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.     

Simultaneous hydrocarbon biodegradation and biosurfactant production by oilfield-selected bacteria

Aims: To study the bacterial diversity associated with hydrocarbon biodegradation potentiality and biosurfactant production of Tunisian oilfields bacteria. Methods and Results: Eight Tunisian hydrocarbonoclastic oilfields bacteria have been isolated and selected for further characterization studies. Phylogenetic analysis revealed that three thermophilic strains belonged to the genera Geobacillus, Bacillus and Brevibacillus, and that five mesophilic strains belonged to the genera Pseudomonas, Lysinibacillus, Achromobacter and Halomonas. The bacterial strains were cultivated on crude oil as sole carbon and energy sources, in the presence of different NaCl concentrations (1, 5 and 10%, w/v), and at 37 or 55°C. The hydrocarbon biodegradation potential of each strain was quantified by GC–MS. Strain C450R, phylogenetically related to the species Pseudomonas aeruginosa, showed the maximum crude oil degradation potentiality. During the growth of strain C450R on crude oil (2%, v/v), the emulsifying activity (E24) and glycoside content increased and reached values of 77 and 1·33 g l^?1, respectively. In addition, the surface tension (ST) decreased from 68 to 35·1 mN m^?1, suggesting the production of a rhamnolipid biosurfactant. Crude biosurfactant had been partially purified and characterized. It showed interest stability against temperature and salinity increasing and important emulsifying activity against oils and hydrocarbons. Conclusions: The results of this study showed the presence of diverse aerobic bacteria in Tunisian oilfields including mesophilic, thermophilic and halotolerant strains with interesting aliphatic hydrocarbon degradation potentiality, mainly for the most biosurfactant produced strains. Significance and Impact of the Study: It may be suggested that the bacterial isolates are suitable candidates for practical field application for effective in situ bioremediation of hydrocarbon‐contaminated sites.     

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.     

The Potential of Bacterial Isolates for Emulsification with a Range of Hydrocarbons

A study was undertaken to investigate the distribution of biosurfactant producing and crude oil degrading bacteria in the oil contaminated environment. This research revealed that hydrocarbon contaminated sites are the potent sources for oil degraders. Among 32 oil degrading bacteria isolated from ten different oil contaminated sites of gasoline and diesel fuel stations, 80% exhibited biosurfactant production. The quantity and emulsification activity of the biosurfactants varied. Pseudomonas sp. DS10‐129 produced a maximum of 7.5 ± 0.4 g/l of biosurfactant with a corresponding reduction in surface tension from 68 mN/m to 29.4 ± 0.7 mN/m at 84 h incubation. The isolates Micrococcus sp. GS2‐22, Bacillus sp. DS6‐86, Corynebacterium sp. GS5‐66, Flavobacterium sp. DS5‐73, Pseudomonas sp. DS10‐129, Pseudomonas sp. DS9‐119 and Acinetobacter sp. DS5‐74 emulsified xylene, benzene, n‐hexane, Bombay High crude oil, kerosene, gasoline, diesel fuel and olive oil. The first five of the above isolates had the highest emulsification activity and crude oil degradation ability and were selected for the preparation of a mixed bacterial consortium, which was also an efficient biosurfactant producing oil emulsifying and degrading culture. During this study, biosurfactant production and emulsification activity were detected in Moraxella sp., Flavobacterium sp. and in a mixed bacterial consortium, which have not been reported before.     

Rhamnolipid Produced from Agroindustrial Wastes Enhances Hydrocarbon Biodegradation in Contaminated Soil

A crude biosurfactant solution was produced by Pseudomonas aeruginosa growing on agroindustrial wastes as the substrate and used to study its effect on hydrocarbon biodegradation by the indigenous soil microflora under laboratory conditions. Two concentrations were studied at first and 1 mg of biosurfactant/g of soil showed to be the most efficient for the total petroleum hydrocarbon reduction, which reached 85% at the first 20 days in soil microcosms. Respirometric and microbial analyses showed that the biosurfactant added did not have toxic effects over the microbial population. The use of a biosurfactant for bioremediation has been limited because of its high cost production. Biosurfactants produced from cost-free by-products combines waste minimization with economic potential bioremediation process.     

Biosurfactant production by Corynebacterium kutscheri from waste motor lubricant oil and peanut oil cake

AIM: Production and characterization of biosurfactant from renewable sources. METHODS AND RESULTS: Biosurfactant production was carried out in 3-l fermentor using waste motor lubricant oil and peanut oil cake. Maximum biomass (9.8 mg ml(-l)) and biosurfactant production (6.4 mg ml(-l)) occurred with peanut oil cake at 120 and 132 h, respectively. Chemical characterization of the biosurfactant revealed that it is a glycolipopeptide with chemical composition of carbohydrate (40%), lipid (27%) and protein (29%). The biosurfactant is able to emulsify waste motor lubricant oil, crude oil, peanut oil, kerosene, diesel, xylene, naphthalene and anthracene; the emulsification activity was comparatively higher than the activity found with Triton X-100. CONCLUSION: This study indicates the possibility of biosurfactant production using renewable, relatively inexpensive and easily available resources like waste motor lubricant oil and peanut oil cake. Emulsification activity found with the biosurfactant against different hydrocarbons showed the possibility of the application of biosurfactants against diverse hydrocarbon pollution. SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained from the study could be useful for large-scale biosurfactant production using economically cheaper substrates. Information obtained in emulsification activity and laboratory-scale experiment on bioremediation inferred that bioremediation of hydrocarbon-polluted sites may be treated with biosurfactants or the bacteria that produces it.     

Biosurfactant Production by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> from Renewable Resources

This study deals with production and characterization of biosurfactant from renewable resources by Pseudomonas aeruginosa. Biosurfactant production was carried out in 3L fermentor using waste motor lubricant oil and peanut oil cake. Maximum biomass (11.6 mg/ml) and biosurfactant production (8.6 mg/ml) occurred with peanut oil cake at 120 and 132 h respectively. Characterization of the biosurfactant revealed that, it is a lipopeptide with chemical composition of protein (50.2%) and lipid (49.8%). The biosurfactant (1 mg/ml) was able to emulsify waste motor lubricant oil, crude oil, peanut oil, kerosene, diesel, xylene, naphthalene and anthracene, comparatively the emulsification activity was higher than the activity found with Triton X-100 (1 mg/ml). Results obtained in the present study showed the possibility of biosurfactant production using renewable, relatively inexpensive and easily available resources. Emulsification activity found with the biosurfactant against different hydrocarbons showed its possible application in bioremediation of environments polluted with various hydrocarbons.     

Effect of biosurfactant and fertilizer on biodegradation of crude oil by marine isolates of Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa

This study was conducted to investigate the effects of fertilizers and biosurfactants on biodegradation of crude oil by three marine bacterial isolates; Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa. Five sets of experiments were carried out in shake flask and microcosm conditions with crude oil as follows: Set 1-only bacterial cells added (no fertilizer and biosurfactant), Set 2-with additional fertilizer only, Set 3-with additional biosurfactant only, Set 4-with added biosurfactant + fertilizer, Set 5-with no bacterial cells added (control), all the above experimental sets were incubated for 168 h. The biosurfactant + fertilizer added Set 4, resulted in maximum crude oil degradation within shake flask and microcosm conditions. Among the three bacterial isolates, P. aeruginosa and biosurfactant produced by this strain resulted in maximum crude oil degradation compared to the other two bacterial strains investigated. Interestingly, when biosurfactant and bacterial cells were used (Set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in Set 4 with added fertilizer + biosurfactant were only 4-5% higher degradation level in shake flask and 3.2-7% in microcosm experiments for all three bacterial strains used. It is concluded that, biosurfactants alone capable of promoting biodegradation to a large extent without added fertilizers, which will reduce the cost of bioremediation process and minimizes the dilution or wash away problems encountered when water soluble fertilizers used during bioremediation of aquatic environments.     

Physiological aspects of hydrocarbon emulsification, metal resistance and DNA profile of biodegrading bacteria isolated from oil polluted sites

The production of biosurfactants was evaluated for seven bacterial strains isolated from different oil contaminated sites by the Emulsification Index using diesel oil as the hydrocarbon source. Minimum Inhibitory Concentrations of Mg²⁺, Cr³⁺ and Cu²⁺ were determined to identify the less sensitive bacteria in order to select the best strains for bioremediation. Plasmid extraction was also performed in order to search for gene sequences involved with biosurfactant synthesis. All strains were able to emulsify diesel oil. Rhodococcus ruber AC239 presented the best index (58%), followed by other Rhodococcus strains. Pseudomonas aeruginosa, R. ruber AC239, AC87 and R. erytropolis AC272 presented smallest sensitivities to heavy metals used, being suitable for use in sites contaminated with high concentrations of them. No plasmid DNA was detected showing that biosurfactant coding genes should be in the chromosomal DNA.     

Isolation and characterization of Halomonas sp. strain C2SS100, a hydrocarbon-degrading bacterium under hypersaline conditions

Aims:  To isolate and characterize an efficient hydrocarbon-degrading bacterium under hypersaline conditions, from a Tunisian off-shore oil field.
Methods and Results:  Production water collected from 'Sercina' petroleum reservoir, located near the Kerkennah island, Tunisia, was used for the screening of halotolerant or halophilic bacteria able to degrade crude oil. Bacterial strain C2SS100 was isolated after enrichment on crude oil, in the presence of 100 g l⁻¹ NaCl and at 37°C. This strain was aerobic, Gram-negative, rod-shaped, motile, oxidase + and catalase +. Phenotypic characters and phylogenetic analysis based on the 16S rRNA gene of the isolate C2SS100 showed that it was related to members of the Halomonas genus. The degradation of several compounds present in crude oil was confirmed by GC–MS analysis. The use of refined petroleum products such as diesel fuel and lubricating oil as sole carbon source, under the same conditions of temperature and salinity, showed that significant amounts of these heterogenic compounds could be degraded. Strain C2SS100 was able to degrade hexadecane (C₁₆). During growth on hexadecane, cells surface hydrophobicity and emulsifying activity increased indicating the production of biosurfactant by strain C2SS100.
Conclusions:  A halotolerant bacterial strain Halomonas sp. C2SS100 was isolated from production water of an oil field, after enrichment on crude oil. This strain is able to degrade hydrocarbons efficiently. The mode of hydrocarbon uptake is realized by the production of a biosurfactant which enhances the solubility of hydrocarbons and renders them more accessible for biodegradation.
Significance and Impact of the Study:  The biodegradation potential of the Halomonas sp. strain C2SS100 gives it an advantage for possibly application on bioremediation of water, hydrocarbon-contaminated sites under high-salinity level.     

Utilization of Drilling Fluid Base Oil Hydrocarbons by Microorganisms Isolated from Diesel-Polluted Soil

Hydrocarbon-degrading microorganisms identified as Pseudomonas luteola, Pseudomonas alcaligenes, Pseudomonas aeruginosa, and Actinomyces sp. were isolated from diesel oil-polluted soils using an enrichment culture technique. The isolates grew luxuriantly on hydrocarbons, including crude oil, diesel, kerosene, engine oil, cyclohexane, and dodecanol. Naphthalene and pyrene were poorly utilized, while there was no growth on benzene. The organisms utilized drilling fluid base oil as the sole source of carbon and energy, with rapid exponential growth at a rate ranging from 0.015 to 0.094 h^?1. The concomitant doubling time was between 7.4 and 45.5 h. Gas chromatographic analyses of the culture revealed reduction in the height of the n-alkane peaks, confirming biodegradation of the compounds. Among the isolates, P. alcaligenes had the highest (99.4%) percentage hydrocarbon degradation. Remarkable (99.2% and 98.7%) hydrocarbon removal was also noted for P. luteola and P. aeruginosa, while the lowest (92.3%) value was recorded in Actinomyces sp. These bacteria with high degradative capacity for hydrocarbons in oil-based drilling fluids would be useful in bioremediation of a tropical environment, polluted with spent drilling mud and drill cuttings.     

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.     

Biosurfactant-Mediated Biodegradation of Polycyclic Aromatic Hydrocarbons—Naphthalene

The present study is aimed at the naphthalene degradation with and without biosurfactant produced from Pseudomonas aeruginosa isolated from oil-contaminated soil. The present study was carried out to isolate the bacterial strains for the naphthalene degradation and also for biosurfactant production. The isolated strains were screened for their ability to degrade the naphthalene by the methods of optimum growth rate test and for the production of biosurfactants by cetyltrimethylammonium bromide, blood agar medium, and thin-layer chromatography. The present study also focused on the effect of biosurfactant for the degradation of naphthalene by isolate-1. Two bacterial strains were isolated and screened, one for biodegradation and another for biosurfactant production. The second organism was identified as Pseudomonas aeruginosa by 16S rRNA analysis. The purified biosurfactant reduces the surface tension of water and also forms stable emulsification with hexadecane and kerosene. The end product of naphthalene degradation was estimated as salicylic acid equivalent by spectrophotometric method. The results demonstrated that Pseudomonas aeruginosa has the potential to produce biosurfactant, which enhances the biodegradation of naphthalene. The study reflects the potential use of biosurfactants for an effective bioremediation in the management of contaminated soils.     

Biosurfactant Production by Azotobacter chroococcum Isolated from the Marine Environment

Preliminary characterization of a biosurfactant-producing Azotobacter chroococcum isolated from marine environment showed maximum biomass and biosurfactant production at 120 and 132 h, respectively, at pH 8.0, 38°C, and 30‰ salinity utilizing a 2% carbon substrate. It grew and produced biosurfactant on crude oil, waste motor lubricant oil, and peanut oil cake. Peanut oil cake gave the highest biosurfactant production (4.6 mg/mL) under fermentation conditions. The biosurfactant product emulsified waste motor lubricant oil, crude oil, diesel, kerosene, naphthalene, anthracene, and xylene. Preliminary characterization of the biosurfactant using biochemical, Fourier transform infrared spectroscopy, and mass spectral analysis indicated that the biosurfactant was a lipopeptide with percentage lipid and protein proportion of 31.3:68.7.     

Inoculation of 1-aminocyclopropane-1-carboxylate deaminase–producing bacteria along with biosurfactant application enhances the phytoremediation efficiency of Medicago sativa in hydrocarbon-contaminated soils

Phytoremediation efficiency of Alfa alfa (Medicago sativa) was evaluated in hydrocarbon-contaminated soil with the combined application of 1-aminocyclopropane-1-carboxylate (ACC) deaminase–producing Bacillus sp. PVMX4 and an isolated biosurfactant from this strain. Results on the plant growth–promoting (PGP) traits of Bacillus sp. PVMX4 revealed that phosphate (P) solubilization, indole-3-acetic acid (IAA) production, and ACC deaminase activity were not affected by low-concentration hydrocarbon amendment in the form of crude oil. Bacillus sp. PVMX4 was able to utilize crude oil as a sole carbon source in mineral salt medium (MSM), and this strain synthesized significant quantities of biosurfactant in growth medium quantified by an emulsification index of 69.2 EI_24% and surface tension reduction of 26.2 mN/m at the end of the experimental period. Biosurfactant, when partially purified and characterized by thin-layer chromatography (TLC) and Fourier transform infrared spectroscopy (FT-IR), revealed it to be a lipopeptide-type biosurfactant. Pilot-scale phytoremediation studies conducted under growth chamber conditions in hydrocarbon-contaminated soil using Medicago sativa along with combined application of ACC deaminase–containing bacteria and biosurfactant recorded 76.4% hydrocarbon degradation.     

Biosurfactant Production by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SNP0614 and its Effect on Biodegradation of Petroleum

An efficient biosurfactant-producing strain was isolated and cultured from Dagang oil field (China) using crude oil as sole source of carbon. Based on partial sequenced 16S rDNA analysis, the isolated strain was identified as Pseudomonas aeruginosa SNP0614. The bacterium P. aeruginosa SNP0614 produced a type of biosurfactant with excessive foam-forming properties. After microbial cultivation at 37°C and 150 rpm for 12 h, the produced biosurfactant was found to reduce the surface tension to 25.4 mN/m with critical micelle concentration (CMC) of 45.0 mg/L. After 20 days of incubation, the biosurfactant exhibited 90% emulsification activity (E₂₄) on crude oil. FTIR spectroscopy of extracted biosurfactant indicated the biosurfactant as lipopeptide. The significant synergistic effect between P. aeruginosa SNP0614 and the mixed oildegrading bacteria resulted in increasing n-alkanes degradation rate by 30%. The strain P. aeruginosa SNP0614 represented as a promising biosurfactant producer and could be applied in a variety of biotechnological and industrial processes, particularly in microbial enhanced oil recovery and the bioremediation of oil pollution.     

Production and characterization of a glycolipid biosurfactant from Bacillus megaterium using economically cheaper sources

Criteria selected for screening of biosurfactant production by Bacillus megaterium were hemolytic assay, bacterial cell hydrophobicity and the drop-collapse test. The data on hemolytic activity, bacterial cell adherence with crude oil and the drop-collapse test confirmed the biosurfactant-producing ability of the strain. Accordingly, the strain was cultured at different temperatures, pH values, salinity and substrate (crude oil) concentration in mineral salt medium to establish the optimum culture conditions, and it was shown that 38°C, 2.0% of substrate concentration, pH 8.0 and 30‰ of salt concentration were optimal for maximum growth and biosurfactant production. Laboratory scale biosurfactant production in a fermentor was done with crude oil and cheaper carbon sources like waste motor lubricant oil and peanut oil cake, and the highest biosurfactant production was found with peanut oil cake. Characterization of partially purified biosurfactant inferred that it was a glycolipid with emulsification potential of waste motor lubricant oil, crude oil, peanut oil, diesel, kerosene, naphthalene, anthracene and xylene.     

Potential for biodegradation of hydrocarbons by microorganisms isolated from Antarctic soils

Seventeen pure aerobic microbial isolates were obtained from soil samples of three regions of Antarctica: Casey Station, Dewart Island and Terra Nova Bay. Most of them were gram positive coryneform bacteria. Isolates were tested for their ability to grow on mineral salt agar plates supplemented with one of the following model n-alkanes or aromatic hydrocarbons: hexane, heptane, paraffin, benzene, toluene, naphthalene and kerosene. Cell hydrophobicity, the ability to produce anionic glycolipids and extracellular emulsifying activity were also determined and assessed on the basis of growth of soil isolates on hydrocarbons. This study revealed degraders with broader abilities to grow on both types of hydrocarbons, good production of glycolipids and emulsifying activity. On this basis, a mixed culture of strains is proposed, which may find application for bioremediation at temperate temperature of soil environments polluted with different hydrocarbons.     

Lysinibacillus sphaericus proved to have potential for the remediation of petroleum hydrocarbons

In this study, the ability of Lysinibacillus sphaericus to degrade aromatic hydrocarbons as well as complex hydrocarbon mixtures, such as diesel oil and oily sludge, was evaluated. L. sphaericus was able to grow when toluene, naphthalene, or phenanthrene were used as a sole carbon source in minimal salt medium. Removal efficiencies of up to 95% were found for C₁₀-C₂₈ hydrocarbons in the biodegradation assays of diesel oil. The biodegradation of oily sludge was evaluated in landfarming-like experiments in the open air and in completely covered containers in the field. After 50 days of treatment, the removal efficiency of total petroleum hydrocarbons in open-air and closed assays was of 84.1% and 60.1%, respectively. Furthermore, L. sphaericus was able to degrade volatile hydrocarbons (benzene, toluene, ethylbenzene, and phenol) in the headspace of closed containers, preventing the emission of these compounds to the atmosphere. L. sphaericus was herein proposed as a promising candidate to be used in bioremediation strategies of petroleum hydrocarbons.     

Isolation and characterization of hydrocarbon-degrading and biosurfactant-producing yeast strains obtained from a polluted lagoon water

Microbial surfactants are environmentally friendly products with amazing properties and spectrum of applications. It is therefore, not surprising that research has increased in recent time with the objectives of sourcing for novel surface-active compounds with dual functions in oil and pharmaceutical industries. Evaluation of hydrocarbon degrading potentials and emulsifying activities indicated that biosurfactants were produced by two newly isolated and promising yeast strains, Saccharomyces cerevisiae and Candida albicans, obtained from a polluted lagoon water. Both strains were able to grow effectively on crude oil and diesel as sole sources of carbon and energy. Growth curves on diesel were obtained to establish the relation between cell growth and biosurfactant production. The growth peak was on the 8th day while the specific growth rate ranged insignificantly (P < 0.05) between 0.46 and 0.48 day⁻¹. Interestingly, biosurfactant was detected on the 2nd day when growth was almost inexistent, with maximal production obtained at stationary/death phase of growth. The partially-purified biosurfactants exhibited antimicrobial activities by completely inhibiting the growth of clinical strains of Escherichia coli and Staphylococcus aureus at all concentrations tested. Although C. albicans appeared to be a better diesel-utilizer and biosurfactant-producer (E₂₄ = 64.2%), the potency of its surfactant was smaller than that of S. cerevisiae. These strains represent a new class of biosurfactant producers that have potential for use in a variety of biotechnological and industrial processes particularly in the pharmaceutical industry.