Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications

Pseudomonas aeruginosa produces and secretes rhamnose-containing glycolipid biosurfactants called rhamnolipids. This review describes rhamnolipid biosynthesis and potential industrial and environmental applications of rhamnolipids. Rhamnolipid production is dependent on central metabolic pathways, such as fatty acid synthesis and dTDP-activated sugars, as well as on enzymes participating in the production of the exopolysaccharide alginate. Synthesis of these surfactants is regulated by a very complex genetic regulatory system that also controls different P. aeruginosa virulence-associated traits. Rhamnolipids have several potential industrial and environmental applications including the production of fine chemicals, the characterization of surfaces and surface coatings, as additives for environmental remediation, and as a biological control agent. Realization of this wide variety of applications requires economical commercial-scale production of rhamnolipids. Received: 4 February 2000 / Received revision: 9 June 2000 / Accepted: 9 June 2000     

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.     

Medium factors on anaerobic production of rhamnolipids by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SG and a simplifying medium for in situ microbial enhanced oil recovery applications

Aerobic production of rhamnolipid by Pseudomonas aeruginosa was extensively studied. But effect of medium composition on anaerobic production of rhamnolipid by P. aeruginosa was unknown. A simplifying medium facilitating anaerobic production of rhamnolipid is urgently needed for in situ microbial enhanced oil recovery (MEOR). Medium factors affecting anaerobic production of rhamnolipid were investigated using P. aeruginosa SG (Genbank accession number KJ995745). Medium composition for anaerobic production of rhamnolipid by P. aeruginosa is different from that for aerobic production of rhamnolipid. Both hydrophobic substrate and organic nitrogen inhibited rhamnolipid production under anaerobic conditions. Glycerol and nitrate were the best carbon and nitrogen source. The commonly used N limitation under aerobic conditions was not conducive to rhamnolipid production under anaerobic conditions because the initial cell growth demanded enough nitrate for anaerobic respiration. But rhamnolipid was also fast accumulated under nitrogen starvation conditions. Sufficient phosphate was needed for anaerobic production of rhamnolipid. SO₄ ^2? and Mg²⁺ are required for anaerobic production of rhamnolipid. Results will contribute to isolation bacteria strains which can anaerobically produce rhamnolipid and medium optimization for anaerobic production of rhamnolipid. Based on medium optimization by response surface methodology and ions composition of reservoir formation water, a simplifying medium containing 70.3 g/l glycerol, 5.25 g/l NaNO₃, 5.49 g/l KH₂PO₄, 6.9 g/l K₂HPO₄·3H₂O and 0.40 g/l MgSO₄ was designed. Using the simplifying medium, 630 mg/l of rhamnolipid was produced by SG, and the anaerobic culture emulsified crude oil to EI₂₄ = 82.5 %. The simplifying medium was promising for in situ MEOR applications.     

Effect of rhamnolipid on biodegradation of hydrocarbons in non-aqueous-phase liquid (NAPL)

Aliphatic and aromatic hydrocarbons are environmental pollutants of serious concern. Their bioavailability is the major limiting factor that makes the bioremediation process slow. Therefore, the present study focuses on biodegradation of non-aqueous-phase liquids (NAPL) by a halophilic consortium (Pseudomonas aeruginosa and Escherichia fergusonii) in presence of rhamnolipid as well as a rhamnolipid-producing Pseudomonas aeruginosa AMB AS7. The study was performed in microcosms, and the residual hydrocarbons after degradation were estimated by gas chromatography. It was found that the degradation of hydrocarbons in NAPL was more in presence of rhamnolipid in comparison with their biotic controls. However, among NAPL, the degradation of phenanthrene (37.5%) and octadecane (47.8%) was found to be more by co-culture of halophilic consortium and rhamnolipid-producing P. aeruginosa AMB AS7. Denaturing gradient gel electrophoresis was performed to determine the viability of different bacterial strains (halophilic and rhamnolipid-producing bacterial strain). Besides, the results also revealed that during NAPL degradation, the cell surface hydrophobicity (CSH) of halophilic consortium increased from 9.12% to 69.55% when added with 100 mg/L of rhamnolipid, whereas CSH of rhamnolipid-producing P. aeruginosa AMB AS7 was constant at 31.9%, even though it produced about 271.8 mg/L of rhamnolipid.     

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     

Glycolipids of the smut fungus Ustilago maydis from cultivation on renewable resources

When grown on vegetable oils and their derivatives, the smut fungus Ustilago maydis (DSM 4500 and ATCC 14826) produces several glycolipids under nitrogen-limiting conditions. With 45 g l⁻¹ sunflower oil fatty acids (technical grade) a yield of 30 g l⁻¹ glycolipid was achieved. The resulting mixture contained predominantly mannosylerythritol lipids together with smaller amounts of cellobiose lipids. The production of the more polar cellobiose lipids was enhanced when glucose was used as carbon source. The molecular structure of the main components of the glycolipid mixture were elucidated by a combination of NMR spectroscopic and mass-spectrometric techniques. Received: 22 June 1998 / Received revision: 11 September 1998 / Accepted: 13 September 1998     

Novel rhamnolipid biosurfactants produced by a polycyclic aromatic hydrocarbon-degrading bacterium Pseudomonas aeruginosa strain NY3

A novel rhamnolipid biosurfactant-producing and Polycyclic Aromatic Hydrocarbon (PAH)-degrading bacterium Pseudomonas aeruginosa strain NY3 was isolated from petroleum-contaminated soil samples. Strain NY3 was characterized by its extraordinary capacity to produce structurally diverse rhamnolipids. A total of 25 rhamnolipid components and 37 different parent molecular ions, representing various metal ion adducts (Na⁺, 2Na⁺ and K⁺), were detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among these compounds are ten new rhamnolipids. In addition to its biosurfactant production, strain NY3 was shown to be capable of efficient degradation of PAHs as well as synergistic improvement in the degradation of high molecular weight PAHs by its biosurfactant. These findings have added novel members to the rhamnolipid group and expanded current knowledge regarding the diversity and productive capability of rhamnolipid biosurfactants from a single specific strain with variation of only one carbon source. Additionally, this paper lays the foundation for improvement in the yield of NY3BS and study of the degradation pathway(s) of PAHs in P. aeruginosa strain NY3.     

Repeated pH-stat fed-batch fermentation for rhamnolipid production with indigenous Pseudomonas aeruginosa S2

Rhamnolipid is one of the most commonly used biosurfactants with the ability to reduce the surface tension of water from 72 to 30 mN/m. An indigenous isolate Pseudomonas aeruginosa S2 possessing excellent ability to produce rhamnolipid was used as a model strain to explore fermentation technology for rhamnolipid production. Using optimal medium and operating conditions (37°C, pH 6.8, and 250 rpm agitation) obtained from batch fermentation, P. aeruginosa S2 was able to produce up to 5.31 g/l of rhamnolipid from glucose-based medium. To further improve the rhamnolipid yield, a pH-stat fed-batch culture was performed by maintaining a constant pH of 6.8 through manipulating glucose feeding. The effect of influent glucose concentration on rhamnolipid yield and productivity was investigated. Using the pH-stat culture, a maximum rhamnolipid concentration (6.06 g/l) and production rate (172.5 ml/h/l) was obtained with 6% glucose in the feed. Moreover, combining pH-stat culture with fill-and-draw operation allowed a stable repeated fed-batch operation for approximately 500 h. A marked increase in rhamnolipid production was achieved, leading to the best rhamnolipid yield of approximately 9.4 g/l during the second repeated run.     

Rhamnolipids: diversity of structures, microbial origins and roles

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: α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate (Rha-Rha-C₁₀-C₁₀), α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoate (Rha-Rha-C₁₀), as well as their mono-rhamnolipid congeners Rha-C₁₀-C₁₀ and Rha-C₁₀. 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.     

Rhamnolipid production by Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene

The potential of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene (vgb) for rhamnolipid production was studied. P. aeruginosa (NRRL B-771) and its transposon mediated vgb transferred recombinant strain, PaJC, were used in the research. The optimization of rhamnolipid production was carried out in the different conditions of cultivation (agitation rate, the composition of culture medium and temperature) in a time-course manner. The nutrient source, especially the carbon type, had a dramatic effect on rhamnolipid production. The PaJC strain and the wild type cells of P. aeruginosa started producing biosurfactant at the stationary phase and its concentration reached maximum at 24 h (838 mg/l⁻¹) and at 72 h (751 mg l⁻¹) of the incubation respectively. Rhamnolipid production was optimal in batch cultures when the temperature and agitation rate were controlled at 30°C and 100 rpm. It reached 8373 mg l⁻¹ when the PaJC cells were grown in 1.0% glucose supplemented minimal media. Genetic engineering of biosurfactant producing strains with vgb may be an effective method to increase its production.     

基因工程微生物合成鼠李糖脂表面活性剂的研究进展

鼠李糖脂是一类重要的生物表面活性剂。相比于化学合成的表面活性剂,其具有更优秀的理化性质及环境友好等特点,被广泛应用于微生物采油、环境污染修复等工程中。目前,鼠李糖脂的工业生产主要采用铜绿假单胞菌这一具有致病性的天然合成菌株,与此同时,受菌株遗传背景的限制,优化发酵过程等方法在产量提升方面遇到了一些瓶颈问题。利用基因工程方法对菌株进行改良有望进一步提高鼠李糖脂生产的安全性、产量、产物性能等多项指标,因此受到了越来越广泛的关注。本文综述了近年来利用基因工程方法优化鼠李糖脂生物合成的最新进展,讨论了异源合成、代谢通路改造、基因表达优化、蛋白质工程、底盘工程等多种策略的应用,并展望了一系列可行的研究方向。     

Production of rhamnolipids in solid-state cultivation using a mixture of sugarcane bagasse and corn bran supplemented with glycerol and soybean oil

Rhamnolipid biosurfactants are attracting attention due to their low toxicity, high biodegradability, and good ecological acceptability. However, production in submerged culture is made difficult by severe foaming problems. Solid-state cultivation (SSC) is a promising alternative production method. In the current work, we report the optimization of rhamnolipid production by Pseudomonas aeruginosa UFPEDA 614 on a solid substrate containing sugarcane bagasse and corn bran. The best rhamnolipid production, 45 g/l of impregnating solution used, was obtained with a 50:50 (m/m) mixture of sugarcane bagasse and corn bran supplemented with an impregnating solution containing 6% (v/v) of each of glycerol and soybean oil. This level is comparable with those of previous studies undertaken in solid-state cultivation; the composition of the biosurfactant is similar, but our medium is cheaper. Our work therefore provides a suitable basis for future studies of the development of an SSC-based process for rhamnolipid production.     

Effects of carbon and nitrogen sources on the proteome of Pseudomonas aeruginosa PA1 during rhamnolipid production

The PA1 strain of Pseudomonas aeruginosa isolated from oil waste produces rhamnolipid, a biodegradable surfactant with applications in several industrial and environmental fields. The metabolic pathway and genetic regulation of rhamnolipid production in P. aeruginosa are poorly understood. Herein, several proteins directly or indirectly related to rhamnolipid production and their genetic regulations were identified by comparative proteomic. We compared the proteome of P. aeruginosa PA1 after fermentation in two different conditions of carbon and nitrogen sources: condition A allowed rhamnolipid production and condition B prevented it. Protein extracts from cellular pellets were compared using 2D-PAGE stained with colloidal Coomassie followed by MALDI-TOF/TOF mass spectrometry. We identified 21 differentially expressed proteins, including those involved in secretion, quorum sensing, oxidative response and metabolism.     

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.     

Enhanced rhamnolipid production in Burkholderia thailandensis transposon knockout strains deficient in polyhydroxyalkanoate (PHA) synthesis

Microbially produced rhamnolipids have significant commercial potential; however, the main bacterial producer, Pseudomonas aeruginosa, is an opportunistic human pathogen, which limits biotechnological exploitation. The non-pathogenic species Burkholderia thailandensis produces rhamnolipids; however, yield is relatively low. The aim of this study was to determine whether rhamnolipid production could be increased in Burkholderia thailandensis through mutation of genes responsible for the synthesis of the storage material polyhydroxyalkanoate (PHA), thereby increasing cellular resources for the production of rhamnolipids. Potential PHA target genes were identified in B. thailandensis through comparison with known function genes in Pseudomonas aeruginosa. Multiple knockout strains for the phbA, phbB and phbC genes were obtained and their growth characteristics and rhamnolipid and PHA production determined. The wild-type strain and an rhamnolipid (RL)-deficient strain were used as controls. Three knockout strains (ΔphbA1, ΔphbB1 and ΔphbC1) with the best enhancement of rhamnolipid production were selected for detailed study. ΔphbB1 produced the highest level of purified RL (3.78 g l⁻¹) compared to the wild-type strain (1.28 g l⁻¹). In ΔphbB1, the proportion of mono-rhamnolipid was also increased compared to the wild-type strain. The production of PHA was reduced by at least 80% in all three phb mutant strains, although never completely eliminated. These results suggest that, in contrast to Pseudomonas aeruginosa, knockout of the PHA synthesis pathway in Burkholderia thailandensis could be used to increase rhamnolipid production. The evidence of residual PHA production in the phb mutant strains suggests B. thailandensis possesses a secondary unelucidated PHA synthesis pathway.

     

Continuous rhamnolipid production using denitrifying Pseudomonas aeruginosa cells in hollow‐fiber bioreactor

Rhamnolipids are high‐value effective biosurfactants produced by Pseudomonas aeruginosa. Large‐scale production of rhamnolipids is still challenging especially under free‐cell aerobic conditions in which the highly foaming nature of the culture broth reduces the productivity of the process. Immobilized systems relying on oxygen as electron acceptor have been previously investigated but oxygen transfer limitation presents difficulties for continuous rhamnolipid production. A coupled system using immobilized cells and nitrate instead of oxygen as electron acceptor taking advantage of the ability of P. aeruginosa to perform nitrate respiration was evaluated. This denitrification‐based immobilized approach based on a hollow‐fiber setup eliminated the transfer limitation problems and was found suitable for continuous rhamnolipid production in a period longer than 1,500 h. It completely eliminated the foaming difficulties related to aerobic systems with a comparable specific productivity of 0.017 g/(g dry cells)‐h and allowed easy recovery of rhamnolipids from the cell‐free medium. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 346–351, 2013     

Rhamnolipid Stimulates Uptake of Hydrophobic Compounds by Pseudomonas aeruginosa

The biodegradation of hexadecane by five biosurfactant-producing bacterial strains (Pseudomonas aeruginosa UG2, Acinetobacter calcoaceticus RAG1, Rhodococcus erythropolis DSM 43066, R. erythropolis ATCC 19558, and strain BCG112) was determined in the presence and absence of exogenously added biosurfactants. The degradation of hexadecane by P. aeruginosa was stimulated only by the rhamnolipid biosurfactant produced by the same organism. This rhamnolipid did not stimulate the biodegradation of hexadecane by the four other strains to the same extent, nor was degradation of hexadecane by these strains stimulated by addition of their own biosurfactants. This suggests that P. aeruginosa has a mode of hexadecane uptake different from those of the other organisms. Rhamnolipid also enhanced the rate of epoxidation of the aliphatic hydrocarbon α,ω-tetradecadiene by a cell suspension of P. aeruginosa. Furthermore, the uptake of the hydrophobic probe 1-naphthylphenylamine by cells of P. aeruginosa was enhanced by rhamnolipid, as indicated by stopped-flow fluorescence experiments. Rhamnolipid did not stimulate the uptake rate of this probe in de-energized cells. These results indicate that an energy-dependent system is present in P. aeruginosa strain UG2 that mediates fast uptake of hydrophobic compounds in the presence of rhamnolipid.     

Microbial rhamnolipid production: a critical re-evaluation of published data and suggested future publication criteria

High production cost and potential pathogenicity of Pseudomonas aeruginosa, commonly used for rhamnolipid synthesis, have led to extensive research for safer producing strains and cost-effective production methods. This has resulted in numerous research publications claiming new non-pathogenic producing strains and novel production techniques many of which are unfortunately without proper characterisation of product and/or producing strain/s. Genes responsible for rhamnolipid production have only been confirmed in P. aeruginosa, Burkholderia thailandensis and Burkholderia pseudomallei. Comparing yields in different publications is also generally unreliable especially when different methodologies were used for rhamnolipid quantification. After reviewing the literature in this area, we strongly feel that numerous research outputs have insufficient evidence to support claims of rhamnolipid-producing strains and/or yields. We therefore recommend that standards should be set for reporting new rhamnolipid-producing strains and production yields. These should include (1) molecular and bioinformatic tools to fully characterise new microbial isolates and confirm the presence of the rhamnolipid rhl genes for all bacterial strains, (2) using gravimetric methods to quantify crude yields and (3) use of a calibrated method (high-performance liquid chromatography or ultra-performance liquid chromatography) for absolute quantitative yield determination.     

Rhamnolipid production in batch and fed-batch fermentation using<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> BYK-2 KCTC 18012P

The optimization of culture conditions for the bacteriumPseudomonas aeruginosa BYK-2 KCTC 18012P, was performed to increase its rhamnolipid production. The optimum level for carbon, nitrogen sources, temperature and pH, for rhamnolipid production in a flask, were identified as 25 g/L fish oil, 0.01% (w/v) urea, 25 and pH 7.0, respectively. Optimum conditions for batch culture, using a 7-L jar fermentor, were 200 rpm of agitation speed and a 2.0 L/min aeration rate. Under the optimum conditions, on fish oil for 216 h, the final cell and rhamnolipid concentrations were 5.3 g/L and 17.0 g/L respectively. Fed-batch fermentation, with different feeding conditions, was carried out in order to increase, cell growth and rhamnolipid production by thePseudomonas aeruginosa, BYK-2 KCTC 18012P. When 2.5 g of fish oil and 100 mL basal salts medium, containing 0.01% (w/v) urea, were fed intermittently during the fermentation, the final cell and rhamnolipid concentrations at 264 h, were 6.1 and 22.7 g/L respectively. The fed-batch culture resulted in a 1.2-fold increase in the dry cell mass and a 1.3-fold increase in rhamnolipid production, compared to the production of the batch culture. The rhamnolipid production-substrate conversion factor (0.75 g/g) was higher than that of the batch culture (0.68 g/g).