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The draft genome of Thermoanaerobacter thermocopriae CM-CNRG TB177 isolated from an oil reservoir in Mexico was determined and annotated. The organism is a thermophilic and strict anaerobe bacterium that produces rhamnolipids, using glucose as a carbon source. The predicted genome size is 2,496,169 bp and 2,550 genes.

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Biosurfactants encompass structurally and chemically diverse molecules with surface active properties, and a broad industrial deployment, including pharmaceuticals. The interest is growing mainly for the low toxicity, biodegradability, and production from renewable sources. In this work, the optimized biosurfactant production by Pseudomonas aeruginosa BM02, isolated from the soil of a mining area in the Brazilian Amazon region was assessed, in addition to its antiviral, antitumor, and antimicrobial activities. The optimal conditions for biosurfactant production were determined using a factorial design, which showed the best yield (2.28 mg/mL) at 25 °C, pH 5, and 1% glycerol. The biosurfactant obtained was characterized as a mixture of rhamnolipids with virucidal properties against Herpes Simplex Virus, Coronavirus, and Respiratory Syncytial Virus, in addition to antimicrobial properties against Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecium), at 50 µg/mL. The antitumor activity of BS (12.5 µg/mL) was also demonstrated, with potential selectivity in reducing the proliferation of breast tumor cells, after 1 min of exposure. These results demonstrate the importance of studying the interconnection between cultivation conditions and properties of industrially important compounds, such as rhamnolipid-type biosurfactant from P. aeruginosa BM02, a promising and sustainable alternative in the development of new antiviral, antitumor, and antimicrobial prototypes.

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Bacillus cereus is a Gram-positive, endospore-forming bacterium well-known as a food pathogen that causes great losses in the food industry, especially in dairy. In this study, rhamnolipid (RL) biosurfactants were evaluated as a bio-based alternative for controlling the growth of vegetative cells and endospores of B. cereus. RLs were tested against 14 B. cereus strains isolated from different types of foodstuffs. The antimicrobial activity against vegetative cells and endospores revealed minimal inhibitory concentration (MIC) values of 0.098 mg/mL for almost all strains tested and minimal bactericidal concentration (MBC) varying between 0.098 and >25 mg/mL. The presence of RLs inhibited endospore germination by more than 99%, reducing by 5.5 log the outgrowth of strain 0426. Scanning and transmission electron microscopy confirmed that exposure to RL causes damage to the structure of endospores. When skim milk was utilized as a food model, RL inhibited the growth of vegetative cells and endospores of B. cereus, showing MBC of 3.13 mg/mL for the vegetative cells of strain 0426. The surfactant also reduced bacterial growth in milk at refrigerator temperature. The results suggest that RLs are promising candidates for the development of novel strategies to control B. cereus in the food industry.

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Biofilm formation has been suggested to play a significant role in the survival of pathogens in food production. Interest in evaluating alternative products of natural origin for disinfectant use has increased. However, there is a lack of information regarding the effects of biosurfactants and organic acids on Salmonella enterica serotype Enteritidis, Escherichia coli, and Campylobacter jejuni biofilms, mainly considering temperatures found in environments of poultry processing, as well as simulating the contact times used for disinfection. The aim of this study was to evaluate the antibiofilm activity of rhamnolipid, malic acid, and citric acid on the adhesion of S. Enteritidis, E. coli, and C. jejuni on polystyrene surfaces at different temperatures (4, 12, and 25 °C), compound concentrations, and times of contact (5 and 10 min), and to analyze the potential use of these compounds to disrupt formed biofilms. All three compounds exhibited antibiofilm activity under all analyzed conditions, both in the prevention and removal of formed biofilms. Contact time was less important than temperature and concentration. The antibiofilm activity of the compounds also varied according to the pathogens involved. In the food industry, compound selection must consider the temperature found in each stage of product processing and the target pathogens to be controlled.

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The interest in nano-sized materials to develop novel products has increased exponentially in the last decade, together with the search for green methods for their synthesis. An alternative to contribute to a more sustainable approach is the use of microbial-derived molecules to assist nanomaterial synthesis. In this sense, biosurfactants (BSs) have emerged as eco-friendly substitutes in nano-sized materials preparation. The inherent amphiphilic and self-assembly character of BSs associated with their low eco-toxicity, biodegradability, biocompatibility, structural diversity, biological activity, and production from renewable resources are potential advantages over chemically-derived surfactants. In nanotechnology, these versatile molecules play multiple roles. In nanoparticle (NP) synthesis, they act as capping and reducing agents and they also provide self-assembly structures to encapsulation, functionalization, or templates and act as emulsifiers in nanoemulsions. Moreover, BSs can also play as active compounds owing to their intrinsic biological properties. This review presents the recent trends in the development of BS-based nanostructures and their biomedical and environmental applications. Fundamental aspects regarding their antimicrobial and anticancer activities are also discussed.

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The present work aimed to give an economical destiny to the produced water, a residue generated by the oil and gas industry by means of producing bioactives such as xanthan gum and ramnolipid. These compounds are often used in combination during enhanced oil recovery strategies. On the other hand, reports on co-culture of bacterial strains that are responsible for their production are rare. This research shows a factorial design method associated with surface response analysis to optimize carbon sources, sucrose and crude glycerin, and fermentation agents for culturing Xanthomonas campestris and Pseudomonas aeruginosa using the described conditions. After the critical point validation resulting in xanthan and ramnolipid production of 8.69 and 4.80 g L-1, quality tests showed an apparent viscosity of 1006 cP with an emulsifying activity abouve 50% for 94 h.

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This study aimed to produce and characterize biosurfactants using the Thermoanaerobacter sp. CM-CNRG TB177 strain isolated from an oil field in Mexico, as well as assessing the influence of different carbon and nitrogen sources on the capacity of the produced surfactant to reduce the surface tension of water. The thin-layer chromatography (TLC) revealed that the obtained extract corresponds to a mono-rhamnolipid; the results of the ultra-performance-liquid chromatography/mass spectrometry (UPLC/MS) analysis revealed that the Thermoanaerobacter sp. CM-CNRG TB177 strain produces a mixture of three rhamnolipids, whose masses correspond to mono-rhamnolipid. The rhamnolipids mixture obtained using 2.5% molasses as carbon source diminished the surface tension of water to 29.67 mNm-1, indicating that the concentration of molasses influenced the capacity of the produced surfactant to reduce the surface tension of water. Also, the microorganism was not capable of growing in the absence of yeast extract as nitrogen source. To the best of our knowledge, the presented results describe for the first time the nature of the biosurfactant produced by a bacterium of the Thermoanaerobacter genus.Key points• Thermoanaerobacter sp. CM-CNRG TB177 produces biosurfactants, and its glycolipid nature is described for the first time.• The HPLC analysis revealed a mixture of three rhamnolipid congeners, and UPLC/MS analysis determined that two of the congeners are the rhamnolipids Rha-C8-C10 and Rha-C12-C10.• The lowest surface tension of 29.67 mNm-1 was obtained with molasses as source of carbon at a 2.5% concentration.

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Foodborne diseases (FBD) are a great problem worldwide, leading millions of people to seek medical help and to significant economic losses for industry. Among the agents implicated in FDB is Bacillus cereus, a Gram-positive, toxigenic and endospore-forming bacterium. In this study, rhamnolipid (RL) biosurfactant, celery oleoresin (OR) and limonene (LN) were evaluated as bio-based alternatives for controlling the growth of vegetative cells and endospores of B. cereus. To address their antimicrobial activity, the compounds were tested separately and in combination. Results demonstrate that, when combined with RL, both OR and LN have lower minimal inhibitory concentration (MIC) values and increased endospore inhibition potential. A percentage of endospore inhibition from 73% to 98%, corresponding to a 2.8-3.6 log reduction in spore outgrowth, was observed. RL inhibited B. cereus growth and endospore germination and potentially enhanced the antimicrobial efficacy of the natural hydrophobic compounds tested.

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BACKGROUND: Antimicrobial resistance poses substantial risks to human health. Thus, there is an urgent need for novel antimicrobial agents, including alternative compounds, such as peptides derived from bacterial toxin-antitoxin (TA) systems. ParELC3 is a synthetic peptide derived from the ParE toxin reported to be a good inhibitor of bacterial topoisomerases and is therefore a potential antibacterial agent. However, ParELC3 is inactive against bacteria due to its inability to cross the bacterial membranes. To circumvent this limitation we prepared and used rhamnolipid-based liposomes to carry and facilitate the passage of ParELC3 through the bacterial membrane to reach its intracellular target - the topoisomerases. METHODS AND RESULTS: Small unilamellar liposome vesicles were prepared by sonication from three formulations that included 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesterol. ParELC3 was loaded with high efficiency into the liposomes. Characterization by DLS and TEM revealed the appropriate size, zeta potential, polydispersity index, and morphology. In vitro microbiological experiments showed that ParELC3 loaded-liposomes are more efficient (29 to 11 µmol·L-1) compared to the free peptide (>100 µmol·L-1) at inhibiting the growth of standard E. coli and S. aureus strains. RL liposomes showed high hemolytic activity but when prepared with POPC and Chol this activity had a significant reduction. Independently of the formulation, the vesicles had no detectable cytotoxicity to HepG2 cells, even at the highest concentrations tested (1.3 mmol·L-1 and 50 µmol·L-1 for rhamnolipid and ParELC3, respectively). CONCLUSION: The present findings suggest the potential use of rhamnolipid-based liposomes as nanocarrier systems to enhance the bioactivity of peptides.

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The objective of this study was to compare the potential of mono-rhamnolipids (mono-RML) and di-rhamnolipids (di-RML) against biofilm formation on carbon steel coupons submitted to oil produced water for 14 days. The antibiofilm effect of the RML on the coupons was analyzed by scanning electron microscopy (SEM) and by epifluorescence microscopy, and the contact angle was measured using a goniometer. SEM analysis results showed that all RML congeners had effective antibiofilm action, as well as preliminary anticorrosion evaluation confirmed that all RML congeners prevented the metal deterioration. In more detail, epifluorescence microscopy showed that mono-RML were the most efficient congeners in preventing microorganism's adherence on the carbon steel metal. Image analyses indicate the presence of 15.9%, 3.4%, and <0.1% of viable particles in di-RML, mono/di-RML and mono-RML pretreatments, respectively, in comparison to control samples. Contact angle results showed that the crude carbon steel coupon presented hydrophobic character favoring hydrophobic molecules adhesion. We calculated the theoretical polarity of the RML congeners and verified that mono-RML (log P = 3.63) presented the most hydrophobic character. This had perfect correspondence to contact angle results, since mono-RML conditioning (58.2°) more significantly changed the contact angle compared with the conditioning with one of the most common surfactants used on oil industry (29.4°). Based on the results, it was concluded that rhamnolipids are efficient molecules to be used to avoid biofilm on carbon steel metal when submitted to oil produced water and that a higher proportion of mono-rhamnolipids is more indicated for this application.

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Biosurfactants are surface-active molecules originated from renewable resources, which are produced by microbial fermentation or chemical/enzymatic catalysis. These molecules present important advantages as compared to petrochemical surfactants, given their resistance to extreme conditions, biodegradability, specificity, and environmental compatibility. Besides that, the high production costs hinder its commercialization. In this way, this article aimed to analyze microbial biosurfactants production, focusing on the optimization of metabolic pathways and production processes, to identify key aspects and provide alternatives to allow a cost-effective production at industrial scale. This was achieved by a broad analysis of biosurfactants properties, applications, and biosynthetic pathways (in terms of yield, cofactors, and energy), in addition to an assessment of production-associated costs. As a result of the present extensive data survey and analysis, key production aspects are disclosed. The metabolic pathway yield analysis demonstrated that production of biosurfactants can be significantly improved (highest theoretical yield was 0.47 gbiosurfactant /gsubstrate ) by the use of biomolecular engineering techniques to generate optimized synthetic pathways. With an alternative proposed pathway for surfactin, yield was improved and imbalance in cofactors and ATP was reduced. Analysis of productive costs indicated that to make rhamnolipids commercial production feasible, the main efforts should focus on lowering substrate costs as well as the identification of energy-efficient unit operations to lower electricity cost, since these parameters accounted for 19.36 and 78.22%, respectively, of the production costs. The data generated by this analysis highlight the need for multidisciplinary collaboration to make rhamnolipids economically feasible, including biomolecular engineering and process intensification.

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The search for novel biosurfactants (Bs) requires the isolation of microorganisms from different environments. The Gulf of Mexico (GoM) is a geographical area active in the exploration and exploitation of hydrocarbons. Recent metagenomic and microbiologic studies in this area suggested a potential richness for novel Bs microbial producers. In this work, nineteen bacterial consortia from the GoM were isolated at different depths of the water column and marine sediments. Bs production from four bacterial consortia was detected by the CTAB test and their capacity to reduce surface tension (ST), emulsion index (EI24), and hemolytic activity. These bacterial consortia produced Bs in media supplemented with kerosene, diesel, or sucrose. Cultivable bacteria from these consortia were isolated and identified by bacterial polyphasic characterization. In some consortia, Enterobacter cloacae was the predominant specie. E. cloacae BAGM01 presented Bs activity in minimal medium and was selected to improve its Bs production using a Taguchi and Box-Behnken experimental design; this strain was able to grow and presented Bs activity at 35 g L-1 of NaCl. This Bs decreased ST to around 34.5 ± 0.56 mNm-1 and presented an EI24 of 71 ± 1.27%. Other properties of this Bs were thermal stability, stability in alkaline conditions, and stability at high salinity, conferring important and desirable characteristics in multiple industries. The analysis of the genome of E. cloacae BAGM01 showed the presence of rhlAB genes that have been reported in the synthesis of rhamnolipids, and alkAB genes that are related to the degradation of alkanes. The bioactive molecule was identified as a rhamnolipid after HPLC derivatization, 1H NMR, and UPLC-QTOF-MS analysis.

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Abstract Rhamnolipid is a potent biodegradable surfactant, which frequently used in pharmaceutical and environmental industries, such as enhanced oil recovery and bioremediation. This study aims to engineer Escherichia coli for the heterologous host production of rhamnolipid, to characterize the rhamnolipid product, and to optimize the production using autoinduction medium and POME (palm oil mill effluent). The construction of genes involved in rhamnolipid biosynthesis was designed in two plasmids, pPM RHLAB (mono-rhamnolipid production plasmid) and pPM RHLABC (di-rhamnolipid production plasmid). The characterization of rhamnolipid congeners and activity using high-resolution mass spectrometry (HRMS) and critical micelle concentration (CMC). In order to estimate rhamnolipid yield, an oil spreading test was performed. HRMS and CMC result show E. coli pPM RHLAB mainly produced mono-rhamnolipid (Rha-C14:2) with 900 mg/L and 35.4 mN/m of CMC and surface tension value, whereas E. coli pPM RHLABC mainly produced di-rhamnolipid (Rha-Rha-C10) with 300 mg/L and 34.3 mN/m of CMC and surface tension value, respectively. The optimum condition to produce rhamnolipid was at 20 h cultivation time, 37 oC, and pH 7. In this condition, the maximum rhamnolipid yield of 1245.68 mg/L using autoinduction medium and 318.42 mg/L using 20% (v/v) of POME. In conclusion, the characteristics of the rhamnolipid by recombinant E. coli is very promising to be used in industries as the most economical way of producing rhamnolipid.

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Aim: This study evaluated the effect of the biosurfactant rhamnolipid on the antimicrobial susceptibility, biofilm growth dynamics and production of virulence factors by Burkholderia pseudomallei. Materials & methods: The effects of rhamnolipid on planktonic and biofilm growth and its interaction with antibacterial drugs were evaluated. Then, its effects on growing and mature biofilms and on protease and siderophore production were assessed. Results: Rhamnolipid did not inhibit B. pseudomallei growth, but significantly enhanced the activity of meropenem and amoxicillin-clavulanate against mature biofilms. Rhamnolipid significantly reduced the biomass of mature biofilms, significantly increased protease production by growing and mature biofilms and siderophore release by growing biofilms. Conclusion: Rhamnolipid enhances the antimicrobial activity against B. pseudomallei, assists biofilm disassembly and alters protease and siderophore production by bacterial biofilms.

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Bacterial adhesion on surfaces is an essential initial step in promoting bacterial mobilization for soil bioremediation process. Modification of the cell surface is required to improve the adhesion of bacteria. The modification of physicochemical properties by rhamnolipid to Pseudomonas putida KT2442, Rhodococcus erythropolis 3586 and Aspergillus brasiliensis ATCC 16404 strains was analysed using contact angle measurements. The surface energy and total free energy of adhesion were calculated to predict the adhesion of both bacteria strains on the A. brasiliensis surface. The study of bacterial adhesion was carried out to evaluate experimental value with the theoretical results. Bacteria and fungi physicochemical properties were modified significantly when treated with rhamnolipid. The adhesion rate of P. putida improved by 16% with the addition of rhamnolipid (below 1 CMC), while the increase of rhamnolipid concentration beyond 1 CMC did not further enhance the bacterial adhesion. The addition of rhamnolipid did not affect the adhesion of R. erythropolis. A good relationship has been obtained in which water contact angle and surface energy of fungal surfaces are the major factors contributing to the bacterial adhesion. The adhesion is mainly driven by acid-base interaction. This finding provides insight to the role of physicochemical properties in controlling the bacterial adhesion on the fungal surface to enhance bacteria transport in soil bioremediation.

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Surfactants represent a billionaire market of amphiphilic molecules with worldwide applications in almost every branch of modern industry. The most common surfactants, available and currently used, are chemically produced. However, there is an urge to replace these chemical compounds with those obtained by mild and green technologies such as microbial biosurfactants produced by fermentative processes. Rhamnolipids are glycolipid biosurfactants that present highly effective surface-active properties and enormous market potential; nevertheless, their production costs remain not competitive. Here, we present a process of rhamnolipid production by static submerged cultivation using membranes of bacterial cellulose as substrate. The mixture of the rhamnolipid congeners was characterized showing effective surface-active properties and high amount of di-rhamnolipids (95.6%). Through this fermentative technology, 15.8 g/L of rhamnolipid was reach using a very simple and low-cost medium. The present process might decrease biosurfactant production cost, avoid foam formation, and finally make rhamnolipid production more viable.

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Rhamnolipids (RMLs) have more effectiveness for specific uses according to their homologue proportions. Thus, the novelty of this work was to compare mono-RMLs and di-RMLs physicochemical properties on microbial enhanced oil recovery (MEOR) applications. For this, RML produced by three strains of Pseudomonas aeruginosa containing different homologues proportion were used: a mainly mono-RMLs producer (mono-RMLs); a mainly di-RMLs producer (di-RMLs), and the other one that produces relatively balanced amounts of mono-RML and di-RML homologues (mono/di-RML). For mono-RML, the most abundant molecules were Rha-C10 C10 (m/z 503.3), for di-RML were RhaRha-C10 C10 (m/z 649.4) and for Mono/di-RML were Rha-C10 C10 (m/z 503.3) and RhaRha-C10 C10 (m/z 649.4). All RMLs types presented robustness under high temperature and variation of salinity and pH, and high ability for oil displacement, foam stability, wettability reversal and were classified as safe for environment according to the European Union Directive No. 67/548/EEC. For all these properties, it was observed a highlight for mono-RML. Mono-RML presented the lowest surface tension (26.40 mN/m), interfacial tension (1.14 mN/m), and critical micellar concentration (CMC 27.04 mg/L), the highest emulsification index (EI24 100%) and the best wettability reversal (100% with 25 ppm). In addition, mono-RML showed the best acute toxicity value (454 mg/L), making its application potential even more attractive. Based on the results, it was concluded that all RMLs homologues studied have potential for MEOR applications. However, results showed that mono-RML stood out and have the best mechanism of oil incorporation in micelles due their most effective surface-active physicochemical features.

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Nanomaterials have emerged as antimicrobial agents due to their unique physical and chemical properties. The development of nanoparticles (NPs) composed of natural biopolymers and biosurfactants have sparked interest, as they can be obtained without the use of complex chemical synthesis and toxic materials. In this study, we develop antimicrobial nanoparticles combining the biopolymer chitosan with the biosurfactant rhamnolipid. Addition of rhamnolipid reduced the size and polydispersity index of chitosan nanoparticles showing a more positive surface charge with improved stability, suggesting that chitosan-free amino groups are predominantly present on the surface of nanoparticles. Antimicrobial activity of chitosan/rhamnolipid nanoparticles (C/RL-NPs) against Staphylococcus strains surpassed that of either single rhamnolipid or chitosan, both in planktonic bacteria and biofilms. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of C/RL-NPs were determined considering the concentration of each individual molecule in NPs. MIC values of 14/19 µg mL-1 and MBC of 29/37 µg mL-1 were observed for S. aureus DSM 1104 and MIC and MBC of 29/37 and 58/75 µg mL-1 were observed against S. aureus ATCC 29213, respectively. For S. epidermidis, MIC and MBC of 7/9 and 14/19 µg mL-1 were noticed. Chitosan and chitosan nanoparticles eliminate the bacteria present in the upper parts of biofilms, while C/RL-NPs were more effective, eradicating most sessile bacteria and reducing the number of viable cells below the detection limit, when NPs concentration of 58/75 µg mL-1 was applied for both S. aureus DSM 1104 and S. epidermidis biofilms. The improved antibacterial efficacy of C/RL-NPs was linked to the increased local delivery of chitosan and rhamnolipid at the cell surface and, consequently, to their targets in Gram-positive bacteria. The combination of chitosan and rhamnolipid offers a promising strategy to the design of novel nanoparticles with low cytotoxicity, which can be exploited in pharmaceutical and food industries.

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Rhamnolipids are glycolipid biosurfactants that are primarily produced by Pseudomonas aeruginosa that have gained a great deal of interest for their numerous industrial applications and environmentally friendly properties. In this study, we explored the potential of waste canola oil as a low-cost and environmentally friendly substrate for the production of rhamnolipids by P. aeruginosa. Four different 23 full factorial designs were used to assess the effect of three independent factors on rhamnolipid production, including carbon source (canola oil and waste canola oil), nitrogen source [(NH4)2SO4 and NaNO3] and production time (7 and 14 days). The highest observed yield was 3585.31 ± 66.24 mg/L when P. aeruginosa was cultured for 14 days with 3% v/v waste canola oil and 4 g/L of NaNO3. The nitrogen source proved to be a crucial factor, as the use of NaNO3 rather than (NH4)2SO4 led to a 30-fold increase in production yield. The observed yield when waste canola oil was used was similar to, and even slightly higher than, that obtained using canola oil. Our results showed that waste canola oil has great potential for use as a carbon source for rhamnolipid production by P. aeruginosa, thus paving the way for the development of a low-cost, efficient, and environmentally friendly bioprocess for the production of rhamnolipids.

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BACKGROUND: Microbial surfactants are multifunctional surface-active molecules that have been overlooked in formulating microbial biopesticides. We report a novel approach using the biosurfactant rhamnolipid (RML) against the destructive cosmopolitan insect pest Bemisia tabaci, as well as the combined action of RML with aerial conidia of two entomopathogenic fungi, Cordyceps javanica and Beauveria bassiana. RML was also tested as a suspension agent to improve the recovery rate of conidia from solid substrate for fungal preparations. RESULTS: The recovery rate of conidia increased dramatically (two to five times) with RML compared with a standard surfactant (Tween 80). Spraying solutions of 0.075% and 0.1% (w/v) RML on B. tabaci third instar nymphs induced 100% mortality within 4 days. Conidial suspensions at 5 × 106 conidia/mL amended with RML at 0.01% or 0.05% markedly increased nymphal mortalities and considerably reduced LC50 . Conidial suspensions of B. bassiana with 0.05% RML added were more effective against whitefly nymphs (87.3% mortality) than C. javanica + RML (51.4% mortality). CONCLUSION: Our results show that this bacterium-based RML improved the recovery rate of hydrophobic conidia, and that mixtures of RML with fungal spore suspensions increased their insecticidal activity. © 2019 Society of Chemical Industry.

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