the plant plasma membrane. Thus, plants can sense bacterial molecules as well as their direct precursors to trigger distinct immune responses.
... In the early stages of biofilm development, a small amount of RLs increases the hydrophobicity of cells, facilitating their attachment to the surface. As the biofilm matures, a high concentration of RLs inhibits cell attachment and microcolony formation, maintaining the complex architecture of the biofilm and enabling the seeding dispersal of motile cells [50,51]. Thus, reducing the levels of RLs under 100-Hz vibration exposure as detected here should enhance biofilm formation, which aligns with the effects found in the previous report [13]. ...
Sound has been shown to impact microbial behaviors. However, our understanding of the chemical and molecular mechanisms underlying these microbial responses to acoustic vibration is limited. In this study, we used untargeted metabolomics analysis to investigate the effects of 100-Hz acoustic vibration on the intra- and extracellular hydrophobic metabolites of P. aeruginosa PAO1. Our findings revealed increased levels of fatty acids and their derivatives, quinolones, and N -acylethanolamines upon sound exposure, while rhamnolipids (RLs) showed decreased levels. Further quantitative real-time polymerase chain reaction experiments showed slight downregulation of the rhlA gene (1.3-fold) and upregulation of fabY (1.5-fold), fadE (1.7-fold), and pqsA (1.4-fold) genes, which are associated with RL, fatty acid, and quinolone biosynthesis. However, no alterations in the genes related to the rpoS regulators or quorum-sensing networks were observed. Supplementing sodium oleate to P. aeruginosa cultures to simulate the effects of sound resulted in increased tolerance of P. aeruginosa in the presence of sound at 48 h, suggesting a potential novel response-tolerance correlation. In contrast, adding RL, which went against the response direction, did not affect its growth. Overall, these findings provide potential implications for the control and manipulation of virulence and bacterial characteristics for medical and industrial applications.
... It contributes to the formation of the biofilm layer by increasing the release of LPSs to the cell surface at low or normal concentrations of rhamnolipids. However, as a result of the overproduction of rhamnolipids, biofilm formation is prevented (Zulianello et al., 2006;Nickzad and Déziel, 2014;Köhler et al., 2010). ...
Pseudomonas aeruginosa (P. aeruginosa), the most pathogenic species among the pseudomonas species, is a bacterium that causes opportunistic infections resulting in significant damage to host tissues. P. aeruginosa, which is resistant to antibiotics, also causes fatal infection in human and animals. Infections caused by P. aeruginosa are difficult to treat due to its rapid proliferation in the environment and its ability to form biofilms that confer resistance to antibiotics. One of the main virulence factors of P. aeruginosa is its direct damage to host tissues, which disrupts the host’s defense mechanisms. P. aeruginosa is a food-borne pathogen often detected in various food groups such as meat, milk, fruit, vegetables, and water. In recent years, there has been a noticeable rise in food-borne contamination with P. aeruginosa. New measures are urgently needed in the treatment of patients with infections due to this agent, since P. aeruginosa can develop resistance to most antibacterials. In this review, general information about P. aeruginosa, which has gained importance for public health, will be given.
... Concerning surface adhesion, Wigneswaran et al. (2016) achieved an increased biofilm formation for a rhamnolipid-producing Pseudomonas putida strain compared to a control strain. Nickzad and Déziel (2014) and Chrzanowski et al. (2012) summarize various studies on the influence of rhamnolipids on biofilm formation, however, this has not been described for cyanobacteria so far. In fully developed biofilms, rhamnolipids also play an important role in maintaining the complex biofilm architecture, such as the formation of channels in the biofilm (Davey et al., 2003). ...
Terrestrial cyanobacteria grow as phototrophic biofilms and offer a wide spectrum of interesting products. For cultivation of phototrophic biofilms different reactor concepts have been developed in the last years. One of the main influencing factors is the surface material and the adhesion strength of the chosen production strain. In this work a flow chamber was developed, in which, in combination with optical coherence tomography and computational fluid dynamics simulation, an easy analysis of adhesion forces between different biofilms and varied surface materials is possible. Hereby, differences between two cyanobacteria strains and two surface materials were shown. With longer cultivation time of biofilms adhesion increased in all experiments. Additionally, the content of extracellular polymeric substances was analyzed and its role in surface adhesion was evaluated. To test the comparability of obtained results from the flow chamber with other methods, analogous experiments were conducted with a rotational rheometer, which proved to be successful. Thus, with the presented flow chamber an easy to implement method for analysis of biofilm adhesion was developed, which can be used in future research for determination of suitable combinations of microorganisms with cultivation surfaces on lab scale in advance of larger processes.
... et al., 2005), and invasion (Laurence et al., 2006). These properties can cause detrimental consequences in industrial and clinical settings due to the opportunistic pathogenicity of these strains (Nickzad and Déziel, 2014). Rhamnolipid synthesis in P. aeruginosa involves a sequential pathway involving three main genes i.e., rhlA, rhlB and rhlC respectively encoding three different enzymes (Soberón-Chávez et al., 2021). ...
Rhamnolipids are one of the most promising eco-friendly green glycolipids for bio-replacements of commercially available fossil fuel-based surfactants. However, the current industrial biotechnology practices cannot meet the required standards due to the low production yields, expensive biomass feedstocks, complicated processing, and opportunistic pathogenic nature of the conventional rhamnolipid producer strains. To overcome these problems, it has become important to realize non-pathogenic producer substitutes and high-yielding strategies supporting biomass-based production. We hereby review the inherent characteristics of Burkholderia thailandensis E264 which favor its competence towards such sustainable rhamnolipid biosynthesis. The underlying biosynthetic networks of this species have unveiled unique substrate specificity, carbon flux control and rhamnolipid congener profile. Acknowledging such desirable traits, the present review provides critical insights towards metabolism, regulation, upscaling, and applications of B. thailandensis rhamnolipids. Identification of their unique and naturally inducible physiology has proved to be beneficial for achieving previously unmet redox balance and metabolic flux requirements in rhamnolipids production. These developments in part are targeted by the strategic optimization of B. thailandensis valorizing low-cost substrates ranging from agro-industrial byproducts to next generation (waste) fractions. Accordingly, safer bioconversions can propel the industrial rhamnolipids in advanced biorefinery domains to promote circular economy, reduce carbon footprint and increased applicability as both social and environment friendly bioproducts.
... One of the necessary conditions for the long-term physiological function of probiotics in the host intestine is the ability to adhere to and colonize the host intestinal mucosal epithelial cells. They play a role after forming a stable flora in certain parts of the intestine, and adhesion is considered a key step in colonization [25]. At present, the in vitro cell adhesion model is widely used by researchers to evaluate the adhesion of LAB. ...
The probiotic role of lactic acid bacteria (LAB) in regulating intestinal microbiota to promote human health has been widely reported. However, the types and quantities of probiotics used in practice are still limited. Therefore, isolating and screening LAB with potential probiotic functions from various habitats has become a hot topic. In this study, 104 strains of LAB were isolated from and identified in traditionally fermented vegetables, fresh milk, healthy infant feces, and other environments. The antibacterial properties—resistance to acid, bile salts, and digestive enzymes—and adhesion ability of the strains were determined, and the biological safety of LAB with better performance was studied. Three LAB with good comprehensive performance were obtained. These bacteria had broad-spectrum antibacterial properties and good acid resistance and adhesion ability. They exhibited some tolerance to pig bile salt, pepsin, and trypsin and showed no hemolysis. They were sensitive to the selected antibiotics, which met the required characteristics and safety evaluation criteria for probiotics. An in vitro fermentation experiment and milk fermentation performance test of Lactobacillus rhamnosus (L. rhamnosus) M3 (1) were carried out to study its effect on the intestinal flora and fermentation performance in patients with inflammatory bowel disease (IBD). Studies have shown that this strain can effectively inhibit the growth of harmful microorganisms and produce a classic, pleasant flavor. It has probiotic potential and is expected to be used as a microecological agent to regulate intestinal flora and promote intestinal health. It can also be used as an auxiliary starter to enhance the probiotic value of fermented milk.
... et al., 2005), and invasion (Laurence et al., 2006). These properties can cause detrimental consequences in industrial and clinical settings due to the opportunistic pathogenicity of these strains (Nickzad and Déziel, 2014). Rhamnolipid synthesis in P. aeruginosa involves a sequential pathway involving three main genes i.e., rhlA, rhlB and rhlC respectively encoding three different enzymes (Soberón-Chávez et al., 2021). ...
... 15 Quorum sensing molecules regulate the self-production of rhamnolipids, which alter cellular interactions. 16 Rhamnose (Rha) sugars can be conjugated to lipid aliphatic chains which can be of varying lengths. One rhamnose (mono−rhamnolipid) can have one aliphatic chain (Rha−C) or two aliphatic chains (Rha−C−C). ...
Different bacterial cell surface associated biomolecules can be analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and coupled with collision induced dissociation (CID) for identification. Pseudomonas aeruginosa is an opportunistic, Gram-negative bacterium that causes acute or chronic biofilm infections. Cells of P. aeruginosa communicate through a system of signaling biomolecules known as quorum sensing (QS). The QS system can result in the production of biosurfactant rhamnolipids known to associate and alter the cellular membrane. MALDI-TOF utilizes a variety of matrices that can interact differently with biomolecules for selective ionization. We examined six common matrices to determine the optimal matrix specific to different molecule classes in P. aeruginosa associated with cell surfaces. Three major molecule classes (quinolones, rhamnolipids, and phospholipids) were observed to ionize selectively with the different matrices tested. Sodiated and protonated adducts differed between matrices utilized in our study. Isobaric ions were identified as different molecule classes depending on the matrix used. We highlight the role of matrix selection in MALDI-TOF identification of molecules within a complex biological mixture.
... Although biofilms can slightly resist chemical exposure through their EPS matrix (Pinto et al. 2020), rhamnolipids are known for their ability to break down biofilms [51], and therefore, we did not expect growth and development of biofilm material to occur on the exposed substrate at a comparable level to the control. Although not significant, loss of C within our biofilms in the rhamnolipid-treated systems led to a decreased C:N ratio. ...
Microbial biofilms have co-evolved with grazing animals, such as gastropods, to develop mutually beneficial relationships. Although microbial biofilms demonstrate resilience and resistance to chemical exposure, pre-existing relationships can be negatively affected by chemical input. In this study, we determined how the grazer, Littorina littorea (common periwinkle sea snail), and a biological surfactant (rhamnolipid) interact on a phototrophic marine biofilm. Biofilms were cultured in 32 twenty-liter buckets at the Queen’s University Marine Laboratory in Portaferry, Northern Ireland on clay tiles that were either exposed to 150 ppm of a rhamnolipid solution or that had no chemical exposure. L. littorea were added into half of the buckets, and biofilms were developed over 14 days. Biofilms exposed to grazing alone demonstrated high tolerance to the disturbance, while those growing on rhamnolipid-exposed substrate demonstrated resistance but experienced slight declines in carbon and stoichiometric ratios. However, when exposed to both, biofilms had significant decreases in stoichiometry and declined in productivity and respiration. This is problematic, as continuing marine pollution increases the likelihood that biofilms will be exposed to combinations of stressors and disturbances. Loss of biofilm productivity within these areas could lead to the loss of an important food source and nutrient cycler within the marine ecosystem.
... Several other effects related to rhamnolipid production may affect the respiratory infections caused by P. aeruginosa, since rhamnolipids can modulate swarming motility, participate in biofilm architecture by promoting the maintenance of channels that diffuse nutrients and oxygen, and mediate biofilm disruption by promoting the seeding dispersal of motile bacteria (48)(49)(50)(51). Furthermore, rhamnolipids increase the bioactivity of the Pseudomonas quinolone signal (PQS) (52), a QS signaling molecule that controls several virulence factors (53), and can be detected in the lungs of CF patients infected with P. aeruginosa (54,55). ...
The opportunistic pathogen Pseudomonas aeruginosa is one of the most common agents of respiratory infections and has been associated with high morbidity and mortality rates. The ability of P. aeruginosa to cause severe respiratory infections results from the coordinated action of a variety of virulence factors that promote bacterial persistence in the lungs. Several of these P. aeruginosa virulence mechanisms are mediated by bacterial lipids, mainly lipopolysaccharide, rhamnolipid, and outer membrane vesicles. Other mechanisms arise from the activity of P. aeruginosa enzymes, particularly ExoU, phospholipase C, and lipoxygenase A, which modulate host lipid signaling pathways. Moreover, host phospholipases, such as cPLA2α and sPLA2, are also activated during the infectious process and play important roles in P. aeruginosa pathogenesis. These mechanisms affect key points of the P. aeruginosa-host interaction, such as: i) biofilm formation that contributes to bacterial colonization and survival, ii) invasion of tissue barriers that allows bacterial dissemination, iii) modulation of inflammatory responses, and iv) escape from host defenses. In this mini-review, we present the lipid-based mechanism that interferes with the establishment of P. aeruginosa in the lungs and discuss how bacterial and host lipids can impact the outcome of P. aeruginosa respiratory infections.
... The synthesis of rhamnolipids is regulated by quorum sensing system RhlRI, based on acylated homoserine lactones (Pearson et al., 1997). Low concentration of rhamnolipids increases the affinity for initial adherence of cells to a surface, but the overproduction of rhamnolipids inhibits the formation of mature biofilms (Nickzad and Déziel, 2014). In this work, the expression of gene cluster rhlABRI was significantly downregulated in the mature biofilm, and it was upregulated in the rpoN mutant. ...
The extracellular matrix is essential for the biofilm formation of food spoilers. Pseudomonas fluorescens PF07 is a previous isolate from spoiled marine fish; however, the genes involved in the extracellular matrix formation of PF07 biofilms remain poorly defined. In this study, PF07 formed a wrinkled macrocolony biofilm through the high production of extracellular matrix. The genes involved in biofilm matrix formation and regulation were screened and identified by RNA-seq-dependent transcriptomic analysis and gene knock-out analysis. The macrocolony biofilms of PF07 grown for 5 days (PF07_5d) were compared with those grown for 1 day (PF07_1d). A total of 1,403 genes were significantly differentially expressed during biofilm formation. These mainly include the genes related to biofilm matrix proteins, polysaccharides, rhamnolipids, secretion system, biofilm regulation, and metabolism. Among them, functional amyloid genes fapABCDE were highly upregulated in the mature biofilm, and the operon fapA-E had a –24/–12 promoter dependent on the sigma factor RpoN. Moreover, the RNA-seq analyses of the rpoN mutant, compared with PF07, revealed 159 genes were differentially expressed in the macrocolony biofilms, and fapA-E genes were positively regulated by RpoN. In addition, the deletion mutants of fapC, rpoN, and brfA (a novel gene coding for an RpoN-dependent transcriptional regulator) were defective in forming mature macrocolony biofilms, solid surface-associated (SSA) biofilms, and pellicles, and they showed significantly reduced biofilm matrices. The fap genes were significantly downregulated in ΔbrfA, as in ΔrpoN. These findings suggest that the functional amyloid Fap is the main component of PF07 biofilm matrices, and RpoN may directly regulate the transcription of fap genes, in conjunction with BrfA. These genes may serve as potential molecular targets for screening new anti-biofilm agents or for biofilm detection in food environments.
... Finally, as the QS communication system is a key factor in bacterial biofilm formation, the two active extracts discovered in this study may potentially act on this complex system and/or on other factors regulated by QS, such as the production of rhamnolipids. This biosurfactant, controlled by the rhl QS system, is involved in the different stages of biofilm formation, particularly in the mediation of cell dispersion [54]. Thus, the present results encourage towards elucidating the potential direct and/or indirect anti-QS activity of these extracts. ...
The organization of bacteria in biofilms is one of the adaptive resistance mechanisms providing increased protection against conventional treatments. Thus, the search for new antibiofilm agents for medical purposes, especially of natural origin, is currently the object of much attention. The objective of the study presented here was to explore the potential of extracts derived from three seaweeds: the green Ulva lactuca, the brown Stypocaulon scoparium, and the red Pterocladiella capillacea, in terms of their antibiofilm activity against P. aeruginosa. After preparation of extracts by successive maceration in various solvents, their antibiofilm activity was evaluated on biofilm formation and on mature biofilms. Their inhibition and eradication abilities were determined using two complementary methods: crystal violet staining and quantification of adherent bacteria. The effect of active extracts on biofilm morphology was also investigated by epifluorescence microscopy. Results revealed a promising antibiofilm activity of two extracts (cyclohexane and ethyl acetate) derived from the green alga by exhibiting a distinct mechanism of action, which was supported by microscopic analyses. The ethyl acetate extract was further explored for its interaction with tobramycin and colistin. Interestingly, this extract showed a promising synergistic effect with tobramycin. First analyses of the chemical composition of extracts by GC–MS allowed for the identification of several molecules. Their implication in the interesting antibiofilm activity is discussed. These findings suggest the ability of the green alga U. lactuca to offer a promising source of bioactive candidates that could have both a preventive and a curative effect in the treatment of biofilms.
... Le rôle des rhamnolipides dans la physiologie de la cellule bactérienne est encore flou, cependant, il a été montré que ces rhamnolipides sont impliqués dans la pathogénicité bactérienne en altérant les jonctions serrées des cellules de l'hôte (Zulianello et al., 2006) ou encore en inhibant l'activité ciliaire des cellules eucaryotes (Hastie et al., 1986). Ces rhamnolipides entrent également en jeu dans la mobilité de type swarming (Caiazza et al., 2005) et ont un rôle important dans l'architecture du biofilm bactérien (Davey et al., 2003;Nickzad & Déziel, 2014). ...
L’endocrinologie microbienne a pour objet d’étude les interactions qui peuvent survenir entre les bactéries et les molécules sécrétées par l’hôte. Pseudomonas aeruginosa et Enterococcus faecalis sont deux pathogènes opportunistes impliqués dans des infections nosocomiales. Leur présence au sein du corps humain, les place donc au contact de molécules eucaryotes, dont les catécholamines (épinéphrine et norépinéphrine) qui sont produites en cas de réponse « combat-fuite » mais aussi lors de trauma, ou d’actes chirurgicaux. Ces substances sont connues pour être capables de moduler la physiologie bactérienne, notamment la virulence et la formation de biofilm. Les travaux menés au cours de cette thèse ont permis de montrer que l’épinéphrine pouvait moduler la physiologie de P. aeruginosa et de souches probiotiques et pathogènes d’E. faecalis. L’épinéphrine stimule, entres autres, la formation de biofilm et l’adhésion et pourrait, chez P. aeruginosa, jouer le rôle de xénosidérophore, favorisant ainsi la mise en place d’une infection par un apport de fer. Nous avons aussi émis l’hypothèse de la présence de senseurs adrénergiques putatifs, respectivement chez P. aeruginosa et E. faecalis, PmrB et VicK. Ces travaux de thèse ont permis d’approfondir les connaissances sur la communication hôte/pathogènes, et de mieux connaître l’effet de l’épinéphrine sur des bactéries présentes au sein du microbiote humain. Ceci pourrait permettre à l’avenir d’aider à la découverte de nouvelles cibles thérapeutiques.
... Rhamnolipids are a glycolipid-type biosurfactant with rhamnose head and β-hydroxyalkanoic acid tail. This structure of rhamnolipids helps to increase cell motility and biofilm formation [8,11,36]. Our preliminary results showed that rhamnolipids can also be produced by Pseudomonas sp. during the biodegradation process. ...
The capabilities of a microbial consortium, composed of Penicillium raperi, Aspergillus flavus, Penicillium glaucoroseum and Pseudomonas spp, for biodegrading of ultraviolet (UV)-pretreated and un-pretreated mixed plastics (polyethylene (PE), polystyrene foam (PS) and polyethylene terephthalate (PET)) in the presence of biosurfactant (rhamnolipid) was examined. The “UV-pretreated & bio-treated + biosurfactant” was found the most effective condition for PS samples with the highest physical weight loss (7.47%), surface degradation, wettability (< 5˚). The highest biofilm formation was observed on PS for “UV-pretreated & bio-treated + biosurfactant” and “UV-pretreated & bio-treated” with OD 595 nm of 1.53 and 1.31 conditions, respectively. Atomic force (AFM) and scanning electron microscopy (SEM) showed considerable surface degradation (cracks and holes) for the PS sample incubated at “UV-pretreated & bio-treated + biosurfactant” condition compared to PE and PET in the same condition. In contrast, PE and PET had a higher biodegradation efficiency only in “UV-pretreated & bio-treated” condition. The utilisation of biosurfactant had negative effects on biodegradation and wettability of PE and PET, due to the consumption of rhamnolipid as food source rather than the plastic itself. Chemical transformation indicated a new peak (C-O) in PS at both “UV-pretreated & bio-treated” and UV-pretreated & bio-treated + biosurfactant) conditions. However, the chemical transformation of PE and PET remained unchanged in all conditions except “UV-pretreated & bio-treated”. Thermogravimetric analysis showed 20 ˚C lower thermal stability of PS incubated at “UV-pretreated & bio-treated + biosurfactant” than other conditions.
... So, further research on low-cost electrode material with a high surface area will be more economical and advantageous. All the previously studied kinetic modelling for MFC-based sensors were preliminary studies and also limited to laboratory scale only under simple conditions and parameters (Mustakeem, 2015) (Behera and Ghangrekar, 2011) (Nickzad and Déziel, 2014). The mathematical models used for the kinetic study were both kinetic models, which can further have divided into two types empirical and mechanistic models. ...
The microbial fuel cell (MFC) sensor is a very promising self-powered self-sustainable system for early warning water quality detection. These sensors are cost-effective, biodegradable, compact in design, and portable in nature are favorable for real-time in situ water quality monitoring. This review represents the mechanism action behind the toxicity detection, optimization strategies, process parameters, role of biofilm, the role of external resistance, hydrodynamic study, and mathematical modeling for improving the performance of the sensor. Additionally, the techno-economic prospect of this MFC-based sensor and its challenges, limitations are addressed to make it economically more favorable for commercial use. The future direction is also explored based on the sensor's disadvantages and limitations. Comprehensively, this review covered all the possible directions of MFC sensor fabrication, their application, recent advancement, prospects challenges, and their possible solutions.
... Their anti-adhesive properties are closely related to the capacity of RLs to inverse the hydrophobic behavior of surfaces into hydrophilic making them unfavorable for hydrophobic bacteria adhesion [12]. In addition, the amphiphilic behavior of RLs molecules acting on cell-to-cell and cell-to-surface interactions may minimize the adherence capability of bacteria into the surface [43]. The observed decrease in contact angle measurements (Fig. 3) and in the initial bacteria adhesion ( Fig. 2A) with RLs-functionalized samples are in accordance with the antiadhesive effect described for RLs. ...
Controlling bacterial biofilm formation on silicone-based bloodstream catheters is of great concern to prevent related-infections. In this study, rhamnolipids (RLs), glycolipid biosurfactants, specifically a RLs mixture and the purified di-RL (RhaRhaC10:0C10:0) were covalently bonded to silicone with the intention of reaching long-lasting antibiofilm surfaces.
RLs mixture and di-RL were identified by an UHPLC-MS method that also allowed the confirmation of compound isolation by automated flash chromatography. Silicone surfaces underwent air-plasma treatment inducing reactive oxygen radicals able to promote the RLs grafting that was confirmed by contact angle, FTIR-ATR and AFM measurements. The antibiofilm activity towards different Gram positive strains was evaluated by colony forming units (CFU) count and confocal laser microscopy. In addition, the protein adsorption and the biocompatibility were also investigated.
RLs were successfully grafted onto silicone and RLs mixture and RhaRhaC10C10:0 functionalized specimens reduced the biofilm formation over 2.3 log units against methicillin sensitive Staphylococcus aureus. Additionally, a decrease of 1 Log units was observed against methicillin resistant S. aureus and S. epidermidis. Functionalized samples showed cytocompatibility towards human dermal fibroblasts, hemocompatibility and no vascular irritation potential. The results mentioned above revealed a synergy between the antimicrobial and the anti-adhesive properties of RLs making these compounds good candidates for the improvement of the medical devices antibiofilm properties.
... For example, pyocyanin, a virulence factor, is also involved in the biofilm formation and iron absorption of bacteria [39,40]. Rhamnolipid and motility are both related to bacterial biofilm formation [41,42]. ...
Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is commonly found in clinical settings and immuno-compromised patients. It is difficult to be eradicated due to its strong antibiotic resistance, and novel inactivation strategies have yet to be developed. Selenium is an essential microelement for humans and has been widely used in dietary supplement and chemoprevention therapy. In this study, the physiological and biochemical effects of sodium selenite on P. aeruginosa PAO1 were investigated. The results showed that 0~5 mM sodium selenite did not impact the growth of PAO1, but increased the lethality rate of PAO1 with antibiotics or H2O2 treatment and the antibiotics susceptibility both in planktonic and biofilm states. In addition, sodium selenite significantly reduced the expression of quorum sensing genes and inhibited various virulence factors of this bacterium, including pyocyanin production, bacterial motilities, and the type III secretion system. Further investigation found that the content of ROS in cells was significantly increased and the expression levels of most genes involved in oxidative stress were up-regulated, which indicated that sodium selenite induced oxidative stress. The RNA-seq result confirmed the phenotypes of virulence attenuation and the expression of quorum sensing and antioxidant-related genes. The assays of Chinese cabbage and Drosophila melanogaster infection models showed that the combination of sodium selenite and antibiotics significantly alleviated the infection of PAO1. In summary, the results revealed that sodium selenite induced oxidative stress and inhibited the quorum sensing system of P. aeruginosa, which in turn enhanced the antibiotic susceptibility and decreased the pathogenicity of this bacterium. These findings suggest that sodium selenite may be used as an effective strategy for adjunct treatment of the infections caused by P. aeruginosa.
... Finally, although the direct effect on in vivo biofilm has yet to be directly demonstrated, RLs are involved in the regulation of in vitro biofilm development, a key virulence function in lung infection, as discussed above. This role of RLs has been reviewed previously [186], and therefore is only briefly discussed here. RLs participate in the regulation of cell-surface hydrophobicity and in the modification of bacterial adhesive interactions [187][188][189]. ...
Driven in part by its metabolic versatility, high intrinsic antibiotic resistance, and a large repertoire of virulence factors, Pseudomonas aeruginosa is expertly adapted to thrive in a wide variety of environments, and in the process, making it a notorious opportunistic pathogen. Apart from the extensively studied chronic infection in the lungs of people with cystic fibrosis (CF), P. aeruginosa also causes multiple serious infections encompassing essentially all organs of the human body, among others, lung infection in patients with chronic obstructive pulmonary disease, primary ciliary dyskinesia and ventilator-associated pneumonia; bacteremia and sepsis; soft tissue infection in burns, open wounds and postsurgery patients; urinary tract infection; diabetic foot ulcers; chronic suppurative otitis media and otitis externa; and keratitis associated with extended contact lens use. Although well characterized in the context of CF, pathogenic processes mediated by various P. aeruginosa virulence factors in other organ systems remain poorly understood. In this review, we use an organ system-based approach to provide a synopsis of disease mechanisms exerted by P. aeruginosa virulence determinants that contribute to its success as a versatile pathogen.
... A search of the Scopus database in 2020 retrieved only three papers published in the last ten years addressing antifouling and biosurfactants. The studies involved analyses of the properties of the cellular surface of bacterial biofilms in seawater [180], the involvement of a rhamnolipid in cell adhesion and the development of biofilms [181], and the antibiofilm activity of biosurfactants produced by bacteria associated with corals in the Gulf of Mannar [182]. These studies focused only on the biofouling aspect of biofilms. ...
The adhesion of organisms to surfaces is an event that occurs in natural and artificial systems and is commonly known as bio-encrustation. The elimination of biofouling through less aggressive and ecological methods has aroused the interest of researchers and industries worldwide. Among the products with the greatest potential for altering surfaces, surfactants act at cell-cell and cell-surface interfaces, reducing surface hydrophobicity, the adherence of microorganisms, and the formation of biofilms. Biological surfactants have stood out for their versatility and proven effectiveness in recent years as valuable tools, especially due to their biodegradable characteristics when compared to synthetic surfactants. Thus, they can transform and modernize biotechnology by providing more robust antifouling technologies. This review discusses the possible roles and applications of synthetic and biological surfactants from different sources, such as biofouling reducing agents, addressing the potential alteration of surfaces, reducing surface tension, biological activities (antimicrobial, antibiofilm, and anti-adherence), biodegradability, and anticorrosion activity. Market trends and the potential uses of these multifunctional biomolecules in biofouling control are also cited, along with examples from the literature using biosurfactants as antifouling agents.
... Hydrophobic D-amino acids are secreted by bacteria prior to dispersal of biofilms and have been shown to inhibit biofilm growth in vitro and in vivo, although their mechanism of action remains unclear (Hochbaum et al., 2011). Some types of bacteria also secrete biosurfactant molecules called rhamnolipids (Nickzad and Déziel, 2014), which inhibit bacterial attachment to surfaces and trigger degradation of the EPS during dispersal. Sugar alcohols like mannitol and erythritol have been shown to stimulate metabolism of bacteria that causes awakening of dormant cells and increased susceptibility to antibiotics (Pace et al., 2019). ...
Fatty-acid signaling molecules can inhibit biofilm formation, signal dispersal events, and revert dormant cells within biofilms to a metabolically active state. We synthesized 2-heptylcyclopropane-1-carboxylic acid (2CP), an analog of cis-2-decenoic acid (C2DA), which contains a cyclopropanated bond that may lock the signaling factor in an active state and prevent isomerization to its least active trans-configuration (T2DA). 2CP was compared to C2DA and T2DA for ability to disperse biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa. 2CP at 125 μg/ml dispersed approximately 100% of S. aureus cells compared to 25% for C2DA; both 2CP and C2DA had significantly less S. aureus biofilm remaining compared to T2DA, which achieved no significant dispersal. 2CP at 125 μg/ml dispersed approximately 60% of P. aeruginosa biofilms, whereas C2DA and T2DA at the same concentration dispersed 40%. When combined with antibiotics tobramycin, tetracycline, or levofloxacin, 2CP decreased the minimum concentration required for biofilm inhibition and eradication, demonstrating synergistic and additive responses for certain combinations. Furthermore, 2CP supported fibroblast viability above 80% for concentrations below 1 mg/ml. This study demonstrates that 2CP shows similar or improved efficacy in biofilm dispersion, inhibition, and eradication compared to C2DA and T2DA and thus may be promising for use in preventing infection for healthcare applications.
... Cationic chitosan is found to rapidly absorb to negative microbial membrane and to affect surface properties such as hydrophilicity/ hydrophobicity and microbial membrane adhesives, which can interfere with hydrophobicity or adhesion mediated by microbial adhesion, thus inhibiting the production of subsequent biofilm [50,51]. RL-CS-NPs displayed superior inhibitory effects than CS-NPs, indicating that RL could also inhibit the development of biofilms due to its ability to alter cell-to-surface and cell-to-cell interactions during early accession and biofilm formation [52]. In addition, the time-based inhibitory activity of RL-CS-NPs has also been observed. ...
Driven by the need to find alternatives to control H. pylori infections , this work describes the development of chitosan-PMLA nanoparticulate systems as carriers for antimicrobial glycolipid. By using a simple ionic gelation method stable nanoparticle was obtained showing an encapsulation efficiency of 73.1 ± 1.3% and an average size of 217.0 ± 15.6 nm for rhamnolipids chitosan-PMLA nanoparticles (RL-CS-NPs). Glycolipid incorporation and particle size were correspondingly corroborated by FT-IR and TEM analysis. Rhamnolipids chitosan nanoparticles (RL-CS-NPs) presented the highest antimicrobial effect towards H. pylori (ATCC 26695) exhibiting a minimal inhibitory concentration of 132 mg/ mL and a biofilm inhibition ability of 99%. Additionally, RL-CS-NPs did not interfere with human fibroblasts viability and proliferation under the tested conditions. The results revealed that the RL-CS-NPs were able to inhibit bacterial growth showing adequate cyto-compatibility and might become, after additional studies, a valuable approach to fight H. pylori biofilm related-infections. ARTICLE HISTORY
... Glycolipids Sophorolipids Antimicrobial activity [5] Antioxidant activity [62] Hypocholestrolemic activity [63] Dermal fibrolast enhancer [64] Antiaging activity [65] Rhamnolipids Antimicrobial activity [66] Antiadhesive and antibiofilm activities [67] Detergency agent [68] Antibacterial activities [69] MEL Antioxidant activity [70] Moisturizing agent [71] Hair flexibility enhancer [72] Lipopeptides Surfactin Detergency agent [73] Emulsifying activity [74] Anticellulite agent [75] 18.4 Dermatological Approach of Biosurfactants 407 was made on the depilated thoracic region of rats. Then the wounded parts were topically applied by several treatments; CICAFLORA a commercial wound healing cream as the positive control, 100% glycerol as the negative control, and 5 mg/ml and 15 mg/ml of gel-based B. subtilis SPB1 biosurfactant. ...
Nowadays, most of the cosmetic products used daily by almost all of the women in all over the world. These cosmetics play an important role in everyone life's especially for beautiful look. But these cosmetics are made up from chemical based ingredients which have many adverse effects such as carcinogenic, allergenic and irritation problems. Moreover, waste of these cosmetics are harmful to our environment due to presence of mercury, lead, arsenic and many more toxic substances. These sector encompasses various social, environmental and economic impacts which could be addressed and developed most efficient and green techniques for formulation of cosmetics. Nowadays, biosurfactants are natural compounds and gaining more attention due to environmental friendly, biodegradable, non‐toxic, hypoallergenic and less side effects. In recent years, cosmetics and dermatological repair products have become more widespread and there is a great demand. In this chapter, we have been discussed the efficiency and potential use of biosurfactants in cosmetics and dermatological repair industry and further reviewed the future challenges and perspectives for cosmaceuticals.
... Components produced by microbes can withstand immune responses and may cross gut or blood-brain barriers [39]. RLs are indeed crucial bacterial products involved in biofilm development, remodeling and disruption, possessing surfactant abilities and enabling the translocation of bacteria to other sites [45,46]. Increased levels of salivary RLs found here, in line with previous results for blood and CSF [21], indicate disruption of the physiological microflora, either with the presence of non-commensal infectious agents (systemically or centrally), or by altered composition and dysbiosis of the commensal bacteria. ...
Alzheimer’s disease (AD) is increasingly affecting the aging population and the estimated prevalence reaches 50 million people worldwide. The need for the discovery of new biomarkers for AD diagnosis is urgent and especially in biological fluids other than cerebrospinal fluid (CSF), as its collection is invasive. Arguments are numerous that chronic bacterial infections might be considered as one of the possible causes of AD. Rhamnolipids (RLs) are bacterial virulence factors, suspicious for dysfunctions and disorders including AD. The aim of this pilot trial was to investigate RLs levels in saliva of Mild Cognitive Impairment (MCI) and AD patients with indirect ELISA. Specifically, salivary RLs were determined in 30 AD patients, 24 MCI patients and 15 cognitively healthy individuals and were found elevated in AD and MCI patients compared to those of the control group. The established biomarkers of AD, tau and Aβ42 amyloid, and the inflammatory markers cyclooxygenases (COX-1 and COX-2) were also determined, to evaluate their possible interdependence from RLs levels. Levels of RLs positively correlate with COX-2 levels and negatively with the mental state according to Mini–Mental State Examination (MMSE) score of donors. Multilinear regression verified the tight interrelation of RLs with COX-2 in saliva of MCI and AD patients. The results of this study stand by the hypothesis of inflammatory involvement in AD and indicate that RLs could be suggested as eventual biomarkers for AD diagnosis using saliva as biological fluid.
... Their production by colonising isolates has been associated with ventilatorassociated pneumonia (VAP) development [174]. While overproduction of RLs impedes biofilm development, low concentrations enhance LPS release to the cell surface, increasing hydrophobicity and affinity for initial adherence of bacteria to a surface [175,176]. The production of an appropriate amount contributes to biofilm architecture by maintaining open the non-colonised channels [177]. ...
Pseudomonas aeruginosa is a dominant pathogen in people with cystic fibrosis (CF) contributing to morbidity and mortality. Its tremendous ability to adapt greatly facilitates its capacity to cause chronic infections. The adaptability and flexibility of the pathogen are afforded by the extensive number of virulence factors it has at its disposal, providing P. aeruginosa with the facility to tailor its response against the different stressors in the environment. A deep understanding of these virulence mechanisms is crucial for the design of therapeutic strategies and vaccines against this multi-resistant pathogen. Therefore, this review describes the main virulence factors of P. aeruginosa and the adaptations it undergoes to persist in hostile environments such as the CF respiratory tract. The very large P. aeruginosa genome (5 to 7 MB) contributes considerably to its adaptive capacity; consequently, genomic studies have provided significant insights into elucidating P. aeruginosa evolution and its interactions with the host throughout the course of infection.
... Rhamnolipids influence the biofilm architecture [26]. Rhamnolipid was found positive in 38 (76 %) of the P. aeruginosa isolates in our study. ...
Background. Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen responsible for burn-wound infection. High incidence, infection severity and increasing resistance characterize P. aeruginosa -induced burn infection.
Purpose. To estimate quorum-sensing (QS)-dependent virulence factors of P. aeruginosa isolates from burn wounds and correlate it to the presence of QS genes.
Methods. A cross-sectional descriptive study included 50 P . aeruginosa isolates from burn patients in Mansoura University Plastic and Burn Hospital, Egypt. Antibiotic sensitivity tests were done. All isolates were tested for their ability to produce biofilm using a micro-titration assay method. Protease, pyocyanin and rhamnolipid virulence factors were determined using skimmed milk agar, King’s A medium and CTAB agar test, respectively. The identity of QS lasR and rhlR genes was confirmed using PCR.
Results. In total, 86 % of isolates had proteolytic activity. Production of pyocyanin pigment was manifested in 66 % of isolates. Altogether, 76 % of isolates were rhamnolipid producers. Biofilm formation was detected in 96 % of isolates. QS lasR and rhlR genes were harboured by nearly all isolates except three isolates were negative for both lasR and rhlR genes and two isolates were positive for lasR gene and negative for rhlR gene. Forty-nine isolates were considered as extremely QS-proficient strains as they produced QS-dependent virulence factors. In contrast, one isolate was a QS deficient strain.
Conclusions. QS affects P. aeruginosa virulence-factor production and biofilm in burn wounds. Isolates containing lasR and rhlR seem to be a crucial regulator of virulence factors and biofilm formation in P. aeruginosa whereas the lasR gene positively regulates biofilm formation, proteolytic activity, pyocyanin production and rhamnolipid biosurfactant synthesis. The QS regulatory RhlR gene affects protease and rhamnolipid production positively.
... Anti-ageing product [120] Cleanser in shampoos [121] Anti-adhesive activity [122] Rhamnolipid/Sophorolipid ...
This article includes an updated review of the classification, uses and side effects of surfactants for their application in the cosmetic, personal care and pharmaceutical industries. Based on their origin and composition, surfactants can be divided into three different categories: (i) synthetic surfactants; (ii) bio-based surfactants; and (iii) microbial biosurfactants. The first group is the most widespread and cost-effective. It is composed of surfactants, which are synthetically produced, using non-renewable sources, with a final structure that is different from the natural components of living cells. The second category comprises surfactants of intermediate biocompatibility, usually produced by chemical synthesis but integrating fats, sugars or amino acids obtained from renewable sources into their structure. Finally, the third group of surfactants, designated as microbial biosurfactants, are considered the most biocompatible and eco-friendly, as they are produced by living cells, mostly bacteria and yeasts, without the intermediation of organic synthesis. Based on the information included in this review it would be interesting for cosmetic, personal care and pharmaceutical industries to consider microbial biosurfactants as a group apart from surfactants, needing specific regulations, as they are less toxic and more biocompatible than chemical surfactants having formulations that are more biocompatible and greener.
Dual-species biofilms formed by Candida albicans and Staphylococcus aureus have high virulence and drug resistance. In this context, biosurfactants produced by Pseudomonas aeruginosa have been widely studied, of which a new derivative (RLmix_Arg) stands out for possible application in formulations. The objective of this study was to evaluate the antibiofilm activity of RLmix_Arg, both alone and incorporated in a gel prepared with Pluronic F-127, against dual-species biofilms of fluconazole-resistant C. albicans (FRCA) and methicillin-resistant S. aureus (MRSA) in impregnated catheters. Broth microdilution tests, MTT reduction assays of mature biofilms, impregnation of RLmix_Arg and its gel in peripheral venous catheters, durability tests and scanning electron microscopy (SEM) were performed. RLmix_Arg showed antimicrobial activity against Candida spp. and S. aureus, by reducing the cell viability of mixed biofilms of FRCA and MRSA, and preventing their formation in a peripheral venous catheter. The incorporation of this biosurfactant in the Pluronic F-127 gel considerably enhanced its antibiofilm activity. Thus, RLmix_Arg has potential application in gels for impregnation in peripheral venous catheters, helping to prevent development of dual-species biofilms of FRCA and MRSA.
Pseudomonas aeruginosa, an increasingly common competitive and biofilm organism in healthcare infection with sophisticated, interlinked and hierarchic quorum systems (Las, Rhl, PQS, and IQS), creates the greatest threats to the medical industry and has rendered prevailing chemotherapy medications ineffective. The rise of multidrug resistance has evolved into a concerning and potentially fatal occurrence for human life.
P. aeruginosa biofilm development is assisted by exopolysaccharides, extracellular DNA, proteins, macromolecules, cellular signaling and interaction. Quorum sensing is a communication process between cells that involves autonomous inducers and regulators. Quorum-induced infectious agent biofilms and the synthesis of virulence factors have increased disease transmission, medication resistance, infection episodes, hospitalizations and mortality. Hence, quorum sensing may be a potential therapeutical target for bacterial illness, and developing quorum inhibitors as an anti-virulent tool could be a promising treatment strategy for existing antibiotics.
Quorum quenching is a prevalent technique for treating infections caused by microbes because it diminishes microbial pathogenesis and increases microbe biofilm sensitivity to antibiotics, making it a potential candidate for drug development. This paper examines P. aeruginosa quorum sensing, the hierarchy of quorum sensing mechanism, quorum sensing inhibition and quorum sensing inhibitory agents as a drug development strategy to supplement traditional antibiotic strategies.
Surfactants are among the most widely produced and used chemicals. During the past decades, these mass chemicals have been produced utilizing nonrenewable resources to a significant extent. The rising demand for sustainable and carbon-neutral products has driven the need to replace petrochemically produced chemicals with biotechnologically produced alternatives based on renewable resources. Among these, microbial rhamnolipid glycolipids are among the most intensively studied class of molecules. Research on rhamnolipids and their applications dates back to over seven decades. An almost overwhelming number of scientific publications present their wide range of possible applications due to their surface-active and environmental properties. This has led to a significant build-up in expectations. The aim of this chapter is to give an answer to the question whether the promise associated with rhamnolipids has come true.
Surfactants can be found and used in most modern industrial sectors, such as the food, cosmetic, and pharmaceutical industries. Synthetically produced surfactants have been shown to have unfavorable effects or low compatibility with products, especially in cosmetic or pharmaceutical products, where the association between the use of some surfactants and the advancement of dermatitis, and eye irritation, among others, have been reported. Biosurfactants (BS) have been widely accepted around the world as a sustainable alternative to synthetic surfactants, as they exhibit the same functionalities in addition to having the potential to synergize with other molecules, further improving the performance of these molecules. Unlike synthetic surfactants, biosurfactants have advantages such as biodegradability and low toxicity. In recent years, BS has been analyzed for cytotoxic effect using various cell lines such as mouse fibroblasts, human embryonic kidney cells, and human epithelial cell lines. The high biodegradability and stability of biosurfactants produced by microorganisms at different temperatures and the low sensitivity to changes in environmental pH are obvious advantages. Therefore, they generally do not pose an ecological threat. The low toxicity and high biodegradability of biosurfactants compared to synthetic surfactants have been reported in recent years and will be exposed in this chapter.KeywordsSurfactantsLow toxicityHigh biodegradabilityNatural molecules
Biosurfactants are classified as microbial molecules that exhibit emulsifying actions and pronounced surfaces. They are amphiphilic with hydrophilic and hydrophobic moieties that tend to mitigate interfacial and surface tension in which they are been formed. Biosurfactants have a large range of chemical structures such as neural lipids, fatty acids, phospholipids, polysaccharide-protein, lipopeptides, and glycolipids. The majority of biosurfactants show low critical micelle concentration and high surface properties; thus, they are promising alternatives for synthetic surfactants. Microbial source biosurfactants have been detected in various studies areas including anticancer treatments, drug delivery systems, and improved oral cavity care, anti-biofilm effect, improving feed digestion, plant-pathogen control, wound healing, dermatological treatments, and antimicrobial activity. The potential usage and commercial uses of microbial-derived biosurfactants in medicine have dramatically increased in the past years. The antifungal, antiviral, antibacterial, anticancer, and wound healing activities of biosurfactants have made them significant compounds in fighting against several pathogens and as biocide agents. Nowadays, most studies on biosurfactant applications have been centered on environmental uses due to their ecology-friendly nature, diversity, suitability for huge-scale selectivity, and production. Nevertheless, biosurfactants are promising bioactive compounds because they have higher versatility, biodegradability, lower toxicity, different biological roles, and structure. In addition, they show a positive killing activity on some types of cells through the lysis of red blood cells and as a bioassay. They have exhibited strong potential in skin compatibility, surface moisturizing effects, and protection which serve as major areas for a better skincare routine. Studies have evaluated the in vitro wound healing potential and antioxidant properties of B. subtilis SPB1 biosurfactant on the wound site in experimental rats. Despite their biological source and potential, very few studies have directed their interest to the wound healing and biocidal property of biosurfactants. Thus, this chapter focuses on the roles of biosurfactants in biocidal and wound healing activity.KeywordsBiosurfactant, biocidal activityWound healingAntimicrobial propertyProbiotic bacteria
Over the years, the food technology has emerged in tandem with changes in dietary trends. New technologies have arisen that not only improve the flavor and shelf life of food goods, but also contain effective additives that are same for customers’ health and provide nutraceutical benefits. As a result, the adoption of natural alternatives to replace synthetic additives is increasing in ongoing research. Biosurfactants have evolved as natural alternatives that can employed as an emulsifying agent in the food processing industry. Biosurfactants of the lipopeptide, glycolipid, glycoprotein, glycolipopeptide types can emulsify a wide range of vegetable oils and fats in food processing with high emulsification index. This biomolecule has a beneficial impact on texture profile analysis and sensory attributes such as color, aroma, and taste evolution in baked goods. The research in this field is still in the lab, and additional research is needed before this natural alternative, biosurfactants, may be used in large industrial sectors.
To protect themselves from environmental challenges, microorganisms are generally formed biofilms. Microbial biofilms present on food industry surfaces are potential sources of contamination, which may lead to food spoilage and disease transmission. The preconditioning of surfaces using microbial surface-active compounds could be an interesting strategy to prevent adhesion of food-borne pathogens to solid surfaces. The biosurfactants, which are surface-active molecules synthetized by microorganisms, have gained attention due to their natural origin and environmental computability. The amphiphilic nature of biosurfactants plays a potential role in the dispersion and disruption of microbial biofilms. In this chapter, we discuss aspects of biofilm characteristics and examine the contribution of biosurfactants to the disruption or inhibition of microbial biofilms especially in food industry.
Biosurfactants are natural surface-active materials created by microorganisms. Biosurfactants can contribute several properties including antibacterial, antioxidant, emulsifying, and antiadhesive activities to the food in which they are incorporated. Biosurfactants have been employed in food formulations to enhance viscosity, extending the shelf life of the products, improving texture and flavor, as well as lowering the calorie value by substituting fat. This chapter summarizes the activities of biosurfactants as antioxidants in food, as well as the features of biosurfactants in boosting food quality and the variables impacting biosurfactant synthesis.
Glycolipids encompass a wide variety of compounds that contain both glycans (i.e., carbohydrates) and lipids. The type of lipid moiety and attached carbohydrate(s) determine the categories and subcategories, which include: fatty acyl glycolipids, glycosphingolipids (cerebrosides, globosides, gangliosides, sulfatides and others), glycoglycerolipids, glycophospholipids (e.g., phosphatidylinositols), glycosylated prenols (e.g., dolichol-phospho-glycans), glycosylated sterols, glycosylated polyketides and saccharolipids. This review provides a brief overview of the types of functions these compounds perform, the structural features of the different categories, and a few examples of how structural diversity is achieved. Although the “glycolipidolome” is formidably large, it is becoming more assessable to characterization as the tools of the “omics” revolution expand to include mass spectrometry and other techniques for structural analysis.
This review discusses the classification, characteristics, and applications of biosurfactants. The biosynthesis pathways for different classes of biosurfactants are reviewed. An in-depth analysis of reported research is carried out emphasizing the synthetic pathways, culture media compositions, and influencing factors on production yield of biosurfactants. The environmental, pharmaceutical, industrial, and other applications of biosurfactants are discussed in detail. A special attention is given to the biosurfactants application in combating the pandemic COVID-19. It is found that biosurfactant production from waste materials can play a significant role in enhancing circular bioeconomy and environmental sustainability. This review also details the life cycle assessment methodologies for the production and applications of biosurfactants. Finally, the current status and limitations of biosurfactant research are discussed and the potential areas are highlighted for future research and development. This review will be helpful in selecting the best available technology for biosynthesis and application of particular biosurfactant under specific conditions.
The application of rhamnolipids in a fungal-cultured biotrickling filter (BTF) has a significant impact on toluene removal. Two BTFs were used; BTF-A, a control bed, and BTF-B fed with rhamnolipids. The effect of empty bed residence times (EBRTs) on toluene bioavailability was investigated. Removal of toluene was carried out at EBRTs of 30 and 60 s and inlet loading rates (LRs) of 23–184 g m⁻³ h⁻¹. At 30 s EBRT, when inlet LR was increased from 23 to 184 g m⁻³ h⁻¹, the removal efficiency (RE) decreased from 93% to 50% for the control bed, and from 94% to 87% for BTF-B. Increasing the EBRT simultaneously with inlet LRs, confirms that BTF-A was diffusion-limited by registering a RE of 62% for toluene inlet LR of 184 g m⁻³ h⁻¹, whereas BTF-B, achieved RE > 96%, confirming a significant improvement in toluene biodegradability. Overall, the best performance was observed at 60 s EBRT and inlet LR of 184 g m⁻³ h⁻¹, providing a maximum elimination capacity (EC) of 176.8 g m⁻³ h⁻¹ under steady-state conditions. While a maximum EC of 114 g m⁻³ h⁻¹ was observed under the same conditions in the absence of rhamnolipids (BTF-A). Measurements of critical micelle concentration showed that 150 mg L⁻¹ of rhamnolipids demonstrated the lowest aqueous surface tension and maximum formation of micelles, while 175 mg L⁻¹ was the optimum dose for fungal growth. Production rate of carbon dioxide, and dissolved oxygen contents highlighted the positive influence of rhamnolipids on adhesive forces, improved toluene mineralization, and promotion of microbial motility over mobility.
Biofouling is a serious consequence in industries that deals with sea cage and water
systems, as well as medical sectors that deal with medical devices associated with the
mucus layer or moist skin. Because of biofouling, there are financial losses as well as
health-related problems. Microbial biofilm formation is a dynamic phenomenon that initiates formation of conditioning layer and adsorption of sessile bacteria. Most of the preventive measures deal either with an impeding conditioning layer by use of extremely smooth surface or coating of toxic metals and/or avoiding the adsorption of sessile bacteria using toxic biocides. In general, most of the methods include employment of toxic metal or substances. Hence, the requirement of green alternative has arises that may act as an ecofriendly antifouling agent. Considering the earlier discussed requirement, biosurfactant can be consider as a potential candidate acting against biofouling. Moreover, biosurfactants are available in vast structural diversity. Production through waste materials has been linked with use of biosurfactant waste disposal and management
In this work, Stacked MFC was prepared by using thermophilic microbes. The fuel cell was tested in three groups. The first group of stacked MFC is dealing with one Brevibacillus species 1 (biosurfactant producing bacteria) and Brevibacillus species 2 (biosurfactant producing bacteria) cultured in Nutrient broth medium. The second group of stacked MFCs is dealing with one Brevibacillus species 1 (biosurfactant producing bacteria) and Brevibacillus species 2 (biosurfactant producing bacteria) cultured Mckeen medium. The third group of stacked MFCs is dealing with Brevibacillus species 1 (biosurfactant producing bacteria) and Brevibacillus species 2 (biosurfactant producing bacteria) cultured in NIT Raipur canteen wastewater. The performance of MFC in terms of voltage and current generation was continuously evaluated for 15 days and biosurfactant production was also analyzed. Biofilm was investigated by SEM (Scanning Electron Microscope) analysis. Both the strain used in this study is biosurfactant-producing bacteria and ability to produce electricity. Unknown stain showing a good result as compared to Brevibacillus. The maximum power density was observed with strain 1 in McKeen media i.e. 64.08 mW cm ⁻².
Chemical surfactants are omnipresent in consumer products, but they are the subject of environmental concerns. For this reason, the complete replacement of petrochemical surfactants by biosurfactants constitutes a holy grail, but this is far from possible in the near future. Although the “biosurfactants revolution” has not yet occurred, mainly due to the higher cost and lower availability of biosurfactants, another reason could explain this fact: poor knowledge of their properties in solution...
Rhamnolipids are a specific class of microbial surfactants, which hold great biotechnological and therapeutic potential. However, their exploitation at the industrial level is hampered because they are mainly produced by the opportunistic pathogen Pseudomonas aeruginosa . The non-human pathogenic bacterium Pantoea ananatis is an alternative producer of rhamnolipid-like metabolites containing glucose instead of rhamnose residues. Herein, we present the isolation, structural characterization, and total synthesis of ananatoside A, a 15-membered macrodilactone-containing glucolipid, and ananatoside B, its open-chain congener, from organic extracts of P. ananatis . Ananatoside A was synthesized through three alternative pathways involving either an intramolecular glycosylation, a chemical macrolactonization or a direct enzymatic transformation from ananatoside B. A series of diasteroisomerically pure (1→2), (1→3), and (1→4)-macrolactonized rhamnolipids were also synthesized through intramolecular glycosylation and their anomeric configurations as well as ring conformations were solved using molecular modeling in tandem with NMR studies. We show that ananatoside B is a more potent surfactant than its macrolide counterpart. We present evidence that macrolactonization of rhamnolipids enhances their cytotoxic and hemolytic potential, pointing towards a mechanism involving the formation of pores into the lipidic cell membrane. Lastly, we demonstrate that ananatoside A and ananatoside B as well as synthetic macrolactonized rhamnolipids can be perceived by the plant immune system, and that this sensing is more pronounced for a macrolide featuring a rhamnose moiety in its native 1C4 conformation. Altogether our results prove that macrolactonization of glycolipids can dramatically interfere with their surfactant properties and biological activity.
Biosurfactant produced from Pseudomonas aeruginosa DSVP20 was evaluated for its potential to disrupt Candida albicans biofilm formed on polystyrene (PS) surfaces in this investigation. P. aeruginosa DSVP20 exhibited optimum production of biosurfactant (5.8 g L-1) after 96 h of growth with an ability to reduce surface tension of the aqueous solution from 72 to 28 mN m-1. Analysis of purified biosurfactant with FT-IR, 1H and 13C NMR and MALDI-TOF MS revealed it to be di-rhamnolipid (RL-2) in nature. Biofilm disrupting ability of RL-2 (0.16 mg mL-1) on Candida cells when checked using XTT reduction assay revealed that about 50 % of the cells remain adhered to 96-well plate after 2 h of treatment, while up to 90 % reduction in pre-formed C. albicans biofilm on PS surface was observed with RL-2 (5.0 mg mL-1) in a dose-dependent manner. Microscopic analyses (SEM and CLSM) further confirm the influence of RL-2 on disruption of Candida biofilm extracellular matrix on PS surface which can be exploited as a potential alternative to the available conventional therapies.
Movement over an agar surface using swarming motility is subject to formidable challenges not encountered during swimming. Bacteria display a great deal of flexibility in coping with these challenges, which include attracting water to the surface, overcoming frictional forces and reducing surface tension. Bacteria that swarm on 'hard' agar surfaces (robust swarmers) display a hyper-flagellated and hyper-elongated morphology. Bacteria requiring a 'softer' agar surface (temperate swarmers) do not exhibit such a dramatic morphology. For polarly flagellated robust swarmers there is good evidence that restriction of flagella rotation somehow signals the induction of a large number of lateral flagella, but this scenario is apparently not relevant to temperate swarmers. Swarming bacteria can be further subdivided by their requirement for multiple stators (Mot proteins) or a stator-associated protein FliL, secretion of essential polysaccharides, cell density-dependent gene regulation including surfactant synthesis, a functional chemotaxis signaling pathway, appropriate c-di-GMP levels, induction of virulence determinants, and various nutritional requirements such as iron limitation or nitrate availability. Swarming strategies are as diverse as the bacteria that utilize them. The strength of these diverse designs is in the vantage point they offer for understanding mechanisms for effective colonization of surface niches, acquisition of pathogenic potential, and identification of environmental signals that regulate swarming. The signature swirling and streaming motion within a swarm is an interesting phenomenon in and of itself, an emergent behavior with properties similar to flocking behavior in diverse systems including birds and fish, providing a convenient new avenue for modeling such behavior.
Yarrowia lipolytica is an ascomycetous dimorphic fungus that exhibits biofilm mode of growth. Earlier work has shown that biosurfactants such as rhamnolipids are efficient dispersants of bacterial biofilms. However, their effectiveness against fungal biofilms (particularly Y. lipolytica) has not been investigated. The aim of this study was to determine the effect of rhamnolipid on a biofilm forming strain of Y. lipolytica. Two chemical surfactants, cetyl-trimethyl ammonium bromide (CTAB) and sodium dodecyl sulphate (SDS) were used as controls for comparison.
The methylene blue dye exclusion assay indicated an increase in fungal cell permeability after rhamnolipid treatment. Microtiter plate assay showed that the surfactant coating decreased Y. lipolytica biofilm formation by 50%. Rhamnolipid treatment disrupted pre-formed biofilms in a more effective manner than the other two surfactants. Confocal laser scanning microscopic studies showed that biofilm formation onto glass surfaces was decreased by 67% after sub-minimum inhibitory concentration (sub-MIC) treatment with rhamnolipids. The disruption of biofilms after rhamnolipid treatment was significant (P<0.05) when compared to SDS and CTAB.
The results indicate a potential application of the biological surfactant to disrupt Y. lipolytica biofilms.
As promising biotechnological products, rhamnolipids (RLs) are the most investigated biosurfactants. Over the years, important
efforts have been spent and an array of techniques has been developed for the isolation of producing bacterial strains and
the characterization of a large variety of RL homologs and congeners. Investigations on RL production by the best known producer,
the opportunistic pathogen Pseudomonas aeruginosa, have shown that production of RLs proceeds through de novo biosynthesis of precursors. Over the last 15 years, the genetic
details underlying RL production in P. aeruginosa have been mostly unraveled, revealing a complex regulatory mechanism controlled by quorum sensing pathways of intercellular
communication. A number of nutritional and cultivation factors affecting RL productivity have also been identified, while
the use of many affordable and renewable raw substrates has been described to optimize the production. Multidisciplinary approaches
are increasingly adopted to develop methods for the safe, cost-effective, and highly efficient production of RLs at the industrial
scale.
We review the environmental role of rhamnolipids in terms of microbial life and activity. A large number of previous research supports the idea that these glycolipids mediate the uptake of hydrophobic substrates by bacterial cells. This feature might be of highest priority for bioremediation of spilled hydrocarbons. However, current evidence confirms that rhamnolipids primarily play a role in surface-associated modes of bacterial motility and are involved in biofilm development. This might be an explanation why no direct pattern of hydrocarbon degradation was often observed after rhamnolipids supplementation. This review gives insight into the current state of knowledge on how rhamnolipids operate in the microbial world.
Multi-species biofilm communities are environments in which complex but ill understood exchanges between bacteria occur. Although monospecies cultures are still widely used in the laboratory, new approaches have been undertaken to study interspecies interactions within mixed communities. This review describes our current understanding of competitive relationships involving nonbiocidal biosurfactants, enzymes, and metabolites produced by bacteria and other microorganisms. These molecules target all steps of biofilm formation, ranging from inhibition of initial adhesion to matrix degradation, jamming of cell-cell communications, and induction of biofilm dispersion. This review presents available data on nonbiocidal molecules and provides a new perspective on competitive interactions within biofilms that could lead to antibiofilm strategies of potential biomedical interest.
In most environments, bacteria reside primarily in biofilms, which are social consortia of cells that are embedded in an extracellular matrix and undergo developmental programmes resulting in a predictable biofilm 'life cycle'. Recent research on many different bacterial species has now shown that the final stage in this life cycle includes the production and release of differentiated dispersal cells. The formation of these cells and their eventual dispersal is initiated through diverse and remarkably sophisticated mechanisms, suggesting that there are strong evolutionary pressures for dispersal from an otherwise largely sessile biofilm. The evolutionary aspect of biofilm dispersal is now being explored through the integration of molecular microbiology with eukaryotic ecological and evolutionary theory, which provides a broad conceptual framework for the diversity of specific mechanisms underlying biofilm dispersal. Here, we review recent progress in this emerging field and suggest that the merging of detailed molecular mechanisms with ecological theory will significantly advance our understanding of biofilm biology and ecology.
Bacteria in nature often exist as sessile communities called biofilms. These communities develop structures that are morphologically
and physiologically differentiated from free-living bacteria. A cell-to-cell signal is involved in the development of Pseudomonas aeruginosa biofilms. A specific signaling mutant, alasI mutant, forms flat, undifferentiated biofilms that unlike wild-type biofilms are sensitive to the biocide sodium dodecyl
sulfate. Mutant biofilms appeared normal when grown in the presence of a synthetic signal molecule. The involvement of an
intercellular signal molecule in the development of P. aeruginosa biofilms suggests possible targets to control biofilm growth on catheters, in cystic fibrosis, and in other environments
where P. aeruginosa biofilms are a persistent problem.
Removal of detrimental biofilms from surfaces exposed in the marine environment remains a challenge. A strain of Bacillus pumilus was isolated from the surface of titanium coupons immersed in seawater in the vicinity of Madras Atomic Power Station (MAPS) on the East coast of India. The bacterium formed extensive biofilms when compared to species such as Bacillus licheniformis, Pseudomonas aeruginosa PAO1 and Pseudomonas aureofaciens. A commercially available rhamnolipid was assessed for its ability to inhibit adhesion and disrupt pre-formed B. pumilus biofilms. The planktonic growth of B. pumilus cells was inhibited by concentrations >1.6mM. We studied the effect of various concentrations (0.05-100mM) of the rhamnolipid on adhesion of B. pumilus cells to polystyrene microtitre plates, wherein the effectiveness varied from 46 to 99%. Biofilms of B. pumilus were dislodged efficiently at sub-MIC concentrations, suggesting the role of surfactant activity in removing pre-formed biofilms. Scanning electron microscopy (SEM) confirmed the removal of biofilm-matrix components and disruption of biofilms by treatment with the rhamnolipid. The results suggest the possible use of rhamnolipids as efficient anti-adhesive and biofilm-disrupting agents with potential applications in controlling biofilms on surfaces.
The microorganisms in biofilms live in a self-produced matrix of hydrated extracellular polymeric substances (EPS) that form their immediate environment. EPS are mainly polysaccharides, proteins, nucleic acids and lipids; they provide the mechanical stability of biofilms, mediate their adhesion to surfaces and form a cohesive, three-dimensional polymer network that interconnects and transiently immobilizes biofilm cells. In addition, the biofilm matrix acts as an external digestive system by keeping extracellular enzymes close to the cells, enabling them to metabolize dissolved, colloidal and solid biopolymers. Here we describe the functions, properties and constituents of the EPS matrix that make biofilms the most successful forms of life on earth.
Rhamnolipids are glycolipidic biosurfactants produced by various bacterial species. They were initially found as exoproducts of the opportunistic pathogen Pseudomonas aeruginosa and described as a mixture of four congeners: alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate (Rha-Rha-C(10)-C(10)), alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxydecanoate (Rha-Rha-C(10)), as well as their mono-rhamnolipid congeners Rha-C(10)-C(10) and Rha-C(10). The development of more sensitive analytical techniques has lead to the further discovery of a wide diversity of rhamnolipid congeners and homologues (about 60) that are produced at different concentrations by various Pseudomonas species and by bacteria belonging to other families, classes, or even phyla. For example, various Burkholderia species have been shown to produce rhamnolipids that have longer alkyl chains than those produced by P. aeruginosa. In P. aeruginosa, three genes, carried on two distinct operons, code for the enzymes responsible for the final steps of rhamnolipid synthesis: one operon carries the rhlAB genes and the other rhlC. Genes highly similar to rhlA, rhlB, and rhlC have also been found in various Burkholderia species but grouped within one putative operon, and they have been shown to be required for rhamnolipid production as well. The exact physiological function of these secondary metabolites is still unclear. Most identified activities are derived from the surface activity, wetting ability, detergency, and other amphipathic-related properties of these molecules. Indeed, rhamnolipids promote the uptake and biodegradation of poorly soluble substrates, act as immune modulators and virulence factors, have antimicrobial activities, and are involved in surface motility and in bacterial biofilm development.
Iron is an essential element for life but also serves as an environmental signal for biofilm development in the opportunistic
human pathogen Pseudomonas aeruginosa. Under iron-limiting conditions, P. aeruginosa displays enhanced twitching motility and forms flat unstructured biofilms. In this study, we present evidence suggesting
that iron-regulated production of the biosurfactant rhamnolipid is important to facilitate the formation of flat unstructured
biofilms. We show that under iron limitation the timing of rhamnolipid expression is shifted to the initial stages of biofilm
formation (versus later in biofilm development under iron-replete conditions) and results in increased bacterial surface motility.
In support of this observation, an rhlAB mutant defective in biosurfactant production showed less surface motility under iron-restricted conditions and developed
structured biofilms similar to those developed by the wild type under iron-replete conditions. These results highlight the
importance of biosurfactant production in determining the mature structure of P. aeruginosa biofilms under iron-limiting conditions.
Like all sessile organisms, surface-attached communities of bacteria known as biofilms must release and disperse cells into the environment to colonize new sites. For many pathogenic bacteria, biofilm dispersal plays an important role in the transmission of bacteria from environmental reservoirs to human hosts, in horizontal and vertical cross-host transmission, and in the exacerbation and spread of infection within a host. The molecular mechanisms of bacterial biofilm dispersal are only beginning to be elucidated. Biofilm dispersal is a promising area of research that may lead to the development of novel agents that inhibit biofilm formation or promote biofilm cell detachment. Such agents may be useful for the prevention and treatment of biofilms in a variety of industrial and clinical settings. This review describes the current status of research on biofilm dispersal, with an emphasis on studies aimed to characterize dispersal mechanisms, and to identify environmental cues and inter- and intracellular signals that regulate the dispersal process. The clinical implications of biofilm dispersal and the potential therapeutic applications of some of the most recent findings will also be discussed.
In this work, two biosurfactant-producing strains, Bacillus subtilis and Bacillus licheniformis, have been characterized. Both strains were able to grow at high salinity conditions and produce biosurfactants up to 10% NaCl. Both extracted-enriched biosurfactants showed good surface tension reduction of water, from 72 to 26-30 mN/m, low critical micelle concentration, and high resistance to pH and salinity. The potential of the two lipopeptide biosurfactants at inhibiting biofilm adhesion of pathogenic bacteria was demonstrated by using the MBEC device. The two biosurfactants showed interesting specific anti-adhesion activity being able to inhibit selectively biofilm formation of two pathogenic strains. In particular, Escherichia coli CFT073 and Staphylococcus aureus ATCC 29213 biofilm formation was decreased of 97% and 90%, respectively. The V9T14 biosurfactant active on the Gram-negative strain was ineffective against the Gram-positive and the opposite for the V19T21. This activity was observed either by coating the polystyrene surface or by adding the biosurfactant to the inoculum. Two fractions from each purified biosurfactant, obtained by flash chromatography, fractions (I) and (II), showed that fraction (II), belonging to fengycin-like family, was responsible for the anti-adhesion activity against biofilm of both strains.
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.
Transposon Tn5-GM-induced mutant strains of Pseudomonas aeruginosa which are unable to produce rhamnolipid biosurfactants and lack rhamnosyltransferase activity have been isolated. The DNA regions flanking the transposon were cloned and used as specific probes for the isolation of the corresponding wild-type genes from a P. aeruginosa wild-type cosmid gene library. Single cosmid clones capable of restoring rhamnolipid synthesis in the mutant strains were isolated and further subcloned and sequenced, resulting in the identification of two genes (rhlAB) which are organized as an operon upstream of the previously identified rhlR regulatory gene. The RhlA protein (32.5 kDa) harbors a putative signal sequence, suggesting that this protein is located in the periplasm, while the RhlB protein (47 kDa) contains at least two putative membrane-spanning domains. The expression of the rhlAB genes was found to be enhanced 20-fold during the stationary phase of growth under conditions of nitrogen limitation, as measured by using rhlA::lacZ fusions. Moreover, the transcriptional activation of the rhlAB genes appears to depend on a functional RhlR regulatory protein. The sequence upstream of the rhlA promoter contains two inverted repeats which define putative binding sites for the RhlR regulator. The controlled expression of the rhlAB genes in Escherichia coli led to the formation of active rhamnosyltransferase. This provides direct evidence for the fact that the rhamnosyltransferase encoding genes have been identified.
A mutant strain (65E12) of Pseudomonas aeruginosa that is unable to produce rhamnolipid biosurfactants and lacks rhamnosyltransferase activity was genetically complemented by using a P. aeruginosa PG201 wild-type gene library. A single complementing cosmid was isolated on the basis of surface tension measurements of subcultures of the transconjugants by using a sib selection strategy. The subcloning of the complementing cosmid clone yielded a 2-kb fragment capable of restoring rhamnolipid biosynthesis, rhamnosyltransferase activity, and utilization of hexadecane as a C source in mutant 65E12. The nucleotide sequence of the complementing 2-kb fragment was determined, and a single open reading frame (rhlR) of 723 bp specifying a putative 28-kDa protein (RhlR) was identified. Sequence homologies between the RhlR protein and some regulatory proteins such as LasR of P. aeruginosa, LuxR of Vibrio fischeri, RhiR of Rhizobium leguminosarum, and the putative activator 28-kDa UvrC of Escherichia coli suggest that the RhlR protein is a transcriptional activator. A putative target promoter which is regulated by the RhlR protein has been identified 2.5 kb upstream of the rhlR gene. Multiple plasmid-based rhlR gene copies had a stimulating effect on the growth of the P. aeruginosa wild-type strain in hexadecane-containing minimal medium, on rhamnolipid production, and on the production of pyocyanin chromophores. Disruption of the P. aeruginosa wild-type rhlR locus led to rhamnolipid-deficient mutant strains, thus confirming directly that this gene is necessary for rhamnolipid biosynthesis. Additionally, such PG201::'rhlR' mutant strains lacked elastase activity, indicating that the RhlR protein is a pleiotropic regulator.
Many microorganisms, especially bacteria, produce biosurfactants when grown on water-immiscible substrates. Biosurfactants are more effective, selective, environmentally friendly, and stable than many synthetic surfactants. Most common biosurfactants are glycolipids in which carbohydrates are attached to a long-chain aliphatic acid, while others, like lipopeptides, lipoproteins, and heteropolysaccharides, are more complex. Rapid and reliable methods for screening and selection of biosurfactant-producing microorganisms and evaluation of their activity have been developed. Genes involved in rhamnolipid synthesis (rhlAB) and regulation (rhlI and rhlR) in Pseudomonas aeruginosa are characterized, and expression of rhlAB in heterologous hosts is discussed. Genes for surfactin production (sfp, srfA, and comA) in Bacillus spp. are also characterized. Fermentative production of biosurfactants depends primarily on the microbial strain, source of carbon and nitrogen, pH, temperature, and concentration of oxygen and metal ions. Addition of water-immiscible substrates to media and nitrogen and iron limitations in the media result in an overproduction of some biosurfactants. Other important advances are the use of water-soluble substrates and agroindustrial wastes for production, development of continuous recovery processes, and production through biotransformation. Commercialization of biosurfactants in the cosmetic, food, health care, pulp- and paper-processing, coal, ceramic, and metal industries has been proposed. However, the most promising applications are cleaning of oil-contaminated tankers, oil spill management, transportation of heavy crude oil, enhanced oil recovery, recovery of crude oil from sludge, and bioremediation of sites contaminated with hydrocarbons, heavy metals, and other pollutants. Perspectives for future research and applications are also discussed.
It has been a decade since multicellularity was proposed as a general bacterial trait. Intercellular communication and multicellular coordination are now known to be widespread among prokaryotes and to affect multiple phenotypes. Many different classes of signaling molecules have been identified in both Gram-positive and Gram-negative species. Bacteria have sophisticated signal transduction networks for integrating intercellular signals with other information to make decisions about gene expression and cellular differentiation. Coordinated multicellular behavior can be observed in a variety of situations, including development of E. coli and B. subtilis colonies, swarming by Proteus and Serratia, and spatially organized interspecific metabolic cooperation in anaerobic bioreactor granules. Bacteria benefit from multicellular cooperation by using cellular division of labor, accessing resources that cannot effectively be utilized by single cells, collectively defending against antagonists, and optimizing population survival by differentiating into distinct cell types.
Surfactants are surface-active compounds capable of reducing surface and interfacial tension at the interfaces between liquids, solids and gases, thereby allowing them to mix or disperse readily as emulsions in water or other liquids. The enormous market demand for surfactants is currently met by numerous synthetic, mainly petroleum-based, chemical surfactants. These compounds are usually toxic to the environment and non-biodegradable. They may bio-accumulate and their production, processes and by-products can be environmentally hazardous. Tightening environmental regulations and increasing awareness for the need to protect the ecosystem have effectively resulted in an increasing interest in biosurfactants as possible alternatives to chemical surfactants. Biosurfactants are amphiphilic compounds of microbial origin with considerable potential in commercial applications within various industries. They have advantages over their chemical counterparts in biodegradability and effectiveness at extreme temperature or pH and in having lower toxicity. Biosurfactants are beginning to acquire a status as potential performance-effective molecules in various fields. At present biosurfactants are mainly used in studies on enhanced oil recovery and hydrocarbon bioremediation. The solubilization and emulsification of toxic chemicals by biosurfactants have also been reported. Biosurfactants also have potential applications in agriculture, cosmetics, pharmaceuticals, detergents, personal care products, food processing, textile manufacturing, laundry supplies, metal treatment and processing, pulp and paper processing and paint industries. Their uses and potential commercial applications in these fields are reviewed.
Little is known about the interaction of biosurfactants with bacterial cells. Recent work in the area of biodegradation suggests that there are two mechanisms by which biosurfactants enhance the biodegradation of slightly soluble organic compounds. First, biosurfactants can solubilize hydrophobic compounds within micelle structures, effectively increasing the apparent aqueous solubility of the organic compound and its availability for uptake by a cell. Second, biosurfactants can cause the cell surface to become more hydrophobic, thereby increasing the association of the cell with the slightly soluble substrate. Since the second mechanism requires very low levels of added biosurfactant, it is the more intriguing of the two mechanisms from the perspective of enhancing the biodegradation process. This is because, in practical terms, addition of low levels of biosurfactants will be more cost-effective for bioremediation. To successfully optimize the use of biosurfactants in the bioremediation process, their effect on cell surfaces must be understood. We report here that rhamnolipid biosurfactant causes the cell surface of Pseudomonas spp. to become hydrophobic through release of lipopolysaccharide (LPS). In this study, two Pseudomonas aeruginosa strains were grown on glucose and hexadecane to investigate the chemical and structural changes that occur in the presence of a rhamnolipid biosurfactant. Results showed that rhamnolipids caused an overall loss in cellular fatty acid content. Loss of fatty acids was due to release of LPS from the outer membrane, as demonstrated by 2-keto-3-deoxyoctonic acid and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and further confirmed by scanning electron microscopy. The amount of LPS loss was found to be dependent on rhamnolipid concentration, but significant loss occurred even at concentrations less than the critical micelle concentration. We conclude that rhamnolipid-induced LPS release is the probable mechanism of enhanced cell surface hydrophobicity.
Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.
Swarming motility plays an important role in surface colonization by several flagellated bacteria. Swarmer cells are specially
adapted to rapidly translocate over agar surfaces by virtue of their more numerous flagella, longer cell length, and encasement
of slime. The external slime provides the milieu for motility and likely harbors swarming signals. We recently reported the
isolation of swarming-defective transposon mutants of Salmonella enterica serovar Typhimurium, a large majority of which were defective in lipopolysaccharide (LPS) synthesis. Here, we have examined
the biofilm-forming abilities of the swarming mutants using a microtiter plate assay. A whole spectrum of efficiencies were
observed, with LPS mutants being generally more proficient than wild-type organisms in biofilm formation. Since we have postulated
that O-antigen may serve a surfactant function during swarming, we tested the effect of the biosurfactant surfactin on biofilm
formation. We report that surfactin inhibits biofilm formation of wild-type S. enterica grown either in polyvinyl chloride microtiter wells or in urethral catheters. Other bio- and chemical surfactants tested
had similar effects.
A mutant strain (65E12) of Pseudomonas aeruginosa that is unable to produce rhamnolipid biosurfactants and lacks rhamnosyltransferase activity was genetically complemented by using a P. aeruginosa PG201 wild-type gene library. A single complementing cosmid was isolated on the basis of surface tension measurements of subcultures of the transconjugants by using a sib selection strategy. The subcloning of the complementing cosmid clone yielded a 2-kb fragment capable of restoring rhamnolipid biosynthesis, rhamnosyltransferase activity, and utilization of hexadecane as a C source in mutant 65E12. The nucleotide sequence of the complementing 2-kb fragment was determined, and a single open reading frame (rhlR) of 723 bp specifying a putative 28-kDa protein (RhlR) was identified. Sequence homologies between the RhlR protein and some regulatory proteins such as LasR of P. aeruginosa, LuxR of Vibrio fischeri, RhiR of Rhizobium leguminosarum, and the putative activator 28-kDa UvrC of Escherichia coli suggest that the RhlR protein is a transcriptional activator. A putative target promoter which is regulated by the RhlR protein has been identified 2.5 kb upstream of the rhlR gene. Multiple plasmid-based rhlR gene copies had a stimulating effect on the growth of the P. aeruginosa wild-type strain in hexadecane-containing minimal medium, on rhamnolipid production, and on the production of pyocyanin chromophores. Disruption of the P. aeruginosa wild-type rhlR locus led to rhamnolipid-deficient mutant strains, thus confirming directly that this gene is necessary for rhamnolipid biosynthesis. Additionally, such PG201::'rhlR' mutant strains lacked elastase activity, indicating that the RhlR protein is a pleiotropic regulator.
Screening test for obtaining growth stimulant (GS) produced by a hydrocarbon-utilizing bacterium, Pseudomonas aeruginosa S7B1, was carried out. In consequence, the anthrone positive substance was most effective on the growth of this strain. Although the growth of this strain on glucose medium had no relation with the addition of GS, the growth on n-hexadecane medium was remarkably stimulated by the addition of GS. This effect of GS seemed to be specific on the growth of P. aeruginosa. GS which had a strong surface activity and emulsifying power was comfirmed to be rhamnolipid.
Two kinds of glycolipids (R-l and R-2) were produced in the culture media by several strains of Pseudomonas aeruginosa when grown on n paraffin (mixture of C12, C13. and C14 fractions). These compounds were isolated through the extraction of the culture broth with ethylacetate and the chromatography on silicic acid column. On the basis of chemical analysis, these lipids were characterized as 2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β- hydroxydecanoyl-β-hydroxydecanoate (R-l) and L-α-rhamnopyranosyl-β- hydroxydecanoyl-β-hydroxydecanoate (R-2). Bactericidal activity of R-2 which is postulated to be a precursor of R-1 was remarked against grampositive bacteria. Further, these compounds also demonstrated mycoplasmacidal and antiviral activities in vitro.
Screening test for obtaining growth stimulant (GS) produced by a hydrocarbon-utilizing bacterium, Pseudomonas aeruginosa S7B1, was carried out. In consequence, the anthrone positive substance was most effective on the growth of this strain. Although the growth of this strain on glucose medium had no relation with the addition of GS, the growth on n-hexadecane medium was remarkably stimulated by the addition of GS. This effect of GS seemed to be specific on the growth of P. aeruginosa. GS which had a strong surface activity and emulsifying power was comfirmed to be rhamnolipid.
Seeding dispersal is an active detachment exhibit in aging Pseudomonas aeruginosa biofilm. Yet, effect factors of this process in the biofilm of clinical isolated mucoid P. aeruginosa strain remain to be better characterized. In our previous work, one mucoid P. earuginosa strain PA17 was isolated from a patient with recurrent pulmonary infection. In this study, confocal scanning laser microscope combined with LIVE/DEAD viability staining revealed that PA17 biofilm exhibited earlier seeding dispersal than non-mucoid PAO1. We further compared the motility and the expression of motility-associated gene during biofilm development between PA17 and PAO1. PA17 was found to be impaired in all three kinds of motility compared to PAO1. Moreover, we investigated the expression of rhamnolipid-associated genes in PA17 and PAO1 biofilm. The expression of these genes was in accordance with the process of seeding dispersal. Our results indicated that rhamnolipid but not motility is associated with the initiation of seeding dispersal of PA17 biofilm.
Bacteria are highly social organisms that communicate via signaling molecules, move collectively over surfaces and make biofilm communities. Nonetheless, our main line of defense against pathogenic bacteria consists of antibiotics-drugs that target individual-level traits of bacterial cells and thus, regrettably, select for resistance against their own action. A possible solution lies in targeting the mechanisms by which bacteria interact with each other within biofilms. The emerging field of microbial social evolution combines molecular microbiology with evolutionary theory to dissect the molecular mechanisms and the evolutionary pressures underpinning bacterial sociality. This exciting new research can ultimately lead to new therapies against biofilm infections that exploit evolutionary cheating or the trade-off between biofilm formation and dispersal.
Bacterial attachment on solid surfaces has various implications in environmental, industrial and medical applications. In this study, the effects of rhamnolipid biosurfactants on initial attachment of bacteria on hydrophilic glass and hydrophobic octadecyltrichlorosilane (OTS) modified glass were evaluated under continuous-flow conditions. The bacteria investigated were three Gram-negative species Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli, and two Gram-positive species Staphylcoccus epidermidis and Bacillus subtilis. Rhamnolipids, at 10 and 200mg/l, significantly reduced the attachment of all but S. epidermidis on both glass and OTS-modified glass. For S. epidermidis rhamnolipids reduced the attachment on OTS-modified glass but not on glass. Studies were further done to identify the mechanism(s) by which rhamnolipids reduced the cell attachment. The following potential properties of rhamnolipids were investigated: inhibition of microbial growth, change of cell surface hydrophobicity, easier detachment of cells already attached to substratum, and modification of substratum surface properties. Results showed that rhamnolipids were ineffective for the latter two effects. Rhamnolipids, up to 200mg/l, inhibited the growth of B. subtilis, S. epidermidis and P. aeruginosa PAO1 but not the growth of E. coli, P. putida and P. aeruginosa E0340. Also, rhamnolipids tended to increase the hydrophobicity of P. aeruginosa PAO1 and E. coli, decrease the hydrophobicity of P. putida and S. epidermidis, and have no clear effect on the hydrophobicity of B. subtillis. These trends however did not correlate with the observed trend of cell attachment reduction. The responsible mechanism(s) remained unknown.
Biofilms are often considered as localized zones of high cell density. Quorum sensing provides a means for control of population processes and has been implicated in the regulation of biofilm activities. We present a role for quorum sensing in programmed detachment and dispersal processes. Biofilms of Pseudomonas
aeruginosa PAO1 and its isogenic homoserine lactone (HSL) mutant P.
aeruginosa PAO-JP2 were grown in batch culture on glass substrata; differences were found in the rate and extent of formation of biofilm. Climax communities were observed for PAO1 at 24 h. These were later accompanied by foaming, a drop in the surface tension of culture media and dispersal of the biofilm, after which no subsequent biofilm accretion occurred. PAO-JP2 cultures reformed biofilm post-detachment and did not foam. Prevention of biofilm reformation in the wild type was related to some component excreted into the culture medium. Rhamnolipid, a biosurfactant regulated by quorum sensing, was detected in PAO1 cultures. When rhamnolipid was added to freshly inoculated substrata, biofilm formation was inhibited. At 20 h, PAO1 biofilms were transferred to medium with added rhamnolipid: biofilm was relatively unaffected. Biofilm events were also studied in medium supplemented with N-butyryl-L-homoserine lactone, which is involved in the regulation of rhamnolipid synthesis. Both strains exhibited similar trends of rapid biofilm formation and dramatic changes in the rate and extent of biofilm accretion. In both cases, there was premature foaming, lowered surface tension and elevated rhamnolipid levels. A role for HSLs in maintenance of biofilm and events leading to dispersion of cells is proposed. This role would encompass dispersion but not necessarily detachment of cells from biofilm and supports a new function for rhamnolipid in pathogenesis, whereby rhamnolipid would promote the dissemination of cells from a nidus of infection.
Microorganisms produce a variety of surface-active agents (or surfactants). These can be divided into low-molecular-weight molecules that lower surface and interfacial tensions efficiently and high-molecular-weight polymers that bind tightly to surfaces. These surfactants, produced by a wide variety of microorganisms, have very different chemical structures and surface properties. It is therefore reasonable to assume that different groups of biosurfactants have different natural roles in the growth of the producing microorganisms. Moreover, as their chemical structures and surface properties are so different, each emulsifier probably provides advantages in a particular ecological niche. Several bioemulsifiers have antibacterial or antifungal activities. Other bioemulsifiers enhance the growth of bacteria on hydrophobic water-insoluble substrates by increasing their bioavailability, presumably by increasing their surface area, desorbing them from surfaces and increasing their apparent solubility. Bioemulsifiers also play an important role in regulating the attachment–detachment of microorganisms to and from surfaces. In addition, emulsifiers are involved in bacterial pathogenesis, quorum sensing and biofilm formation. Recent experiments indicate that a high-molecular-weight bioemulsifier that coats the bacterial surface can be transferred horizontally to other bacteria, thereby changing their surface properties and interactions with the environment.
The toxicity of the glycolipid ofPs. aeruginosa to mice has to be ascribed to the fact that it causes hemolysis by dissolving materials from the cell wall structure of the
red blood cells.
Nearly 12 years after promising results suggested that antibiofilm agents might be developed into novel therapeutics, there are no such products on the market. In our opinion, the reasons for this have been predominantly economic. Recent developments, however, suggest that there could still be emerging opportunities for the developments of such products.
How bacteria regulate, assemble and rotate flagella to swim in liquid media is reasonably well understood. Much less is known about how some bacteria use flagella to move over the tops of solid surfaces in a form of movement called swarming. The focus of bacteriology is changing from planktonic to surface environments, and so interest in swarming motility is on the rise. Here, I review the requirements that define swarming motility in diverse bacterial model systems, including an increase in the number of flagella per cell, the secretion of a surfactant to reduce surface tension and allow spreading, and movement in multicellular groups rather than as individuals.
Solid surfaces in contact with water have been found to be biofouled due to the attachment of various organisms. For better understanding of the biofilm formation, the important initial stage of bacterial attachment was investigated with Pseudomonas aeruginosa PAO1 as a model microorganism. Effects of the biosurfactant rhamnolipids and the shear conditions were particularly examined.
A highly reproducible procedure was employed. The procedure involved monitoring and counting the number of attached cells on glass walls of the flow chambers, through which a PAO1 suspension was circulated and, subsequently, a saline solution was passed for washing. The experiments were made under different circulation rates (exerting different shear on the bacteria) and rhamnolipid concentrations.
Reproducibility of the procedure was confirmed. The velocity profiles near the flow chamber wall were determined. Rhamnolipids, even at a very low concentration of 13 mg/l, were found to deter the bacterial attachment substantially. Prewashing the cells with a 100 mg/l rhamnolipid solution, however, did not affect the attachment significantly. As for the effect of shear, the PAO1 attachment showed an increasing-then-decreasing trend in the range investigated, i.e., 1.0 to 26 mN/m(2) shear stresses at the chamber wall. The diffusion-limited transport of cells to the chamber wall might have contributed to, but could not fully explain, the increasing attachment observed in the very low shear range (up to 3.5-5.0 mN/m(2)).
As compared to static systems, the flow chamber systems significantly improved the reproducibility of initial attachment results. Flow chamber systems were more suitable for experimental investigations of bacterial attachment to surfaces. Rhamnolipids were found to be potent antifoulants for PAO1 attachment on glass. The initial cell attachment increased with increasing shear at the very low shear range (up to 3.5-5.0 mN/m(2)), but the attachment could be minimized with further increase of the shear.
Many of the virulence factors produced by the opportunistic human pathogen Pseudomonas aeruginosa are quorum-sensing (QS) regulated. Among these are rhamnolipids, which have been shown to cause lysis of several cellular components of the human immune system, e.g. monocyte-derived macrophages and polymorphonuclear leukocytes (PMNs). We have previously shown that rhamnolipids produced by P. aeruginosa cause necrotic death of PMNs in vitro. This raises the possibility that rhamnolipids may function as a 'biofilm shield'in vivo, which contributes significantly to the increased tolerance of P. aeruginosa biofilms to PMNs. In the present study, we demonstrate the importance of the production of rhamnolipids in the establishment and persistence of P. aeruginosa infections, using an in vitro biofilm system, an intraperitoneal foreign-body model and a pulmonary model of P. aeruginosa infections in mice. Our experimental data showed that a P. aeruginosa strain, unable to produce any detectable rhamnolipids due to an inactivating mutation in the single QS-controlled rhlA gene, did not induce necrosis of PMNs in vitro and exhibited increased clearance compared with its wild-type counterpart in vivo. Conclusively, the results support our model that rhamnolipids are key protective agents of P. aeruginosa against PMNs.
Biofilms might result in healthcare-associated infections and substantially impact healthcare delivery. Bacteriophage (phage) has been used to treat infectious diseases in humans and there is interest in phage to control biofilms. Phages propagate in their bacterial host and many phages produce depolymerases that hydrolyze biofilm extracellular polymers. Drawbacks of phage to consider include narrow host range, bacterial resistance to phage and phage-encoded virulence genes that can incorporate into the host bacterial genome. The immune system might inactivate phage, and impure phage preparations could contain endotoxin. Phage mixtures or engineered phages could provide effective strategies to overcome these obstacles. Lytic bacteriophages could become a new class of anti-biofilm agents.
In this study, the effect of a purified rhamnolipid biosurfactant on the hydrophobicity of octadecane-degrading cells was investigated to determine whether differences in rates of octadecane biodegradation resulting from the addition of rhamnolipid to four strains of Pseudomonas aeruginosa could be related to measured differences in hydrophobicity. Cell hydrophobicity was determined by a modified bacterial adherence to hydrocarbon (BATH) assay. Bacterial adherence to hydrocarbon quantitates the preference of cell surfaces for the aqueous phase or the aqueous-hexadecane interface in a two-phase system of water and hexadecane. On the basis of octadecane biodegradation in the absence of rhamnolipid, the four bacterial strains were divided into two groups: the fast degraders (ATCC 15442 and ATCC 27853), which had high cell hydrophobicities (74 and 55% adherence to hexadecane, respectively), and the slow degraders (ATCC 9027 and NRRL 3198), which had low cell hydrophobicities (27 and 40%, respectively). Although in all cases rhamnolipid increased the aqueous dispersion of octadecane at least 10(4)-fold, at low rhamnolipid concentrations (0.6 mM), biodegradation by all four strains was initially inhibited for at least 100 h relative to controls. At high rhamnolipid concentrations (6 mM), biodegradation by the fast degraders was slightly inhibited relative to controls, but the biodegradation by the slow degraders was enhanced relative to controls. Measurement of cell hydrophobicity showed that rhamnolipids increased the cell hydrophobicity of the slow degraders but had no effect on the cell hydrophobicity of the fast degraders. The rate at which the cells became hydrophobic was found to depend on the rhamnolipid concentration and was directly related to the rate of octadecane biodegradation.(ABSTRACT TRUNCATED AT 250 WORDS)
The objective of this research was to evaluate the effect of low concentrations of a rhamnolipid biosurfactant on the in situ biodegradation of hydrocarbon entrapped in a porous matrix. Experiments were performed with sand-packed columns under saturated flow conditions with hexadecane as a model hydrocarbon. Application of biosurfactant concentrations greater than the CMC (the concentration at which the surfactant molecules spontaneously form micelles or vesicles [0.03 mM]) resulted primarily in the mobilization of hexadecane entrapped within the sand matrix. In contrast, application of biosurfactant concentrations less than the CMC enhanced the in situ mineralization of entrapped hexadecane; however, this effect was dependent on the choice of bacterial isolate. The two Pseudomonas isolates tested, R4 and ATCC 15524, were used because they exhibit different patterns of biodegradation of hexadecane, and they also differed in their physical response to rhamnolipid addition. ATCC 15524 cells formed extensive multicell aggregates in the presence of rhamnolipid while R4 cells were unaffected. This behavior did not affect the ability of the biosurfactant to enhance the biodegradation of hexadecane in well-mixed soil slurry systems but had a large affect on the extent of entrapped hexadecane biodegradation in the sand-packed-column system that was used in this study.
The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface in response to appropriate environmental signals. We report the isolation and characterization of mutants of Pseudomonas aeruginosa PA14 defective in the initiation of biofilm formation on an abiotic surface, polyvinylchloride (PVC) plastic. These mutants are designated surface attachment defective (sad ). Two classes of sad mutants were analysed: (i) mutants defective in flagellar-mediated motility and (ii) mutants defective in biogenesis of the polar-localized type IV pili. We followed the development of the biofilm formed by the wild type over 8 h using phase-contrast microscopy. The wild-type strain first formed a monolayer of cells on the abiotic surface, followed by the appearance of microcolonies that were dispersed throughout the monolayer of cells. Using time-lapse microscopy, we present evidence that microcolonies form by aggregation of cells present in the monolayer. As observed with the wild type, strains with mutations in genes required for the synthesis of type IV pili formed a monolayer of cells on the PVC plastic. However, in contrast to the wild-type strain, the type IV pili mutants did not develop microcolonies over the course of the experiments, suggesting that these structures play an important role in microcolony formation. Very few cells of a non-motile strain (carrying a mutation in flgK) attached to PVC even after 8 h of incubation, suggesting a role for flagella and/or motility in the initial cell-to-surface interactions. The phenotype of these mutants thus allows us to initiate the dissection of the developmental pathway leading to biofilm formation.
Liquid chromatography/mass spectrometry using electrospray ionisation was used to analyse rhamnolipids produced by a Pseudomonas aeruginosa strain with mannitol or naphthalene as carbon source. Identification and quantification of 28 different rhamnolipid congeners was accomplished using a reverse-phase C(18) column and a 30 min chromatographic run. Isomeric rhamnolipids that were not chromatographically resolved could be identified by interpretation of their mass spectra and their relative proportions estimated. The most abundant rhamnolipid produced on mannitol contained two rhamnoses and two 3-hydroxydecanoic acid groups. The most abundant rhamnolipid produced from naphthalene contained two rhamnoses and one 3-hydroxydecanoic acid group.
Two rapid and simple methods for the characterisation and quantification of rhamnolipids produced by a growing culture of the Pseudomonas aeruginosa strain 57RP were developed. Two rhamnolipids were purified and their response factors determined. The various rhamnolipids produced were then measured using liquid chromatography/mass spectrometry. The culture supernatants were injected directly, without prior purification, in a HPLC equipped with a C(18) reverse-phase column. The complete profile of rhamnolipid congeners produced during a 2 week cultivation period was monitored. In order to shorten the analysis time, another method was developed which did not require chromatographic separation of the rhamnolipids prior to their detection. Quantification of rhamnolipids using the direct infusion method gave results very similar to those obtained with HPLC separation. These two methods were very well correlated with the standard colorimetric orcinol method. The rhamnolipid profiles obtained show that the various rhamnolipid congeners are secreted simultaneously, and that their relative proportion remained unchanged throughout the cultivation period.
Biofilms can be defined as communities of microorganisms attached to a surface. It is clear that microorganisms undergo profound changes during their transition from planktonic (free-swimming) organisms to cells that are part of a complex, surface-attached community. These changes are reflected in the new phenotypic characteristics developed by biofilm bacteria and occur in response to a variety of environmental signals. Recent genetic and molecular approaches used to study bacterial and fungal biofilms have identified genes and regulatory circuits important for initial cell-surface interactions, biofilm maturation, and the return of biofilm microorganisms to a planktonic mode of growth. Studies to date suggest that the planktonic-biofilm transition is a complex and highly regulated process. The results reviewed in this article indicate that the formation of biofilms serves as a new model system for the study of microbial development.
Microorganisms produce a variety of surface-active agents (or surfactants). These can be divided into low-molecular-weight molecules that lower surface and interfacial tensions efficiently and high-molecular-weight polymers that bind tightly to surfaces. These surfactants, produced by a wide variety of microorganisms, have very different chemical structures and surface properties. It is therefore reasonable to assume that different groups of biosurfactants have different natural roles in the growth of the producing microorganisms. Moreover, as their chemical structures and surface properties are so different, each emulsifier probably provides advantages in a particular ecological niche. Several bioemulsifiers have antibacterial or antifungal activities. Other bioemulsifiers enhance the growth of bacteria on hydrophobic water-insoluble substrates by increasing their bioavailability, presumably by increasing their surface area, desorbing them from surfaces and increasing their apparent solubility. Bioemulsifiers also play an important role in regulating the attachment-detachment of microorganisms to and from surfaces. In addition, emulsifiers are involved in bacterial pathogenesis, quorum sensing and biofilm formation. Recent experiments indicate that a high-molecular-weight bioemulsifier that coats the bacterial surface can be transferred horizontally to other bacteria, thereby changing their surface properties and interactions with the environment.