Effect of addition ofPseudomonas aeruginosa UG2 inocula or biosurfactants on biodegradation of selected hydrocarbons in soil

Summary A laboratory study was undertaken to assess the effect of adding eitherPseudomonas aeruginosa UG2 cells or the biosurfactants produced by this m microorganism on the biodegradation of a hydrocarbon mixture in soil at 20°C over a 2-month incubation period. The addition of 100 g of UG2 biosurfactants per g soil significantly enhanced the degradation of tetradecane, hexadecene and pristane but not 2-methylnaphthalene, the most water-soluble of the hydrocarbons. Addition of UG2 cells at densities of 10⁶, 10⁷, and 10⁸ per g soil did not have a significant effect on biodegradation of the hydrocarbon mixture.     

Phenanthrene mineralization by Pseudomonas sp. UG14

A phenanthrene-mineralizing Pseudomonas sp., designated UG14, was isolated from creosote-contaminated soil. It contained two plasmids, of approximately 77 kb and 76 kb, the smaller of which contained DNA sequences that hybridized with probes specific for ndoB and xylE, genes involved in catabolism of aromatic hydrocarbons. At initial phenanthrene concentrations of 10, 50, 200 and 1000 mg/l broth, 27%, 19%, 7.7% and 3.3%, respectively, of the [9-¹⁴C]phenanthrene was recovered as ¹⁴CO₂ after 36 days' incubation at 30°C. Most ¹⁴C-label was converted to a water-soluble metabolite tentatively identified as 1-hydroxy-2-naphthoic acid. Rhamnolipid biosurfactants produced by P. aeruginosa UG2 enhanced mineralization of 50 mg phenanthrene/l by Pseudomonas sp. UG14. With the biosurfactant at 0, 25 and 250 mg rhamnose equivalents/l, 6.5%, 8.2% and 9.8%, respectively, of the phenanthrene was mineralized after 35 days.M.A. Providenti, H. Lee and J.T. Trevors are with the Department of Environmental Biology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada; C.W. Greer is with the National Research Council Canada, Biotechnology Research Institute, 6100 Royalmount Ave, Montreal, Quebec, H4P 2R2, Canada.     

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.     

Isolation and characterization of acetonitrile utilizing bacteria

Summary Bacteria utilizing high concentrations of acetonitrile as the sole carbon source were isolated and identified asChromobacterium sp. andPseudomonas aeruginosa. Maximum growth was attained after 96 h of incubation andP. aeruginosa grew slightly faster thanChromobacterium sp. The strains were able to grow and oxidize acetonitrile at concentrations as high as 600 mM. However, higher concentrations inhibited growth and oxygen uptake. Degradation studies with (¹⁴C)acetonitrile indicated 57% of acetonitrile was degraded byPseudomonas aeruginosa as compared to 43% byChromobacterium. The isolates utilized different nitrile compounds as carbon substrates.     

Biosurfactant production by two isolates ofPseudomonas aeruginosa

Two strains of biosurfactant-producing bacteria, identified asPseudomonas aeruginosa, were isolated from injection water and crude oil-associated water in Venezuelan oil fields. Both biosurfactants resembled rhamnolipids and produced stable emulsions of heavy and extra-heavy crude oils, reducing the surface tension of water from 72 to 28 dynes/cm. Tenso-active properties of the biosurfactants were not affected by pH, temperature, salinity or Ca²⁺ or Mg²⁺ at concentrations in excess of those found in many oil reservoirs in Venezuela.     

Biosurfactants,an help in the biodegradation of hexadecane? The case of <Emphasis Type="Italic">Rhodococcus</Emphasis> and <Emphasis Type="Italic">Pseudomonas</Emphasis> strains

The roles of the extracellular biosurfactants produced by two bacterial strains, Pseudomonas aeruginosa GL1 and Rhodococcus equi Ou2, in hexadecane uptake and biodegradation were compared. For this purpose, cell hydrophobicity and production of glycolipidic biosurfactants were evaluated during bacterial growth on hexadecane, as well the effects of these biosurfactants on culture supernatants properties i.e., surface and interfacial tensions, and emulsification and pseudosolubilization capacities. The results showed that the role of biosurfactants was different in these two strains and was directly related to the hydrophobicity of the bacterial cells concerned. Extracellular biosurfactants produced by strain R. equi Ou2 had only a minor role in hexadecane degradation. Direct interfacial accession appeared to be the main mechanism for hexadecane uptake by the hydrophobic cells of strain R. equi Ou2. On the contrary, the biosurfactants produced by P. aeruginosa GL1 were required for growth on hexadecane, and their pseudosolubilization capacity rather than their emulsification capacity was involved in substrate degradation, allowing uptake from hexadecane micelles by the hydrophilic cells of this bacterium. The roles of biosurfactants thus differ widely among bacteria degrading hydrophobic compounds. J.-P. Vandecasteele—in retirement     

Biosurfactant production and hydrocarbon-degradation by halotolerant and thermotolerant Pseudomonas sp.

A hydrocarbon degrading and biosurfactant producing, strain DHT2, was isolated from oil-contaminated soil. The organism grew and produced biosurfactant when cultured in variety of substrates at salinities up to 6 g l⁻¹ and temperatures up to 45°C. It was capable of utilizing crude oil, fuels, alkanes and PAHs as carbon source across the wide range of temperature (30–45°C) and salinity (0–6%). Over the range evaluated, the salinity and temperature did not influence the degradation of hydrocarbon and biosurfactant productions. Isolate DHT2 was identified as Pseudomonas aeruginosa by analysis of 16S rRNA sequences (100% homology) and biochemical analysis. PCR and DNA hybridization studies revealed that enzymes involved in PAH metabolism were related to the naphthalene dioxygenase pathway. Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by DHT2 during growth on both, water miscible and immiscible substrates, including PAH. The biosurfactants lowered the surface tension of medium from 54.9 to 30.2 dN/cm and formed a stable emulsion. The biosurfactant produced by the organism emulsified a range of hydrocarbons with hexadecane as best substrate and toluene was the poorest. These findings further indicate that the isolate could be useful for bioremediation and bio-refining application in petroleum industry.     

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

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

Effects of metals on elastase fromPseudomonas aeruginosa SES-938-1

Among the numerous virulance factors produced byPseudomonas aeruginosa, elastase is the one most often associated with pathogenesis. In this study, effects of various metal ions on elastase from a new isolate ofP. aeruginosa (Strain SES-938-1) was investigated. Crude elastase was prepared from culture supernatant via salting out by ammonium sulfate, and then desalting and concentrating the sample using a centricon microconcentrator. Activities were measured at 450 nm usingN-succinyl-l-(ala)₃-p-nitroanilide as the substrate. The metal chelating agents EDTA and EGTA inhibited thePseudomonas elastase, which shows that the enzyme is a typical metalloproteinase. At a 10-mM concentration, Mn²⁺, Ni²⁺, and Zn²⁺ strongly inhibited the elastase, whereas Mg²⁺ effect was negligable. There was a gradual decrease in the enzyme activity in accordance with an increase in the concentration of metal ions.     

Characterization of a diesel-degrading bacterium, <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> IU5, isolated from oil-contaminated soil in Korea

A diesel-degrading bacterium (strain IU5) isolated from oil-contaminated soil was characterized in this study. Fatty acid and 16s rDNA sequence analysis identified IU5 as a strain of Pseudomonas aeruginosa, and growth curve experiments identified the bacterium’s optimum conditions as pH 7 and 30 °C. P. aeruginosa IU5 degraded up to 60 of applied diesel (8500 mg/kg) over 13 days in a soil-slurry phase. In addition, this strain was able to grow on many other petroleum hydrocarbons as sole carbon sources, including crude oil, gasoline, benzene, toluene, xylene, and even PAHs such as naphthalene, phenanthrene and pyrene. Therefore, P. aeruginosa IU5 may be useful for bioremediation of soils and groundwater contaminated with a variety of hydrocarbons.     

Selective control of cyanobacteria by surfactin-containing culture broth of Bacillus subtilis C1

Of several types of chemical surfactants and biosurfactants, only the culture broth of Bacillus subtilis C1 containing surfactin at 10 mg l^–1 completely inhibited the growth of Microcystis aeruginosa, a bloom-forming cyanobacterium in highly eutrophic lakes. The broth with 10 mg surfactin l^–1 also removed 85% of the maximally grown M. aeruginosa (chlorophyll-a concentration, 1000 g l^–1) within 2 d, and the removal efficiency was enhanced by Ca²⁺ over 1 mM. The growth of Anabaena affinis, another bloom-forming cyanobacterium, was also inhibited about 70% with surfactin at 10 mg l^–1 broth. However, the effect of the broth was delayed over 3 d in the green algae, Chlorella vulgaris and Scenedesmus sp., and was negligible in a diatom, Navicula sp., indicating the potential for the selective control of cyanobacterial blooms.     

On the control of HAB species using low biosurfactant concentrations

Biosurfactants have been suggested as a method to control harmful algal blooms (HABs), but warrant further and more in-depth investigation. Here we have investigated the algicidal effect of a biosurfactant produced by the bacterium Pseudomonas aeruginosa on five diverse marine and freshwater HAB species that have not been tested previously. These include Alexandrium minutum (Dinophycaee), Karenia brevis (Dinophyceae), Pseudonitzschia sp. (Bacillariophyceae), in marine ecosystems, and Gonyostomum semen (Raphidophyceae) and Microcystis aeruginosa (Cyanophyecae) in freshwater. We examined not only lethal but also sub-lethal effects of the biosurfactant. In addition, the effect of the biosurfactant on Daphnia was tested. Our conclusions were that very low biosurfactant concentrations (5 μg mL⁻¹) decreased both the photosynthesis efficiency and the cell viability and that higher concentrations (50 μg mL⁻¹) had lethal effects in four of the five HAB species tested. The low concentrations employed in this study and the diversity of HAB genera tested suggest that biosurfactants may be used to either control initial algal blooms without causing negative side effect to the ecosystem, or to provoke lethal effects when necessary.     

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.     

Effect of alginate encapsulation and selected disinfectants on survival of and phenanthrene mineralization byPseudomonas sp UG14Lr in creosote-contaminated soil

The survival and phenanthrene-mineralizing ability of free and alginate-encapsulatedPseudomonas sp UG14Lr cells were examined in a creosote-contaminated soil. Alginate encapsulation adversely affected both survival and phenanthrene mineralization. This was postulated to be due to concentration of water-soluble toxic compounds in the alginate beads. Toxicity studies showed that the concentrated water-soluble fraction of the creosote-contaminated soil may be toxic toPseudomonas sp UG14Lr in soil with a low moisture content. Survival of alginate-encapsulated cells improved with increasing soil moisture content. Free cells survived well at a steady population of 10⁸ CFU g^–1 dry soil for 28 days in the creosote-contaminated soil. However, phenanthrene mineralization was not improved compared to the uninoculated control. This was attributed to the existence of indigenous phenanthrene-mineralizing microorganisms already present in this contaminated soil. The effect of calcium hypochlorite and Germiphene on survival of and phenanthrene mineralization by free and alginate-encapsulatedPseudomonas sp UG14Lr cells in creosote-contaminated soil was also studied. Addition of 0.1% (w/w dry soil) calcium hypochlorite reduced the introduced free cells to below detection limits (10 CFU g^–1 dry soil) within 14 days, while Germiphene had no effect on cell numbers. Phenanthrene mineralization by free cells was not adversely affected by treatment with calcium hypochlorite or Germiphene. Survival of alginate-encapsulated cells after treatment with disinfectants was as poor as that without disinfection. The results show that alginate encapsulation may not be a suitable formulation for introduction ofPseudomonas sp UG14Lr into creosote-contaminated soils.     

Chitinase-mediated destructive antagonistic potential of Pseudomonas aeruginosa GRC1 against Sclerotinia sclerotiorum causing stem rot of peanut

Pseudomonas aeruginosa GRC₁ exhibited strong antagonistic activity against Sclerotinia sclerotiorum, in vitro and in vivo. Scanning electron microscopic (SEM) studies showed morphological abnormalities such as perforation, lysis and fragmentation of hyphae of S. sclerotiorum caused by P. aeruginosa GRC₁. This strain produced extracellular chitinase enzyme, the role of which was clearly demonstrated through Tn5 mutagenesis. Bacterization of peanut seeds with GRC₁ resulted in increased seed germination and reduced stem-rot of peanut in S. sclerotiorum-infested soil by 97%. Other vegetative and yield plant parameters such as nodules per plant, pods and grain yield per plant were enhanced with a statistical significance in comparison to control. Neomycin resistant (GRC₁^neo+) bacterium was a good root colonizer and frequently isolated from rhizosphere of peanut plants. These findings showed P. aeruginosa GRC₁ as a potential biocontrol agent against S. sclerotiorum.     

Production of extracellular emulsifying agent byPseudomonas aeruginosa UG1

Summary Twenty-three bacterial strains were isolated from oil-contaminated soil samples. Of these, 20 displayed some ability to effect oil dispersion and they were screened quantitatively for the ability to emulsify 0.5% (v/v) reference oil. One strain, identified asPseudomonas aeruginosa UG1, produced extracellular material that emulsified reference oil, hexadecane and 2-methylnaphthalene at concentrations as high as 6% (v/v) in nutrient broth. Emulsification activity increased during a 10 day incubation period at 30°C. The activity was not influenced by pH over the range 5 to 9. The emulsifying agent was precipitated by cold ethanol. The highest emulsifying activity was detected in the extracellular fraction precipitated between 30 and 50% (v/v) ethanol. A linear relationship was observed between emulsifier concentration (mg/ml) and emulsifying activity. Genetic analysis showed that thePseudomonas aeruginosa UG1 strain did not carry extrachromosomal plasmids, suggesting that the gene(s) coding for emulsifying activity was carried on the chromosome.     

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.     

N2-Succinylornithine in ornithine catabolism of Pseudomonas aeruginosa

Most Pseudomonas aeruginosa PAO mutants which were unable to utilize l-arginine as the sole carbon and nitrogen source (aru mutants) under aerobic conditions were also affected in l-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N²-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N²-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N²-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase (l-ornithine:succinyl-CoA N²-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.Abbreviations OAT ornithine 5-aminotransferase - SOAT N²-succinylornithine 5-aminotransferase - Oru ornithine utilization - Aru arginine utilization     

The effects of oxalates produced by Salsola tragus on the phosphorus nutrition of Stipa pulchra

Oxalic acid is produced by some species of plants and mycorrhizal fungi and it may solubilize unavailable soil phosphorus (P) bound by cations (Ca⁺⁺, Al⁺⁺, Fe⁺⁺⁺). Field and greenhouse experiments were conducted to show whether oxalate produced by the annual Salsola tragus or added oxalic acid would solubilize P from the inorganic-bound soil P pool, making it available for uptake by Stipa pulchra. Oxalate could be leached in the laboratory from the senescent canopy of Salsola, and leaching by rainfall was hypothesized to be a source of potential increased soil P under the Salsola canopy. Both oxalate leached from the canopy of Salsola and added oxalic acid increased the availability of soil P in greenhouse experiments. The source of the increase in available soil P in the greenhouse experiment was shown to be the inorganic-bound P pool, as the total P concentration of the soil decreased with increasing oxalate. There were significant increases in Stipa shoot P in response to Salsola leachates and in response to added oxalate in the greenhouse studies. These results suggest an important role for oxalate in P cycling. On disturbed sites where Salsola invades it may act to facilitate the establishment of later seral species like Stipa by creating a nutrient island of available P.     

Algicidal activity of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa

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