Improved production of biosurfactant with newly isolated Pseudomonas aeruginosa S2

An indigenous strain Pseudomonas aeruginosa S2 (P. aeruginosa S2), isolated from diesel-contaminated soil, produced extracellular surface-active material identified as rhamnolipid. Due to its excellent surface activity, rhamnolipid is known to be well-suited for stimulating the bioremediation efficiency of oil contaminated sites. To improve production yield of rhamnolipid with P. aeruginosa S2, various carbon and nitrogen sources were screened to select favorable ones leading to better biosurfactant production yield. It was found that using 4% glucose could attain better rhamnolipid yield, while 50 mM NH4NO3 appeared to be the most preferable nitrogen source. Meanwhile, the effect of carbon to nitrogen ratio (C/N ratio) on rhamnolipid yield was also investigated, and the optimal C/N ratio was identified as approximately 11.4. Moreover, response surface methodology (RSM) was applied to optimize the trace element concentration for rhamnolipid production. Results from two-level design indicate that concentrations of MgSO4 and FeSO4 were the most significant factors affecting rhamnolipid production. Using steepest ascent method and RSM analysis, an optimal medium composition was determined, giving a rhamnolipid production yield of 2.37 g/L in 100 h at 37 degrees C and 200 rpm agitation. Scale-up production of rhamnolipid in a well-controlled 5 L jar fermentor using the optimal medium and operating condition (at 37 degrees C and pH 6.8) further elevated the biosurfactant production yield to 5.31 g/L (in 97 h), which is over 2-fold higher than the best results obtained from shake-flask tests.     

Structural and physicochemical characterization of crude biosurfactant produced by Pseudomonas aeruginosa SP4 isolated from petroleum-contaminated soil

Pseudomonas aeruginosa strain SP4, isolated from petroleum-contaminated soil in Thailand, was used to produce a biosurfactant from a nutrient broth with palm oil as the carbon source. The key components of the crude biosurfactant were fractionated by using HPLC-ELSD technique. With the use of ATR-FTIR spectroscopy, in combination with (1)H NMR and MS analyses, chemical structures of the fractionated components of the crude biosurfactant were identified as rhamnolipid species. When compared to synthetic surfactants, including Pluronic F-68, which is a triblock nonionic surfactant containing poly(ethylene oxide) and poly(propylene oxide), and sodium dodecyl sulfate, the crude biosurfactant showed comparable physicochemical properties, in terms of the surface activities. The crude biosurfactant reduced the surface tension of pure water to 29.0 mN/m with a critical micelle concentration of approximately 200 mg/l, and it exhibited good thermal and pH stability. The crude biosurfactant also formed stable water-in-oil microemulsions with crude oil and various types of vegetable oils, but not with short-chain hydrocarbons.     

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     

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.     

Characterization of glycolipid biosurfactant from Pseudomonas aeruginosa CPCL isolated from petroleum‐contaminated soil

Aims: To isolate and characterize the biosurfactant‐producing micro‐organism from petroleum‐contaminated soil as well as to determine the biochemical properties of the biosurfactant. Methods and Results: A novel rhamnolipid‐producing Pseudomonas aeruginosa (GenBank accession number GQ241355 ) strain was isolated from a petroleum‐contaminated soil. Surface active compound was separated by solvent extraction of the acidified culture supernatant. The extract was able to reduce the surface tension of water from 72 to 44 mN m^?1 at a critical micelle concentration of 11·27 ± 1·85 mg l^?1. It showed better activity (based on microdilution method) against Gram‐positive (≤ 31 mg ml^?1) bacteria and filamentous fungi (≤ 50 mg ml^?1) than Gram‐negative bacteria (≥ 125 mg ml^?1) with mild toxicity (HC₅₀– 38 ± 8·22 μg ml^?1) to red blood cells. Fourier transform infrared spectroscopy revealed the presence of aliphatic chain, hydroxyl groups, ester and glycosidic bonds. Presence of nineteen rhamnolipid homologues with variation in chain length and saturation was revealed from liquid chromatography coupled to mass spectrometry with electrospray ionization. Conclusion: The results indicate that the isolated biosurfactant has a novel combination of rhamnolipid congeners with unique properties. Significance and Impact of the Study: This study provides a biosurfactant, which can be used as a biocontrol agent against phytopathogens (Fusarium proliferatum NCIM 1105 and Aspergillus niger NCIM 596) and exploited for biomedical applications.     

Modulation of the physical properties of dielaidoylphosphatidylethanolamine membranes by a dirhamnolipid biosurfactant produced by Pseudomonas aeruginosa

Rhamnolipids are bacterial biosurfactants produced by Pseudomonas spp. These compounds have been shown to present several interesting biological activities, restricting the growth of Bacillus subtilis and showing zoosporicidal activity on zoosporic phytopathogens. It has been suggested that the interaction with the membrane could be the ultimate responsible for these actions. Therefore, it is of great interest to get insight into the molecular mechanism of the interaction of purified rhamnolipids with the various phospholipid components of biological membranes. In this paper we report on the phase behaviour of mixtures of dielaidoylphosphatidylethanolamine (DEPE) with a purified dirhamnolipid (DiRL) fraction from Pseudomonas aeruginosa, as studied by a number of physical techniques such as differential scanning calorimetry, FTIR, small angle X-ray (SAX) diffraction and dynamic light scattering. Our data indicate that the presence of DiRL counteracts the tendency of DEPE to form vesicular aggregates of large size, forming vesicles of smaller diameter which most probably have a lower lamellarity index. The partial phase diagram obtained from calorimetric data shows a complex behaviour with a solid-phase immiscibility. X-ray diffraction shows that DiRL has a bilayer stabilizing effect, impeding formation of the inverted hexagonal-HII phase of DEPE. The presented data are discussed focussing into how DiRL/DEPE interactions could help to explain the membrane perturbing activities of this biosurfactant.     

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.     

Pilot plant production of rhamnolipid biosurfactant by Pseudomonas aeruginosa

Rhamnolipid biosurfactants were continuously produced with Pseudomonas aeruginosa on the pilot plant scale. Production and downstream processing elaborated on the laboratory scale were adapted to the larger scale. Differences in performance resulting from the scale-up are discussed. A biosurfactant concentration of approximately 2.25 g liter-1 was achieved. The biosurfactant yield on glucose was 77 mg g-1 h-1, and the productivity was 147 mg liter-1 h-1, corresponding to a daily production of 80 g of biosurfactant. The first enrichment step consisted of an adsorption chromatography which was followed by an anion-exchange chromatography. The resulting product was 90% pure, and the overall recovery of active material was above 60% with the downstream processing used.     

Characteristics of rhamnan isolated from preparations of Pseudomonas aeruginosa lipopolysaccharides

A polysaccharide isolated from the degraded lipopolysaccharides of P. aeruginosa serogroup O7 (Lányi--Bergan classification) was characterized by liquid chromatography, acid hydrolysis, and 1H and 13C NMR spectroscopy. It has molecular mass 15,000 and represents mainly a rhamnan of the structure----2)-alpha-D-Rha-(1----3)-alpha-D-Rha-(1----3)-alpha-D-Rha-(1 ----, identical to the structure of O-specific polysaccharides of Pseudomonas aeruginosa pvs morsprunorum and cerasi. Some minor constituents, such as glucose, mannose, an unknown sugar, and phosphate, are found in the polysaccharide preparation as well. Distribution of the rhamnan in some other P. aeruginosa serogroups is discussed and its identity to the common polysaccharide antigen of P. aeruginosa is suggested.     

Kinetics of biosurfactant production by Pseudomonas aeruginosa strain BS2 from industrial wastes

Summary Batch kinetic studies were carried out on rhamnolipid biosurfactant production from synthetic medium, industrial wastes viz. distillery and whey waste as substrates. The results indicated that the specific growth rates ( _max) and specific product formation rates (V _max) from both the wastes are comparatively better than the synthetic medium, revealing that both the industrial wastes (distillery and whey) can be successfully utilized as substrates for biosurfactant production.     

Correlation of nitrogen metabolism with biosurfactant production by Pseudomonas aeruginosa

A direct relationship between increased glutamine synthetase activity and enhanced biosurfactant production was found in Pseudomonas aeruginosa grown in nitrate and Proteose Peptone media. A chloramphenicol-tolerant strain showed a twofold increase in biosurfactant production and glutamine synthetase activity. Increased ammonium and glutamine concentrations repressed both phenomena.     

Pilot plant production of rhamnolipid biosurfactant by Pseudomonas aeruginosa.

Rhamnolipid biosurfactants were continuously produced with Pseudomonas aeruginosa on the pilot plant scale. Production and downstream processing elaborated on the laboratory scale were adapted to the larger scale. Differences in performance resulting from the scale-up are discussed. A biosurfactant concentration of approximately 2.25 g liter-1 was achieved. The biosurfactant yield on glucose was 77 mg g-1 h-1, and the productivity was 147 mg liter-1 h-1, corresponding to a daily production of 80 g of biosurfactant. The first enrichment step consisted of an adsorption chromatography which was followed by an anion-exchange chromatography. The resulting product was 90% pure, and the overall recovery of active material was above 60% with the downstream processing used.     

Correlation of nitrogen metabolism with biosurfactant production by Pseudomonas aeruginosa.

A direct relationship between increased glutamine synthetase activity and enhanced biosurfactant production was found in Pseudomonas aeruginosa grown in nitrate and Proteose Peptone media. A chloramphenicol-tolerant strain showed a twofold increase in biosurfactant production and glutamine synthetase activity. Increased ammonium and glutamine concentrations repressed both phenomena.     

Heterologous production of Pseudomonas aeruginosa EMS1 biosurfactant in Pseudomonas putida

A new bacterial strain isolated from activated sludge, identified as Pseudomonas aeruginosa EMS1, produced a biosurfactant when grown on acidified soybean oil as the sole carbon source. An optimum biosurfactant production of 5 g/L was obtained with the following medium composition: 2% acidified soybean oil, 0.3% NH4NO3, 0.03% KH2PO4, 0.03% K2HPO4, 0.02% MgSO4.7H2O and 0.025% CaCl2.2H2O, with shaking at 200 rpm for an incubation period of 100 h at 30 degrees C. The production of the biosurfactant was found to be a function of cell growth, with maximum production occurring during the exponential phase. Hemolysis of erythrocytes and thin-layer chromatography studies revealed that the secreted biosurfactant was rhamnolipid. To overcome the complex environmental regulation with respect to rhamnolipid biosynthesis, and to replace the opportunistic pathogen P. aeruginosa with a safe industrial strain, attempts were made to achieve rhamnolipid production in a heterologous host, Pseudomonas putida, using molecular cloning of rhlAB rhamnosyltransferase genes with the rhlRI quorum sensing system, assuming that a functional rhamnosyltransferase would catalyze the formation of rhamnosyl-6-hydroxydecanoyl-6-hydroxydecanoate (mono-rhamnolipid) in P. putida. It was shown that rhamnolipid can be produced in the heterologous strain, P. putida, when provided with the rhamnosyltransferase genes.     

Isolation and structural characterization of biosurfactant produced by an alkaliphilic bacterium Cronobacter sakazakii isolated from oil contaminated wastewater

Production of biosurfactant from an alkaliphilic bacterium Cronobacter sakazakii (accession no. JN398668) was screened by haemolytic assay, emulsifying activity and surface tension measurement. Biosurfactant, comprised of total sugars (73.3%), reducing sugars (1.464%), protein (11.9%), uronic acid (15.98%) and sulfate (6.015%), showed low viscosity with pseudoplastic rheological behavior and exhibited significant emulsification activity with oils and hydrocarbons. A series of low and mid range mass peaks (m/z) corresponding to mono-, di-, tri- and oligosaccharides were detected in the positive ion reflector mode of MALDI TOF-TOF MS. GC-MS analysis revealed composition of monosaccharide moieties (w/w) viz. glucose (14%), mannose (24%), galactose (14%), xylose (20%) and arabinose (1.9%). ¹H NMR, FT-IR and EDX analyses confirmed the characteristic various functional groups, bonds and elements respectively. Thermostability (up to 260 °C) and CI (0.456) were determined by TG and DSC analyses. Inherent properties of biosurfactant make it a potential candidate for bioremediation of oil and hydrocarbons.     

Characterization of new biosurfactant produced by Klebsiella sp. Y6-1 isolated from waste soybean oil

To obtain predominant bacteria degrading crude oil, we isolated some bacteria from waste soybean oil. Isolated bacterial strain had a marked tributyrin (C4:0) degrading activity as developed clear zone around the colony after incubation for 24h at 37 degrees C. It was identified as Klebsiella sp. Y6-1 by analysis of 16S rRNA gene. Crude biosurfactant was extracted from the culture supernatant of Klebsiella sp. Y6-1 by organic solvent (methanol:chloroform:1-butanol) after vacuum freeze drying and the extracted biosurfactant was purified by silica gel column chromatography. When the purified biosurfactant dropped, it formed degrading zone on crude oil plate. When a constituent element of the purified biosurfactant was analyzed by TLC and SDS-PAGE, it was composed of peptides and lipid. The emulsification activity and stability of biosurfactant was measured by using hydrocarbons and crude oil. The emulsification activity and stability of the biosurfactant showed better than the chemically synthesized surfactant. It reduced the surface tension of water from 72 to 32 mN/m at a concentration of 40 mg/l.     

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.     

Transformation by extracellular DNA produced by Pseudomonas aeruginosa

Most Pseudomonas aeruginosa strains are capable of producing extracellular DNA. Very closely linked chromosomal markers (leu+ and trp+) were co-transferred to P. aeruginosa PAO1819 (leu9001, trp9008) by the extracellular DNA produced by P. aeruginosa strains IFO3445 and PAO1 at a frequency of 10(-7) to 10(-8). Treatment of the extracellular DNA with DNase, heating at 95 C or sonication completely destroyed its transforming ability. The R plasmid in the extracellular DNA produced by P. aeruginosa IFO3445 (RP4) or PAO2142 (RLb679) could be transferred to Escherichia coli ML4901 or P. aeruginosa PAO1819. The resultant transformants showed identical resistance patterns in the respective donors, and the sizes of the DNAs of RLb679 and RP4 isolated from the transformants were the same as those in the respective donors. These results demonstrate that the extracellular DNA contains both chromosomal DNA and plasmid DNA, and that it exhibits transforming ability. This implies that transformation by the extracellular DNA produced by P. aeruginosa may occur in nature and this seems to be of clinical importance in view of the spread of R plasmids among pathogens.     

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.