Physicochemical characterization and antimicrobial properties of rhamnolipids produced by Pseudomonas aeruginosa 47T2 NCBIM 40044

Pseudomonas aeruginosa 47T2, grown in submerged culture with waste frying oil as a carbon source, produced a mixture of rhamnolipids with surface activity. Up to 11 rhamnolipid homologs (Rha-Rha-C(8)-C(10); Rha-C(10)-C(8)/Rha-C(8)-C(10);Rha-Rha-C(8)-C(12:1); Rha-Rha-C(10)-C(10); Rha-Rha-C(10)-C(12:1); Rha-C(10)-C(10); Rha-Rha-C(10)-C(12)/Rha-Rha-C(12)-C(10); Rha-C(10)-C(12:1)/Rha-C(12:1)-C(10); Rha-Rha-C(12:1)-C(12); Rha-Rha-C(10)-C(14:1); Rha-C(10)-C(12)/Rha-C(12)-C(10)) were isolated from cultures of P. aeruginosa 47T2 from waste frying oil and identified by HPLC-MS analysis. This article deals with the production, isolation, and chemical characterization of the rhamnolipid mixture RL(47T2). The physicochemical and biological properties of RL(47T2) as a new product were also studied. Its surface tension decreased to 32.8 mN/m; and the interfacial tension against kerosene to 1 mN/m. The critical micellar concentration for RL(47T2) was 108.8 mg/mL. The product showed excellent antimicrobial properties. Antimicrobial activity was evaluated according to the minimum inhibitory concentration (MIC), the lowest concentration of an antimicrobial agent that inhibits development of visible microbial growth. Low MIC values were found for bacteria Serratia marcescens (4 microg/mL), Enterobacter aerogenes (8 microg/mL), Klebsiella pneumoniae (0.5 microg/mL), Staphylococcus aureus and Staphylococcus epidermidis (32 microg/mL), Bacillus subtilis (16 microg/mL), and phytopathogenic fungal species: Chaetonium globosum (64 microg/mL), Penicillium funiculosum (16 microg/mL), Gliocadium virens (32 microg/mL) and Fusarium solani (75 microg/mL).     

Molecular and structural characterization of the biosurfactant produced by Pseudomonas aeruginosa DAUPE 614

Pseudomonas aeruginosa DAUPE 614 produced rhamnolipids (3.9gL(-1)) when cultivated on a medium containing glycerol and ammonium nitrate. These rhamnolipids reduced the surface tension of water to 27.3mNm(-1), with a critical micelle concentration of 13.9mgL(-1). The maximum emulsification index against toluene was 86.4%. The structure of the carbohydrate moiety of the glycolipid was determined by gas chromatography-mass spectroscopy (GC-MS) analysis allied to electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) 1D, 2D (13)C, (1)H spectroscopy. The hydroxyl fatty acids were analyzed by GC-MS as hydroxy-acetylated fatty acid methyl ester derivatives. The positions of the fatty acids in the lipid moiety were variable, with 6 mono-rhamnolipid homologues (Rha-C(10)-C(10); Rha-C(10)-C(8); Rha-C(8)-C(10); Rha-C(10)-C(12:1); Rha-C(12)-C(10); Rha-C(10)-C(12)) and 6 di-rhamnolipid homologues (Rha(2)-C(10)-C(10); Rha(2)-C(10)-C(8); Rha(2)-C(8)-C(10); Rha(2)-C(10)-C(12:1); Rha(2)-C(12)-C(10); Rha(2)-C(10)-C(12)). The ratio of Rha(2)-C(10)-C(10) to Rha-C(10)-C(10) was higher than has been reported in previous studies. Our methodology allowed us to distinguish between the isomeric pairs Rha-C(10)-C(8)/Rha-C(8)-C(10), Rha-C(10)-C(12)/Rha-C(12)-C(10), Rha(2)-C(10)-C(8)/Rha(2)-C(8)-C(10) and Rha(2)-C(12)-C(10)/Rha(2)-C(10)-C(12). For each isomeric pair, the congener with the shorter chain adjacent to the sugar was always more abundant than the congener with longer chain.     

Structural characterization of rhamnolipid produced by Pseudonomas aeruginosa strain FIN2 isolated from oil reservoir water

Biosurfactant-producing microorganisms inhabiting oil reservoirs are of great potential in industrial applications. Yet, till now, the knowledge about the structure and physicochemical property of their metabolites are still limited. The aim of this study was to purify and structurally characterize the biosurfactant from Pseudomonas aeruginosa strain FIN2, a newly isolated strain from an oil reservoir. The purification was conducted by silica gel column chromatography followed by pre-RP HPLC and the structural characterization was carried out by GC–MS combined with MS/MS. The results show that the biosurfactant produced by FIN2 is rhamnolipid in nature and its four main fractions were identified to be Rha-C10-C10(46.1 %), Rha–Rha-C10-C10(20.1 %), Rha-C8-C10 (7.5 %) and Rha-C10-C12:1(5.5 %), respectively. Meanwhile, the rarely reported rhamnolipid congeners containing β-hydroxy fatty acids of C6, C9, C10:1 and C11 were also proved to be present in the rhamnolipid mixture produced. The rhamnolipid mixture exhibited a strong surface activity by lowering the surface tension of distilled water to 28.6 mN/m with a CMC value of 195 mg/l.     

发酵碳源对铜绿假单胞菌NY3所产鼠李糖脂结构及性能的影响

为研究发酵碳源对铜绿假单胞菌NY3所产鼠李糖脂结构及性能的影响,从鼠李糖脂的结构组分、性能和应用效果等方面展开研究。薄层实验证明两种鼠李糖脂均含有单糖脂和双糖脂。液质分析发现以橄榄油作碳源时,鼠李糖脂中双糖脂(Rha-Rha-C5-C6:1和Rha-Rha-C8-C8:2)比例更大,约为73.09%。而地沟油作碳源时,单糖脂(Rha-C10-C10和Rha-C16-C16:2)的比例更高,约为76.91%。橄榄油和地沟油为碳源的鼠李糖脂的临界胶束浓度(CMC)分别为55 mg/L和80mg/L。相同投加量时,前者乳化性和乳化稳定性均优于后者。NY3菌降解含油污泥时,投加双糖脂含量高的鼠李糖脂会使C16-C30直链烷烃的去除率更高。     

Chemical structure, surface properties and biological activities of the biosurfactant produced by Pseudomonas aeruginosa LBI from soapstock

Pseudomonas aeruginosa LBI isolated from petroleum-contaminated soil produced rhamnolipids (RL(LBI)) when cultivated on soapstock as the sole carbon source. HPLC-MS analysis of the purified culture supernatant identified 6 RL homologues (%): R(2) C(10) C(10) 28.9; R(2) C(10) C(12:1) 23.0; R(1) C(10) C(10) 23.4; R(2) C(10) C(12) 11.3; R(2) C(10) C(12) 7.9; R(2) C(10) C(12) 5.5. To assess the potential antimicrobial activity of the new rhamnolipid product, RL(LBI), its physicochemical properties were studied. RL(LBI) had a surface tension of 24 mN m(-1) and an interfacial tension of 1.31 mN m(-1); the cmc was 120 mg l(-1). RL(LBI) produced stable emulsions with hydrocarbons and vegetable oils. This product showed good antimicrobial behaviour against bacteria: MIC for Bacillus subtilis, Staphylococcus aureus and Proteus vulgaris was 8 mg l(-1), for Streptococcus faecalis 4 mg l(-1), and for Pseudomonas aeruginosa 32 mg l(-1). RL(LBI) was active against phytopathogenic fungal species, MIC values of 32 mg l(-1) being found against Penicillium, Alternaria, Gliocadium virens and Chaetonium globosum. Due to its physicochemical properties and antimicrobial behaviour, RL(LBI) could be used in bioremediation treatment and in the food, cosmetic and pharmaceutical industries.     

Oil wastes as unconventional substrates for rhamnolipid biosurfactant production by Pseudomonas aeruginosa LBI

Oil wastes were evaluated as alternative low-cost substrates for the production of rhamnolipids by Pseudomonas aeruginosa LBI strain. Wastes obtained from soybean, cottonseed, babassu, palm, and corn oil refinery were tested. The soybean soapstock waste was the best substrate, generating 11.7 g/L of rhamnolipids with a surface tension of 26.9 mN/m, a critical micelle concentration of 51.5 mg/L, and a production yield of 75%. The monorhamnolipid RhaC(10)C(10) predominates when P. aeruginosa LBI was cultivated on hydrophobic substrates, whereas hydrophilic carbon sources form the dirhamnolipid Rha(2)C(10)C(10) predominantly.     

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.

     

Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials

This study was aimed at the development of economical methods for higher yields of biosurfactant by suggesting the use of low-cost raw materials. Two oil-degrading strains, Pseudomonas aeruginosa GS9-119 and DS10-129, were used to optimize a substrate for maximum rhamnolipid production. Among the two strains, the latter produced maxima of 4.31, 2.98, and 1.77 g/L rhamnolipid biosurfactant using soybean oil, safflower oil, and glycerol, respectively. The yield of biosurfactant steadily increased even after the bacterial cultures reached the stationary phase of growth. Characterization of rhamnolipids using mass spectrometry revealed the presence of dirhamnolipids (Rha-Rha-C(10)-C(10)). Emulsification activity of the rhamnolipid biosurfactant produced by P. aeruginosa DS10-129 was greater than 70% using all the hydrocarbons tested, including xylene, benzene, hexane, crude oil, kerosene, gasoline, and diesel. P. aeruginosa GS9-119 emulsified only hexane and kerosene to that level.     

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.     

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.     

Evaluation of critical nutritional parameters and their significance in the production of rhamnolipid biosurfactants from Pseudomonas aeruginosa BS‐161R

Eleven biosurfactant producing bacteria were isolated from different petroleum‐contaminated soil and sludge samples. Among these 11 isolates, two were identified as promising, as they reduced the surface tension of culture medium to values below 27 mN m^?1. Besides biosurfactant production property, they exhibited good flocculating activity. Microbacterium sp. was identified as a new addition to the list of biosurfactant and bioflocculant‐producers. Optimization of various conditions for rhamnolipid production was carried out for one of the promising isolate, Pseudomonas aeruginosa BS‐161R. Bioglycerol (2.5%), as a cheap renewable carbon source, attained better rhamnolipid yield, while sodium nitrate appeared to be the preferable nitrogen source. The optimum carbon to nitrogen (C/N) and carbon to iron (C/Fe) ratios achieved were 15 and 28,350, respectively, which favored rhamnolipid production. Physical parameters like pH, temperature, and agitation speed also affected the production of rhamnolipids. Results from shake flask optimization indicated that the concentration of bioglycerol, sodium nitrate, and iron were the most significant factors affecting rhamnolipid production, which was supported by the results of central composite rotatable design. After optimization of the culture conditions, the production of rhamnolipids increased by ninefold from 0.369 to 3.312 g L^?1. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Biosurfactant production by a new Pseudomonas putida strain

Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by the newly isolated and promising strain Pseudomonas putida 21BN. The biosurfactants were identified as rhamnolipids, the amphiphilic surface-active glycolipids usually secreted by Pseudomonas spp. Their production was observed when the strain was grown on soluble substrates, such as glucose or on poorly soluble substrates, such as hexadecane, reaching values of 1.2 g l(-1). When grown on hexadecane as the sole carbon source the biosurfactant lowered the surface tension of the medium to 29 mN m(-1) and formed stable and compact emulsions with emulsifying activity of 69%.     

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.     

Chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste

The aim of this work was to study chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste. The culture produced two biosurfactants, a and b, which showed strong activity and were identified as L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate or Rha-Rha-C10-C10 and L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydodecanoate or Rha-Rha-C10-C12, respectively. Both compounds exhibited higher surfactant activities tested by the drop collapse test than several artificial surfactants such as SDS and Tween 80. Rhamnolipid a showed significant antiproliferative activity against human breast cancer cell line (MCF-7) at minimum inhibitory concentration (MIC) at 6.25 microg/mL while rhamnolipid b showed MIC against insect cell line C6/36 at 50 microg/mL.     

Production of biosurfactant using different hydrocarbons by Pseudomonas aeruginosa EBN-8 mutant

The present investigation dealt with the use of previously isolated and studied gamma-ray mutant strain Pseudomonas aeruginosa EBN-8 for the production of biosurfactant by using different hydrocarbon substrates viz. n-hexadecane, paraffin oil and kerosene oil, provided in minimal medium, as the sole carbon and energy sources. The batch experiments were conducted in 250 mL Erlenmeyer flasks, containing 50 mL minimal salt media supplemented with 1% (w/v) hydrocarbon substrate, inoculated by EBN-8 and incubated at 37 degrees C and 100 rpm in an orbital shaker. The sampling was done on 24 h basis for 10 d. The surface tension of cell-free culture broth decreased from 53 to 29 mN/m after 3 and 4 d of incubation when the carbon sources were paraffin oil and n-hexadecane, respectively. The largest reduction in interfacial tension from 26 to 0.4 mN/m was observed with n-hexadecane, while critical micelle dilution was obtained as 50 x CMC for paraffin oil as carbon source. When grown on n-hexadecane and paraffin oil, the EBN-8 mutant strain gave 4.1 and 6.3 g of the rhamnolipids/L, respectively. These surface-active substances subsequently allowed the hydrocarbon substrates to disperse readily as emulsion in aqueous phase.     

Identification and production of a rhamnolipidic biosurfactant by a Pseudomonas species

 A glycolipid-producing bacterium, Pseudomonas aeruginosa GL1, was isolated from the soil contaminated with polycyclic aromatic hydrocarbons (PAH) from a manufactured gas plant. The glycolipid produced was characterized in detail by chromatographic procedures as a mixture of four rhamnolipids, consisting of different associations of rhamnose and hydroxy fatty acids: the main component was monorhamnosyl di-3-hydroxydecanoic acid. The rhamnolipid composition presented marked analogies with a defined part of P. aeruginosa outer membrane lipopolysaccharides (lipopolysaccharide band A). Rhamnolipid production was stimulated under conditions of nitrogen limitation. Glycerol yielded higher productions than did hydrophobic carbon sources. Cell hydrophobicity decreased during growth on glycerol and on n-hexadecane whereas glycolipid production increased. P. aeruginosa GL1 was found to be unable to grow on a variety of 2, 3 and 4 cycle PAH. However, it was shown to persist after at least 12 subcultures in a bacterial population growing on a mixture of pure PAH, suggesting a physiological role for rhamnolipid as a means to enhance PAH availability in a mutualistic PAH-degrading bacterial community. Received: 4 July 1995/Received revision: 7 September 1995/Accepted: 13 September 1995     

Chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste

The aim of this work was to study chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste. The culture produced two biosurfactants, a and b, which showed strong activity and were identified as L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate or Rha-Rha C10-C10 and L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydodecanoate or Rha-Rha C(10)-C(12), respectively. Both compounds exhibited higher surfactant activities tested by the drop collapse test than several artificial surfactants such as SDS and Tween 80. Rhamnolipid a showed significant antiproliferative activity against human breast cancer cell line (MCF-7) at minimum inhibitory concentration (MIC) at 6.25 microg/mL while rhamnolipid b showed MIC against insect cell line C6/36 at 50 microg/mL.     

Liquid chromatography/mass spectrometry analysis of mixtures of rhamnolipids produced by Pseudomonas aeruginosa strain 57RP grown on mannitol or naphthalene.

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.     

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.     

废弃食用油脂生物合成鼠李糖脂研究进展

碳源的成本过高限制了鼠李糖脂的工业化生产及应用,废弃食用油脂作为一种廉价易得的碳源,越来越多的研究者开始关注用它发酵生产鼠李糖脂.废弃食用油脂的种类、投加量对鼠李糖脂的产量、结构、性质均会产生影响,目前研究中用废弃食用油脂作碳源,鼠李糖脂产量最高可达24.61g/L、表面张力最低达到24mN/m、产物CMC最低可达40.19mg/L.此外,本文还总结了菌株、氮源、微量元素、pH、溶氧及培养方式等因素对废弃食用油脂生产鼠李糖脂的影响,并展望了利用废弃食用油脂生产鼠李糖脂实现产业化的重点研究方向.