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.     

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.     

Hydrocarbon assimilation and biosurfactant production in Pseudomonas aeruginosa mutants.

We isolated transposon Tn5-GM-induced mutants of Pseudomonas aeruginosa PG201 that were unable to grow in minimal media containing hexadecane as a carbon source. Some of these mutants lacked extracellular rhamnolipids, as shown by measuring the surface and interfacial tensions of the cell culture supernatants. Furthermore, the concentrated culture media of the mutant strains were tested for the presence of rhamnolipids by thin-layer chromatography and for rhamnolipid activities, including hemolysis and growth inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the conditions tested, lacked the capacity to take up 14C-labeled hexadecane, and did not grow in media containing individual alkanes with chain lengths ranging from C12 to C19. However, growth on these alkanes and uptake of [14C]hexadecane were restored when small amounts of purified rhamnolipids were added to the cultures. Mutant 59C7 was unable to grow in media containing hexadecane, nor was it able to take up [14C]hexadecane. The addition of small amounts of rhamnolipids restored growth on alkanes and [14C]hexadecane uptake. In glucose-containing media, however, mutant 59C7 produced rhamnolipids at levels about twice as high as those of the wild-type strain. These results show that rhamnolipids play a major role in hexadecane uptake and utilization by P. aeruginosa.     

Isomerization and biodegradation of beta-cypermethrin by Pseudomonas aeruginosa CH7 with biosurfactant production

Pseudomonas aeruginosa CH7, isolated from activated sludge, was able not only to isomerize and degrade beta-cypermethrin but also to utilize it as the sole source of carbon and energy for growth and produce biosurfactant. The strain effectively degraded beta-cypermethrin with inocula biomass of 0.1-0.2 g L⁻¹ at 25-35 °C, pH 6-9, and a final concentration of beta-cypermethrin 25-900 mg L⁻¹. Via response surface methodology analysis, we found the optimal condition was 29.4 °C, pH 7.0, and inocula biomass of 0.15 g L⁻¹; under these conditions, about 90% of the beta-cypermethrin could be degraded within 12 days. Noticeably, biosurfactant was detected in the MSM culture of strain CH7, suggesting that the biosurfactant (rhamnolipid) could potentially enhance the degradation of beta-cypermethrin by promoting the dissolution, adsorption, and absorption of the hydrophobic compounds. Therefore, CH7 may serve as a promising strain in the bioremediation of wastewater and soil polluted by beta-cypermethrin.     

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.     

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.     

Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source.

Rsan-ver, a strain of Pseudomonas aeruginosa isolated at this department, was used for the development of a continuous process for biosurfactant production. The active compounds were identified as rhamnolipids. A final medium for production was designed in continuous culture by means of medium shifts, since the formation of surface-active compounds was decisively influenced by the composition and concentration of the medium components. In the presence of yeast extract, biosurfactant production was poor. For the nitrogen-source nitrate, which was superior to ammonium, an optimum carbon-to-nitrogen ratio of ca. 18 existed. The iron concentration needed to be minimized to 27.5 micrograms of FeSO4 X 7H2O per g of glucose. A carbon-to-phosphate ratio below 16 yielded the maximum production of rhamnolipids. The final productivity dilution rate diagram indicated that biosurfactant production was correlated to low growth rates (dilution rate below 0.15 h-1). With a medium containing 18.2 g of glucose liter-1, a biosurfactant concentration (expressed as rhamnolipids) of up to 1.5 g liter-1 was obtained in the cell-free culture liquid.     

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.     

Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source

Rsan-ver, a strain of Pseudomonas aeruginosa isolated at this department, was used for the development of a continuous process for biosurfactant production. The active compounds were identified as rhamnolipids. A final medium for production was designed in continuous culture by means of medium shifts, since the formation of surface-active compounds was decisively influenced by the composition and concentration of the medium components. In the presence of yeast extract, biosurfactant production was poor. For the nitrogen-source nitrate, which was superior to ammonium, an optimum carbon-to-nitrogen ratio of ca. 18 existed. The iron concentration needed to be minimized to 27.5 micrograms of FeSO4 X 7H2O per g of glucose. A carbon-to-phosphate ratio below 16 yielded the maximum production of rhamnolipids. The final productivity dilution rate diagram indicated that biosurfactant production was correlated to low growth rates (dilution rate below 0.15 h-1). With a medium containing 18.2 g of glucose liter-1, a biosurfactant concentration (expressed as rhamnolipids) of up to 1.5 g liter-1 was obtained in the cell-free culture liquid.     

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.     

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.     

Pyocyanine production by Pseudomonas aeruginosa.

Dextrose enhanced the growth of P. aeruginosa but suppressed the biosynthesis of pyocyanine. The preformed pigment could be released from dead cells. Pigmentation was not correlated directly with number of viable organisms in the culture. High concentration of maltose likewise inhibited pyocyanine production. Maltose contained in medium used for pyocyanine production by P. aeruginosa should be kept in low concentration or omitted.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa.

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Dependence of Pseudomonas aeruginosa continous culture biosurfactant production on nutritional and environmental factors

Summary Continuous culture studies with Pseudomonas aeruginosa were performed in order to establish nutritional and environmental conditions necessary for high production of biosurfactants. Empirical adjustments of the mineral medium formulation showed that better yields of the active compounds, rhamnolipids, are obtained by minimizing the concentration of the respective salts of magnesium, calcium, potassium, sodium and the trace elements. Improvements in performance were more evident when the intial substrate concentration, glucose, was increased up to 73 gl^-1. Further, the ranges for pH (6.2 to 6.4) and temperature (32° to 34°C) that yield high biosurfactant biosynthesis were established. Concerning the physiological state of the microorganism, rhamnolipid formation was restricted to specific growth rates lower than D=0.14 h^-1. By applying the conditions elaborated up to 300 mg rhamnose l^-1 h^-1 (equivalent to 685 mg rhamnolipid l^-1 h^-1) were obtained in a continuous production process.     

Regulation of Pseudomonas aeruginosa chemotaxis by the nitrogen source.

The regulation of amino acid chemotaxis by nitrogen was investigated in the gram-negative bacterium Pseudomonas aeruginosa. The quantitative capillary tube technique was used to measure chemotactic responses of bacteria to spatial gradients of amino acids and other attractants. Chemotaxis toward serine, arginine, and alpha-aminoisobutyrate was sharply dependent on the form in which nitrogen was presented to the bacteria. Bacteria grown on mineral salts-succinate with potassium nitrate gave responses to amino acids that were 2 to 3 times those of cells grown on ammonium sulfate and 10 to 20 times those of cells grown in mineral salts-succinate with Casamino Acids as the nitrogen source. A combination of ammonium sulfate and glutamate was as effective as Casamino Acids in depressing serine taxis. The threshold concentration for alpha-aminoisobutyrate taxis was consistently lower in nitrate-grown bacteria than in ammonia-grown bacteria. Responsiveness to sodium succinate, however, was not subject to regulation by nitrogen, and glucose chemotaxis was inhibited, rather than enhanced, in nitrate-grown bacteria. These results indicate that chemotaxis of P. aeruginosa toward amino acids is subject to regulation by nitrogen and that this regulation probably is expressed at the level of the chemoreceptors or transducers.     

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.     

Influence of different magnetites on properties of magnetic Pseudomonas aeruginosa immobilizates used for biosurfactant production

During the last decades, whole‐cell immobilization has been used successfully in many bioprocesses. In particular, it is aimed at implementing continuous production processes, reaching higher production rates, and reusing the biocatalyst. In some cases, effective retention of immobilizates in the bioprocess is not feasible by membranes or sieves due to pore plugging or undesired losses of immobilizates. In the present publication, it is reported about the investigation of magnetic immobilizates of Pseudomonas aeruginosa for application in continuous biosurfactant production of rhamnolipids by foam fractionation and retention of entrained immobilizates by high‐gradient magnetic separation from foam. Different materials and methods were tested with respect to important parameters, such as stability, diffusion properties or magnetic separation. Good magnetic separation of immobilizates was achieved at 5% (w/w) magnetite loading. Best results in terms of homogeneous embedding, good diffusion properties, and stability enhancement vis‐à‐vis pure alginate beads was achieved with alginate beads with embedded Bayoxide® magnetite or MagPrep® silica particles. Although polyurethane immobilizates showed higher stabilities compared with alginate beads, rhamnolipid diffusion in immobilizates was superior in magnetic alginate beads. Regarding bead production, smaller immobilizates were achieved with suspension polymerization compared to droplet extrusion by the JetCutting® technology. In total, magnetic immobilizates are a promising tool for an easier handling of biocatalysts in a continuous biological production process, but they have to be adapted to the current production task.© 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.