Structure,properties and applications of rhamnolipids produced by Pseudomonas aeruginosa L2-1 from cassava wastewater

The properties and applications of rhamnolipid surfactants produced by Pseudomonas aeruginosa L2-1 from cassava wastewater added with waste cooking oil (CWO) as low-cost substrate, were investigated and compared with the commercial rhamnolipid mixture JBR599 (Jeneil Biosurfactant Co., Saukville, USA). The rhamnolipids produced by strain L2-1 were characterized by high performance liquid chromatography–mass spectrometry. Sixteen different rhamnolipid congeners were detected, with Rha-C₁₀-C₁₀ and Rha-Rha-C₁₀-C₁₀ being the most abundant. The L2-1 rhamnolipids from CWO showed similar or better tensioactive properties than those from JBR599, with a minimal surface tension of 30 mN/m and a critical micelle concentration (CMC) of 30 mg/l. The L2-1 biosurfactants formed stable emulsions with several hydrocarbons and showed excellent emulsification of soybean oil (100%). These rhamnolipids removed 69% of crude oil present in contaminated sand samples at the CMC and presented antimicrobial activity against Bacillus cereus (32 μg/ml), Micrococcus luteus (32 μg/ml) and Staphylococcus aureus (128 μg/ml). These results demonstrate that the rhamnolipids produced in CWO can be useful for industrial applications, such as the bioremediation of oil spills.     

Algicidal activity of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa

The algicidal activity of the rhamnolipid biosurfactants (the mixture of Rha-Rha-C₁₀-C₁₀ and Rha-C₁₀-C₁₀) produced by Pseudomonas aeruginosa was investigated in the present paper. The results indicated that the biosurfactants had potential algicidal effects on the harmful algal bloom (HAB) species, Heterosigma akashiwo. The growth of H. akashiwo was strongly inhibited in medium containing rhamnolipids (0.4–3.0 mg L⁻¹); moreover, the rhamnolipids showed strong lytic activity toward H. akashiwo at higher concentrations (≥4.0 mg L⁻¹). In addition, the effects of the rhamnolipids on the growth of Gymnodinium sp. and Prorocentrum dentatum, another two kinds of HAB species, were also studied. Compared with the dramatic algicidal effect on H. akashiwo, the cells of P. dentatum were inhibited or lysed at higher concentrations (1.0–10.0 mg L⁻¹), while the cells of Gymnodinium sp. were not suppressed with the same treatment, indicating the rhamnolipids had the potential for the selective control of HABs.Morphometric analysis at ultrastructural level by transmission electron micrographs indicated that the extent of ultrastructural damage of the alga was severe at high concentrations of rhamnolipids and during extended periods of contact. The first response occurred in the plasma membrane which partly disintegrated. The lack of membrane facilitated the rhamnolipid biosurfactants into the cells and allowed damage to other organelles, which resulted in the injury of chloroplast, vacuolization of mitochondria and deformation of the cristae, disruption of nuclear membrane and condensation of chromatin in nucleus, suggesting that the lytic activity of rhamnolipids was mainly due to their powerful surfactivity and their tendency to cohere on the surface of phospholipids bimolecular layer of the cells and further destroyed the layers, and then the structure of quasi-membrane configurations inside the cells was disintegrated, following by the irreversible damage to the ultrastructure and the loss of the function of organelles, consequently leading the cells to lyse.     

Rhamnolipid production by a novel thermophilic hydrocarbon-degrading Pseudomonas aeruginosa AP02-1

Thermophilic bacterial cultures were isolated from a hot spring environment on hydrocarbon containing mineral salts media. One strain identified as Pseudomonas aeruginosa AP02-1 was tested for the ability to utilize a range of hydrocarbons both n-alkanes and polycyclic aromatic hydrocarbons as sole carbon source. Strain AP02-1 had an optimum growth temperature of 45°C and degraded 99% of crude oil 1% (v/v) and diesel oil 2% (v/v) when added to a basal mineral medium within 7 days of incubation. Surface activity measurements indicated that biosurfactants, mainly glycolipid in nature, were produced during the microbial growth on hydrocarbons as well as on both water-soluble and insoluble substrates. Mass spectrometry analysis showed different types of rhamnolipid production depending on the carbon substrate and culture conditions. Grown on glycerol, P. aeruginosa AP02-1 produced a mixture of ten rhamnolipid homologues, of which Rha-Rha-C₁₀-C₁₀ and Rha-C₁₀-C₁₀ were predominant. Rhamnolipid-containing culture broths reduced the surface tension to ≈28 mN and gave stable emulsions with a number of hydrocarbons and remained effective after sterilization. Microscopic observations of the emulsions suggested that hydrophobic cells acted as emulsion-stabilizing agents.     

Synergistic extraction using sweep-floc coagulation and acidification of rhamnolipid produced from industrial lignocellulosic hydrolysate in a bioreactor using sequential (fill-and-draw) approach

Sequential fill-and-draw fermentation strategy provides an approach to increase the productivity by replenishing nutrients and minimizing the toxic effects of by-products. In the present work, the same strategy was adopted using lignocellulosic industrial rice-straw C₆ hydrolysate stream to produce rhamnolipids from Achromobacter sp. (PS1) in a 6 L bioreactor with a working-volume of 2 L. The production results showed overall rhamnolipid production of 22.03 g/L in 15 days observed at par with 19.35 g/L obtained under shake flask conditions in 18 days. At each sequential feed (2 % sugars), a rise in dissolved oxygen (D.O) concentration was observed in the range between 60–53 % which declined to 47–39 % with consecutive depletion in sugar concentration under no D.O control. For maximum extraction of rhamnolipids from culture broth, the synergistic effect of sweep floc-coagulation using FeCl₃ at 0.4 % (w/v) followed by its acidification and solvent extraction was adopted which resulted in maximum recovery of 97.5 % compared to 89.05 % recovery obtained in simply acidification followed by solvent extraction. The characterization of partially purified biosurfactant using tandem-MS revealed six-congeners, Rha-C₁₀-C₁₀ and Rha-Rha-C₁₀-C₁₀ being the most abundant. Oil recovery of 92.21 % from motor-oil impregnated sand using crude rhamnolipid further added the value to the biosurfactant.     

Rhamnolipids are conserved biosurfactants molecules: implications for their biotechnological potential

A range of isolates of Pseudomonas aeruginosa from widely different environmental sources were examined for their ability to synthesise rhamnolipid biosurfactants. No significant differences in the quantity or composition of the rhamnolipid congeners could be produced by manipulating the growth conditions. Sequences for the rhamnolipid genes indicated low levels of strain variation, and the majority of polymorphisms did lead to amino acid sequence changes that had no evident phenotypic effect. Expression of the rhlB and rhlC rhamnosyltransferase genes showed a fixed sequential expression pattern during growth, and no significant up-regulation could be induced by varying producer strains or growth media. The results indicated that rhamnolipids are highly conserved molecules and that their gene expression has a rather stringent control. This leaves little opportunity to manipulate and greatly increase the yield of rhamnolipids from strains of P. aeruginosa for biotechnological applications.     

Purification and structural characterization of glycolipid biosurfactants fromPseudomonas aeruginosa YPJ-80

Glycolipids produced byPseudomonas aeruginosa YPJ-80 were characterized by chromatographic and spectroscopic techniques as a mixture of two rhamnolipids. For recovery of glycolipids from the culture broth, various isolation methods including ultrafiltration, adsorption and solvent extraction were compared. Ultrafiltration showed the best results in terms of glycolipids recovery. Further purification for spectroscopic analysis was carried out by adsorption chromatography and preparation thin layer chromatography. From the spectroscopic analysis, such as IR spectroscopy, FAB-MS,¹H-NMR and¹³C-NMR and hydrolysis analysis, the glycolipids were identified as L-α-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate and 2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate. Monorhamnolipid and dirhamnolipid lowered the surface tension of water to 28.1 mN/m and 29.3 mN/m, respectively.     

Directed microbial biosynthesis of deuterated biosurfactants and potential future application to other bioactive molecules

Deuterated rhamnolipids were produced using strain AD7 of Pseudomonas aeruginosa, which was progressively adapted to increasing levels of deuterium in D₂O and carbon substrates. Fourteen different deuterated rhamnolipid structures, including structural isomers, were produced which is similar to normal protonated structures. There were two main products monorhamnolipid Rha-C₁₀-C₁₀ and dirhamnolipid Rha₂-C₁₀-C₁₀. The levels of deuteration varied from 16% with 25% D₂O + h-glycerol to 90% with 100% D₂O + d-glycerol. When d-tetradecane was used with H₂O, virtually all the deuterium appeared in the lipid chains while using h-tetradecane + D₂O led to the majority of deuterium in the sugars. The adaptation to growth in deuterium appeared to be metabolic since no genetic changes could be found in the key rhamnolipid biosynthetic genes, the rhamnosyl transferases RhlB and RhlC. Deuterated sophorolipids were similarly produced using Candida bombicola and Candida apicola although in this case, no adaptation process was necessary. Up to 40 different sophorolipids were produced by these yeasts. However, unlike the rhamnolipids, use of D₂O did not lead to any deuteration of the lipid chains, but direct incorporation into the lipid was achieved using d-isostearic acid. The results from these experiments show the feasibility of producing deuterated bioactive compounds from microorganisms coupled with the possibility of manipulating the pattern of labelling through judicious use of different deuterated substrates.     

Characterization of rhamnolipid production by Burkholderia glumae

Aims: To investigate if Burkholderia glumae can produce rhamnolipids, define a culture medium for good production yields, analyse their composition and determine their tensioactive properties. Methods and Results: Burkholderia glumae AU6208 produces a large spectrum of mono‐ and di‐rhamnolipid congeners with side chains varying between C₁₂‐C₁₂ and C₁₆‐C₁₆, the most abundant being Rha‐Rha‐C₁₄‐C₁₄.The effects on rhamnolipid production of the cultivation temperature, nitrogen and carbon source were investigated. With urea as the nitrogen source and canola oil as the carbon source, a production of 1000·7 mg l^?1 was reached after 6 days. These rhamnolipids display a critical micelle concentration of 25–27 mg l^?1 and decrease the interfacial tension against hexadecane from 40 to 1·8 mN m^?1. They also have excellent emulsifying properties against long chain alkanes. Conclusions: Burkholderia glumae AU6208 can produce considerable amounts of rhamnolipids. They are produced as diversified mixtures of congeners. Their side chains are longer than those normally produced by those of Pseudomonas aeruginosa. They also present excellent tensioactive properties. Significance and Impact of the Study: In contrast with the classical rhamnolipid producer Ps. aeruginosa, B. glumae is not a pathogen to humans. This work shows that the industrial production of rhamnolipids with this species could be easier than with Ps. aeruginosa.     

Production and characterisation of a biosurfactant isolated from Pseudomonas aeruginosa UW-1

Pseudomonas aeruginosa UW-1 produced 17–24 g L⁻¹ rhamnolipid in vegetable oil-containing media in shake flask cultures in 13 days. In time course studies of growth and rhamnolipid production in a salts medium containing 6% canola oil, total bacterial count reached 2.6 × 10¹⁰ CFU ml⁻¹ after 48 h and a maximum rhamnolipid yield of 24.3 g L⁻¹ was obtained after 9 days. Rhamnolipid components were purified and separated by chloroform-methanol extraction and TLC chromatography. The major rhamnolipid components were characterised as L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate and L-rhamnosyl-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate by nuclear magnetic resonance and mass spectrometry. The components were separated preparatively by silica gel column chromatography. The recovered monorhamnosyl fraction contained no dirhamnosyl moiety while the recovered dirhamnosyl fraction contained 5% of the monorhamnosyl moiety when analyzed by HPLC. The ratio of mono- to dirhamnosyl components produced by P. aeruginosa UW-1 was determined by HPLC to be 4 : 1 by weight. Purified mono- and dirhamnosyl components had the same CMC value of 40 μg ml⁻¹ and decreased the surface tension of water to 27.7 and 30.4 dynes cm⁻¹, respectively. Received 04 April 1997/ Accepted in revised form 15 July 1997     

Characterization of rhamnolipid biosurfactants produced by recombinant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain DAB with removal of crude oil

Objectives

To improve rhamnolipid production and its potential application in removal of crude oil, the recombinant Pseudomonas aeruginosa strain DAB was constructed to enhance yield of rhamnolipids.

Results

Strain DAB had a higher yield of 17.3 g rhamnolipids l^?1 in the removal process with crude oil as the sole carbon source than 10 g rhamnolipids l^?1 of wild-type strain DN1, where 1% crude oil was degraded more than 95% after 14 days cultivation. These rhamnolipids reduced the surface tension of water from 72.92 to 26.15 mN m^?1 with CMC of 90 mg l^?1. The predominant rhamnolipid congeners were Rha–C₁₀–C₁₀ and Rha–Rha–C₁₀–C₁₀ detected by MALDI-TOF MS analysis with approx. 70% relative abundance, although a total of 21 rhamnolipid congeners were accumulated.

Conclusion

Increasing the copy number of rhlAB genes efficiently enhanced the production of rhamnolipids by the recombinant P. aeruginosa DAB and thus presents a promising application for the bioremediation process.

     

Cadmium effects on transcriptional expression of rhlB/rhlC genes and congener distribution of monorhamnolipid and dirhamnolipid in Pseudomonas aeruginosa IGB83

While variable production of the biosurfactant, rhamnolipid, by Pseudomonas aeruginosa has been shown to be dependent on growth conditions, no research has evaluated potential relationships between rhamnolipid production and the presence of heavy metals. The current investigation evaluates the influence of Cd²⁺ on rhamnolipid synthesis. Cultures grown in the presence of 0.45 and 0.89 mM Cd²⁺ were monitored for rhlB/rhlC expression, rhamnolipid yield, and the ratio of monorhamnolipid (RL1) and dirhamnolipid (RL2) produced. Results show a Cd-induced enhancement of rhlB expression in mid-stationary phase (53 h). In addition, sustained production of rhamnolipid through late stationary growth phase (96 h) was observed for Cd-amended cultures, unlike Cd-free control cultures that ceased rhamnolipid production by mid-stationary growth phase. Most significant was an observed increase in the ratio of RL2 to RL1 congeners produced by cultures grown in the presence of Cd²⁺. Previous results have shown that the complexation constant for RL2–Cd is several orders of magnitude larger than that of RL1–Cd thus the preferential production of RL2 in the presence of Cd²⁺ impacts its bioavailability and toxicity both for the cell and in the surrounding environment.     

Rhamnolipids produced by Pseudomonas: from molecular genetics to the market

Rhamnolipids are biosurfactants with a wide range of industrial applications that entered into the market a decade ago. They are naturally produced by Pseudomonas aeruginosa and some Burkholderia species. Occasionally, some strains of different bacterial species, like Pseudomonas chlororaphis NRRL B-30761, which have acquired RL-producing ability by horizontal gene transfer, have been described. P. aeruginosa, the ubiquitous opportunistic pathogenic bacterium, is the best rhamnolipids producer, but Pseudomonas putida has been used as heterologous host for the production of this biosurfactant with relatively good yields. The molecular genetics of rhamnolipids production by P. aeruginosa has been widely studied not only due to the interest in developing overproducing strains, but because it is coordinately regulated with the expression of different virulence-related traits by the quorum-sensing response. Here, we highlight how the research of the molecular mechanisms involved in rhamnolipid production have impacted the development of strains that are suitable for industrial production of this biosurfactant, as well as some perspectives to improve these industrial useful strains.     

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically-engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To engineer Escherichia coli for the heterologous production of di-rhamnolipids, which are important biosurfactants but mainly produced by opportunistic pathogen Pseudomonas aeruginosa.

Results

The codon-optimized rhlAB and rhlC genes originating from P. aeruginosa and Burkholderia pseudomallei were combinatorially expressed in E. coli to produce di-rhamnolipids with varied congeners compositions. Genes involved in endogenous upstream pathways (rhamnose and fatty acids synthesis) were co-overexpressed with rhlAB–rhlC, resulting in variations of rhamnolipids production and congeners compositions. Under the shake-flask condition, co-overexpression of rfbD with rhlAB–rhlC increased rhamnolipids production (0.64 ± 0.02 g l^?1) than that in strain only expressing rhlAB–rhlC (0.446 ± 0.009 g l^?1), which was mainly composed of di-rhamnolipids congeners Rha–Rha–C₁₀–C₁₀.

Conclusion

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically engineered E. coli strains were achieved via combiniations of mono-/di-rhamnolipids synthesis modules and endogenous upstream modules.

     

Structural characterization and surface activities of biogenic rhamnolipid surfactants from Pseudomonas aeruginosa isolate MN1 and synergistic effects against methicillin-resistant Staphylococcus aureus

The aim of present work was to study chemical structures and biological activities of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa MN1 isolated from oil-contaminated soil. The results of liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that total rhamnolipids (RLs) contained 16 rhamnolipid homologues. Di-lipid RLs containing C₁₀-C₁₀ moieties were by far the most predominant congeners among mono-rhamnose (53.29?%) and di-rhamnose (23.52?%) homologues. Mono-rhamnolipids form 68.35?% of the total congeners in the RLs. Two major fractions were revealed in the thin layer chromatogram of produced RLs which were then purified by column chromatography. The retardation factors (R _f) of the two rhamnolipid purple spots were 0.71 for RL1 and 0.46 for RL2. LC-MS/MS analysis proved that RL1 was composed of mono-RLs and RL2 consisted of di-RLs. RL1 was more surface-active with the critical micelle concentration (CMC) value of 15?mg/L and the surface tension of 25 mN/m at CMC. The results of biological assay showed that RL1 is a more potent antibacterial agent than RL2. All methicillin-resistant Staphylococcus aureus (MRSA) strains were inhibited by RLs that were independent of their antibiotic susceptibility patterns. RLs remarkably enhanced the activity of oxacillin against MRSA strains and lowered the minimum inhibitory concentrations of oxacillin to the range of 3.12?C6.25???g/mL.     

Palm oil utilization for the simultaneous production of polyhydroxyalkanoates and rhamnolipids by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Direct utilization of palm oil for the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids was demonstrated using Pseudomonas aeruginosa IFO3924. By secreted lipase, palm oil was hydrolyzed into glycerol and fatty acids. Fatty acids became favorable carbon sources for cell growth and PHA production via β-oxidation and glycerol for rhamnolipid production via de novo fatty acid synthesis. Both PHA and rhamnolipid syntheses started after the nitrogen source was exhausted and cell growth ceased. PHA synthesis continued until all fatty acids were exhausted, and at that time, PHA content in the cells reached a maximum, but stopped despite the remaining glycerol (<2g/l). In contrast, rhamnolipid synthesis continued until glycerol was exhausted.     

Analysis of rhamnolipid biosurfactants by methylene blue complexation

Rhamnolipids, produced by Pseudomonas aeruginosa, represent an important group of biosurfactants having various industrial, environmental, and medical applications. Current methods for rhamnolipid quantification involve the use of strong hazardous acids/chemicals, indirect measurement of the concentration of sugar moiety, or require the availability of expensive equipment (HPLC-MS). A safer, easier method that measures the whole rhamnolipid molecules would significantly enhance strain selection, metabolic engineering, and process development for economical rhamnolipid production. A semi-quantitative method was reported earlier to differentiate between the rhamnolipid-producing and non-producing strains using agar plates containing methylene blue and cetyl trimethylammonium bromide (CTAB). In this study, a rapid and simple method for rhamnolipid analysis was developed by systematically investigating the complexation of rhamnolipids and methylene blue, with and without the presence of CTAB. The method relies on measuring the absorbance (at 638 nm) of the rhamnolipid−methylene blue complex that partitions into the chloroform phase. With P. aeruginosa fermentation samples, the applicability of this method was verified by comparison of the analysis results with those obtained from the commonly used anthrone reaction technique.     

Structure identification of natural rhamnolipid mixtures by fast atom bombardment tandem mass spectrometry

Two rhamnobiose-lipid preparations have been studied by fast atom bombardment (FAB) tandem mass spectrometry. The principal rhanobiose-lipids contain the -hydroxydecanoyl--hydroxydecanoate Rha-Rha-C₁₀-C₁₀ and the -hydroxytetradecanoyl--hydroxytetradecanoate Rha-Rha-C₁₄-C₁₄. Both preparations contain minor components which are heterogenous in -hydroxy fatty acid composition. FAB ionization of rhamnobiose-lipids in the presence of Na⁺ shows the formation of both [M + Na]⁺, [M + 2Na - H]⁺, [M + 3Na - 2H]⁺ and [M - H]^– ions. Tandem mass spectrometry of the [M + 2Na - H]⁺ and [M - H]^– ions give information about the sequence of the building blocks. Particularly, heterogeneity in -hydroxy fatty acid composition is determined for the principal components and all the minor components present in the preparations.     

Expression of genes involved in rhamnolipid synthesis in Pseudomonas aeruginosa PAO1 in a bioreactor cultivation

There is a growing demand for economic bioprocesses based on sustainable resources rather than petrochemical-derived substances. Particular attention has been paid to rhamnolipids—surface-active glycolipids—that are naturally produced by Pseudomonas aeruginosa. Rhamnolipids have gained increased attention over the past years due to their versatile chemical and biological properties as well as numerous biotechnological applications. However, rhamnolipid synthesis is tightly governed by a complex growth-dependent regulatory network. Quantitative comprehension of the molecular and metabolic mechanisms during bioprocesses is key to manipulating and improving rhamnolipid production capacities in P. aeruginosa. In this study, P. aeruginosa PAO1 was grown under nitrogen limitation with sunflower oil as carbon and nitrate as nitrogen source in a batch fermentation process. Gene expression was monitored using quantitative PCR over the entire time course. Until late deceleration phase, an increase in relative gene expression of the las, rhl, and pqs quorum-sensing regulons was observed. Thereafter, expression of the rhamnolipid synthesis genes, rhlA and rhlC, as well as the las regulon was downregulated. RhlR was shown to remain upregulated at the late phase of the fermentation process.     

Accumulation of unusual sterile transcripts of TCRβ in mouse hybridoma, murine tumour and non-human primate marmoset tumour

Accumulation of immunoglobulin Ig RNA (from several loci viz., C_H, C_α, J_k-C_k and S_μ during Igμ isotype switching) in B cells and T cell receptor (TCR) RNAs (α, β andγ) in T cells of unusual sizes emanating from germline and rearranged genes were reported to accumulate in human and mouse (and murine too). The precise mechanism and function of these sterile RNA species are yet to be delineated. Similar accumulation of RNA species of unusual sizes were identified with DNA-RNA hybridization and isolation of cDNA employing with DNA and antibody probes in mouse hybridoma, murine tumour, non-human primate marmoset tumour and human leukemic cells.