Efficiency of the EPS emulsifier produced by <Emphasis Type="Italic">Ochrobactrum anthropi</Emphasis> in different hydrocarbon bioremediation assays

Ochrobactrum anthropi strain AD2 was isolated from the waste water treatment plant of an oil refinery and was identified by analysis of the sequence of the gene encoding 16S rDNA. This bacterium produced exopolysaccharides in glucose nutrient broth media supplemented with various hydrocarbons (n-octane, mineral light and heavy oils and crude oils). The exopolysaccharide AD2 (EPS emulsifier) synthesized showed a wide range of emulsifying activity but none of them had surfactant activity. Yield production varied from 0.47 to 0.94 g of EPS l⁻¹ depending on the hydrocarbon added. In the same way, chemical composition and emulsification activity of EPS emulsifier varied with the culture conditions. Efficiency of the EPS emulsifier as biostimulating agent was assayed in soil microcosms and experimental biopiles. The AD2 biopolymer was added alone or combined with commercial products frequently used in oil bioremediation such as inorganic NPK fertilizer and oleophilic fertilizer (S200 C). Also, its efficiency was tested in mixture with activated sludge from an oil refinery. In soil microcosms supplemented with S200 C + EPS emulsifier as combined treatment, indigenous microbial populations as well as hydrocarbon degradation was enhanced when compared with microcosms treated with NPK fertilizer or EPS emulsifier alone. In the same way EPS emulsifier stimulated the bioremediation effect of S200 C product, increasing the number of bacteria and decreasing the amount of hydrocarbon remained. Finally, similar effects were obtained in biopile assays amended with EPS emulsifier plus activated sludge. Our results suggest that the bioemulsifier EPS emulsifier has interesting properties for its application in environment polluted with oil hydrocarbon compounds and may be useful for bioremediation purposes.     

Production of an extracellular polysaccharide with emulsifier properties by Penicillium citrinum

Penicillium citrinum produced a glycolipid with emulsifier properties during cultivation on mineral medium with 1% (v/v) olive oil as carbon source. The emulsifier production was growth-associated and reached maximal activity at 60 h of cultivation. The production yield (Y _p/s) was 0.54 and the best emulsifying activity was observed for xylene and diesel oil when compared to other carbohydrates tested. The emulsifier was shown to be stable to a wide range of pH and temperature values and was shown to contain D-galactose, D-glucose and D-xylose (8.2:1.0:5.3) with a total carbohydrate content of 43%. The presence of salts stimulated the emulsification activity, suggesting potential for its application in industrial waste or marine remediation.     

The potential for hydrocarbon biodegradation and production of extracellular polymeric substances by aerobic bacteria isolated from a Brazilian petroleum reservoir

Extracellular polymeric substances (EPS) can contribute to the cellular degradation of hydrocarbons and have a huge potential for application in biotechnological processes, such as bioremediation and microbial enhanced oil recovery (MEOR). Four bacterial strains from a Brazilian petroleum reservoir were investigated for EPS production, emulsification ability and biodegradation activity when hydrocarbons were supplied as substrates for microbial growth. Two strains of Bacillus species had the highest EPS production when phenanthrene and n-octadecane were offered as carbon sources, either individually or in a mixture. While Pseudomonas sp. and Dietzia sp., the other two evaluated strains, had the highest hydrocarbon biodegradation indices, EPS production was not detected. Low EPS production may not necessarily be indicative of an absence of emulsifier activity, as indicated by the results of a surface tension reduction assay and emulsification indices for the strain of Dietzia sp. The combined results gathered in this work suggest that a microbial consortium consisting of bacteria with interdependent metabolisms could thrive in petroleum reservoirs, thus overcoming the limitations imposed on each individual species by the harsh conditions found in such environments.     

Isolation of a bioemulsifier from Candida lipolytica

The yeast Candida lipolytica produced an inducible extracellular emulsification activity when it was grown with a number of water-immiscible carbon substrates. Negligible emulsification activity was produced by this yeast when it was grown with glucose as the carbon substrate. In hexadecane-supplemented cultures, emulsification activity was first detected after 36 h of growth, with maximum production after 130 h. A water-soluble emulsification activity was partially purified by repeated solvent extractions of the culture filtrate. This emulsifier, which we named liposan, was primarily composed of carbohydrate. Maximum emulsification activity was obtained when the ratio of hexadecane to liposan was 50:1. Maximum emulsification activity was obtained from pH 2 to 5. Liposan was heat stable to temperatures up to 70 degrees C, with a 60% loss in activity after heating for 1 h at 100 degrees C. Liposan effected stable oil-in-water emulsions with a variety of hydrocarbons.     

Isolation of a bioemulsifier from Candida lipolytica.

The yeast Candida lipolytica produced an inducible extracellular emulsification activity when it was grown with a number of water-immiscible carbon substrates. Negligible emulsification activity was produced by this yeast when it was grown with glucose as the carbon substrate. In hexadecane-supplemented cultures, emulsification activity was first detected after 36 h of growth, with maximum production after 130 h. A water-soluble emulsification activity was partially purified by repeated solvent extractions of the culture filtrate. This emulsifier, which we named liposan, was primarily composed of carbohydrate. Maximum emulsification activity was obtained when the ratio of hexadecane to liposan was 50:1. Maximum emulsification activity was obtained from pH 2 to 5. Liposan was heat stable to temperatures up to 70 degrees C, with a 60% loss in activity after heating for 1 h at 100 degrees C. Liposan effected stable oil-in-water emulsions with a variety of hydrocarbons.     

Hydrocarbon emulsification and enhanced crude oil degradation by lauroyl glucose ester

Enzymatically synthesized lauroyl glucose emulsified different hydrophobic substrates when assayed spectrophotometrically. Stable emulsions were formed with triglycerides as well as with hydrocarbons. There was a linear relation between the concentration of lauroyl glucose (50-450 microg) and emulsification activity under the assay conditions when tested with aromatic and aliphatic hydrocarbons. This sugar ester was able to emulsify the aromatic hydrocarbons benzene, toluene and xylene. Long chain alkanes (n-decane and n-hexadecane) as well as brominated long chain alkanes (1-bromodecane and 1-bromohexadecane) were efficiently emulsified. The effect of lauroyl glucose ester on degradation of crude oil by a known oil-degrading Rhodococcus species was also investigated. The culture showed enhanced degradation of crude oil when lauroyl glucose ester was used as an emulsifier. It degraded 70% of the aliphatic fraction of Bombay High crude oil in the presence of the sugar ester at a concentration of 200mg l(-1) as compared to 50% without the emulsifier.     

Cell surface properties of five polycyclic aromatic compound-degrading yeast strains

To investigate the effects of physiological properties on polycyclic aromatic compound (PAH) degradation, the surface tension and emulsification activities, and cell surface hydrophobicity of five PAH-degrading yeast isolates were compared to Saccharomyces cerevisiae from cultures grown with glucose, hexadecane, or naphthalene as carbon sources. The cell surface hydrophobicity values for the five yeast strains were significantly higher than for S. cerevisiae for all culture conditions, although these were highest with hexadecane and naphthalene. Strains with higher hydrophobicity showed higher rates of naphthalene and phenanthrene degradation, indicating that increased cell hydrophobicity might be an important strategy in PAH degradation for the five strains. Emulsification activities increased for all five yeast strains with naphthalene culturing, although no relationship existed between emulsification activity and PAH degradation rate. Surface tensions were not markedly reduced with naphthalene culturing.     

Bioemulsifier production by Bacillus stearothermophilus VR-8 isolate

Bacillus stearothermophilus produced an extracellular bioemulsifier during growth in a medium containing 4% crude oil. Over the temperature range of 45° to 70°C, maximum recovery (0·6 g 1^-1) occurred at 50°C. The emulsifier had its greatest activity on benzene, among the hydrocarbons tested. Acetone precipitated, dialysed emulsifier contained 46% protein, 16% carbohydrate and 10% lipid. The emulsification activity was stable over a broad range of temperature (50–80°C), pH (2–8) and salt concentration (5% NaCl, 5% CaCl₂ and 1% MgCl₂). Thus, this emulsifier was found to be better than liposan (showing emulsifying activity between pH 2–5 and stable up to 70°C) in terms of pH and temperature stability. Additionally, it was also salt tolerant, suggesting its potential use in crude oil tank clean-up and enhanced oil recovery.     

Production of an extracellular emulsifier by a gram negative bacterium

Summary Gram negative bacterial isolated PCB-106 produced significant amounts of bioemulsifiers in the late log phase of growth during the shake cultivation in minimal medium with ethanol (115 units/ml). About 3 fold higher emulsification activity was achieved by manipulating the medium components. The emulsifier was found to be more effective towards the mixture of aliphatic and aromatic than individual hydrocarbons.     

银耳粗多糖对桉叶油的乳化性研究

对不同浓度、温度、pH、NaCl浓度条件下,银耳粗多糖对桉叶油的乳化能力,以及乳化体系的稳定性进行了研究。结果表明,银耳粗多糖对桉叶油的乳化性随浓度增大上升,在浓度1%时达到最大,随后呈下降趋势;在pH 4.0~8.0之间;有较好的乳化性及乳化稳定性,在此范围以外,随pH的升高、降低,银耳粗多糖的乳化和乳化性均呈下降趋势;随着温度和NaCl浓度升高,银耳粗多糖的乳化活性和乳化稳定性有所降低。     

The mannoprotein of Saccharomyces cerevisiae is an effective bioemulsifier.

The mannoprotein which is a major component of the cell wall of Saccharomyces cerevisiae is an effective bioemulsifier. Mannoprotein emulsifier was extracted in a high yield from whole cells of fresh bakers' yeast by two methods, by autoclaving in neutral citrate buffer and by digestion with Zymolase (Miles Laboratories; Toronto, Ontario, Canada), a beta-1,3-glucanase. Heat-extracted emulsifier was purified by ultrafiltration and contained approximately 44% carbohydrate (mannose) and 17% protein. Treatment of the emulsifier with protease eliminated emulsification. Kerosene-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 5% sodium chloride or up to 50% ethanol in the aqueous phase. In the presence of a low concentration of various solutes, emulsions were stable to three cycles of freezing and thawing. An emulsifying agent was extracted from each species or strain of yeast tested, including 13 species of genera other than Saccharomyces. Spent yeast from the manufacture of beer and wine was demonstrated to be a possible source for the large-scale production of this bioemulsifier.     

The mannoprotein of Saccharomyces cerevisiae is an effective bioemulsifier

The mannoprotein which is a major component of the cell wall of Saccharomyces cerevisiae is an effective bioemulsifier. Mannoprotein emulsifier was extracted in a high yield from whole cells of fresh bakers' yeast by two methods, by autoclaving in neutral citrate buffer and by digestion with Zymolase (Miles Laboratories; Toronto, Ontario, Canada), a beta-1,3-glucanase. Heat-extracted emulsifier was purified by ultrafiltration and contained approximately 44% carbohydrate (mannose) and 17% protein. Treatment of the emulsifier with protease eliminated emulsification. Kerosene-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 5% sodium chloride or up to 50% ethanol in the aqueous phase. In the presence of a low concentration of various solutes, emulsions were stable to three cycles of freezing and thawing. An emulsifying agent was extracted from each species or strain of yeast tested, including 13 species of genera other than Saccharomyces. Spent yeast from the manufacture of beer and wine was demonstrated to be a possible source for the large-scale production of this bioemulsifier.     

Studies on bioemulsifier production by Acinetobacter strains isolated from healthy human skin

AIMS: In recent years, interest has been growing in the search for novel bioemulsifiers. Many bacterial genera including Acinetobacter have been reported to produce bioemulsifiers. The present study aims to screen Acinetobacter isolates from healthy human skin for bioemulsifier production. Methods and Results: Acinetobacter junii SC14 produced maximum bioemulsifier in the presence of almond oil during stationary growth phase at 37 degrees C and pH 7.2. Partially purified, nondialysable bioemulsifier from SC14 was a proteoglycan. The protein and polysaccharide fractions resulted in 95.2% reconstitution of the emulsification activity. The role of esterase in the release of cell-bound emulsifier and the contribution of capsular polysaccharide to the emulsification activity were observed. CONCLUSION: Acinetobacter strains from human skin exhibited better emulsification activity than that by burn wound or soil isolates, owing to the inherent differences in chemical microenvironment of their habitats. SIGNIFICANCE AND IMPACT OF THE STUDY: Investigation of skin commensals, especially acinetobacters, would lead to the discovery of novel bioemulsifiers with interesting properties. Attempts of screening and strain improvement directed towards skin commensals will open up new avenues for strains producing bioemulsifier on a commercial scale.     

Characterization of hydrocarbon emulsification and solubilization occurring during the growth of Endomycopsis lipolytica on hydrocarbons

A comparative characterization of the phenomena of hydrocarbon emulsification and solubilization taking place during the growth of Endomycopsis lipolytica on n-alkanes and alkenes was made. Evidence was obtained for the cellular production of different factors involved in emulsification and solubilization of hydrocarbons. It was shown that the production of these factors closely followed cell growth. The inducible nature of the alkane solubilizing factor was demonstrated using actidione as inhibitor. Whereas emulsifying factor was demonstrated using actidione as inhibitor. Whereas emulsifying factors showed a broad affinity to some particular hydrocarbons, solubilizing factor was found to be highly specific for the particular hydrocarbon on which the cells were grown. The emulsifying factor was heat stable whereas the solubilizing factor was highly unstable even at ?4°C. Metal-ion chelating agents strongly inhibited the activity of both of the factors. A crude isolate of the alkane emulsifying factor was obtained and its peptide characteristics were demonstrated. Using EDTA as an inhibitor for the emulsification–solubilization activity, evidence was obtained for the predominent role played by the emulsification–solubilization mechanism in the uptake of alkane by yeast cells.     

Preparation and in vivo toxicity study of solid lipid microparticles as carrier for pulmonary administration

The purpose of this research was to investigate the effects of processing conditions on the characteristics of solid lipid microparticles (SLM) with a potential application as carriers for pulmonary administration. Compritol (5.0% wt/wt) SLM dispersions were prepared by rotor-stator homogenization, at different surfactant concentrations and emulsification times. The SLM were characterized, in terms of morphology and size, after lyophilization and sterilization by autoclaving process. In vivo assessment was carried out in rats by intratracheal instillation of either placebo or SLM dispersion, and by bronchoalveolar lavage for cytological analysis. Mean particle size of 4 to 5 μm was achieved using 0.3% and 0.4% (wt/wt) of emulsifier (Poloxamer 188) and emulsification times of 2 and 5 minutes. The particles showed spherical shape and smooth surface. The morphology of microparticles, the size, and the size distribution were not substantially modified after lyophilization and sterilization. Total cell counts showed no significant differences between placebo and SLM 0.5% or 2.5% groups. Regarding cytology, percentage of polymorphonuclear neutrophils and macrophages did not significantly differ between groups. These results suggest that a single intratracheal administration of the SLMs does not induce a significant inflammatory airway response in rats and that the SLMs might be a potential carrier for encapsulated drug via the pulmonary route.     

Enhancement of emulsifier production by <Emphasis Type="Italic">Curvularia lunata</Emphasis> in cadmium,zinc and lead presence

The influence of cadmium, zinc and lead on fungal emulsifier synthesis and on the growth of filamentous fungus Curvularia lunata has been studied. Tolerance to heavy metals established for C. lunata was additionally compared with the sensitivity exhibited by strains of Curvularia tuberculata and Paecilomyces marquandii—fungi which do not secrete compounds of emulsifying activity. Although C. lunata, as the only one out of all studied fungi, exhibited the lowest tolerance to heavy metals when grown on a solid medium (in conditions preventing emulsifier synthesis), it manifested the highest tolerance in liquid culture - in conditions allowing exopolymer production. Cadmium, zinc and lead presented in liquid medium up to a concentration of 15 mM had no negative effect on C. lunata growth and stimulated emulsifier synthesis. In the presence of 15 mM of heavy metals, both the emulsifier and 24-h-old growing mycelium exhibited maximum sorption capacities, which were determined as 18.2 ± 2.67, 156.1 ± 10.32 mg g⁻¹ for Cd²⁺, 22.2 ± 3.40, 95.2 ± 14.21 mg g⁻¹ for Zn²⁺ and 51.1 ± 1.85, 230.0 ± 28.47 mg g⁻¹ for Pb²⁺ respectively. The results obtained by us in this work indicate that the emulsifier acts as a protective compound increasing the ability of C. lunata to survive in heavy metal polluted environment. Enhancement of exopolymer synthesis in the presence of Cd²⁺, Zn²⁺ and Pb²⁺ may also suggest, at least to some extent, a metal-specific nature of emulsifier production in C. lunata. Due to accumulation capability and tolerance to heavy metals, C. lunata mycelium surrounded by the emulsifier could be applied for toxic metal removal.     

Anionic lipopeptides from Bacillus mojavensis I4 as effective antihypertensive agents: Production,characterization, and identification

A new isolated Bacillus mojavensis strain I4 was found as producer of biosurfactants by different screening methods, such as parafilm M test, hemolytic activity, oil displacement test, emulsification index, surface tension, and lipase production assay. Enhanced biosurfactants production was obtained using glucose and glutamic acid as carbon and nitrogen sources, respectively. The optimal production of the biosurfactants was obtained by using a C/N ratio of 17, pH of 7.0, and temperature of 37°C. The surface tension was reduced to 29 mN/m and the emulsification index E24 of 62% was achieved after 72 h of culture. The purified biosurfactants showed stability with regard to surface tension reduction and emulsification in a wide range of temperatures (4–120°C), pH (4–10), and salinity (2–12% of NaCl). The thin‐layer chromatography showed that the produced biosurfactants were lipopeptides. The biosurfactants were characterized as a group of anionic lipopeptides with zeta potential measurement. Chromatographic characterization using HPLC revealed that I4 lipopeptides contained numerous isoforms and surfactin was the major component. Moreover, the I4 lipopeptides showed interesting angiotensin‐converting enzyme‐inhibitory activity.     

Production of a peptidoglycolipid bioemulsifier by Pseudomonas aeruginosa grown on hydrocarbon

A strain of Pseudomonas aeruginosa isolated from a polluted soil was found to produce an extracellular bioemulsifier when cultivated on hexadecane as sole carbon source. The emulsifier was precipitated with acetone and redissolved in sterile water. Dodecane, crude oil and kerosene were found to be good substrates for emulsification by the bioemulsifier. Growth and bioemulsifier production reached the optimal levels on the fourth and fifth day, respectively. Emulsifying activity was observed over a pH range of 3.5 to 10.0 with a maximum at pH 7.0. The activity of the bioemulsifier was heat stable up to 70 degrees C while about 50 percent of its activity was retained at 100 degrees C. The components of the bioemulsifier were determined, it was found to contain carbohydrate, protein and lipid. The protein complex was precipitated with ammonium sulphate and fractionated on a Sephadex G-100. Gel electrophoresis of the bioemulsifier showed a single band whose molecular weight was estimated as 14,322 Da. The bioemulsifier was classified as a peptidoglycolipid. Certain strains of P. aeruginosa produce peptidoglycolipid in place of rhamnolipid.     

Emulsifying activity in thermophilic and extremely thermophilic microorganisms

Thermophilic and extremely thermophilic enrichments from several different environments produced cell-associated emulsifiers as did several pure cultures ofArchaea. The bioemulsifiers were effective over a wide range of pH, at NaCl concentrations up to 200 g L^–1, and at temperatures up to 80°C. The emulsifying activity in cell-free extracts ofMethanobacterium thermoautotrophicum was a cell-associated protein with a molecular weight greater than 5000 Da. This emulsifier formed viscous emulsions, but did not reduce the surface tension of water or the interfacial tension between water and hexadecane. The emulsifier had the greatest activity with alkanes with carbon numbers greater than 10. The characteristics of the bioemulsifier fromM. thermoautotrophicum makes it suitable for use in saline or thermophilic oil reservoirs as a mobility control agent or in well-bore clean up processes.     

DNA encapsulation by an air-agitated, liquid-liquid mixer

Smooth and spherical alginate microspheres and nylon-membrane bound microcapsules were formed in an air-agitated, liquid-liquid mixer by emulsification/internal gelation and interfacial polymerization respectively. The mean diameter of the alginate microspheres ranged from 100 to 800 mum, and was controlled by process modifications. Increase in emulsifier concentration, gas flowrate, and emulsification time resulted in smaller microsphere size as did a decrease in liquid height. Increase in the dispersed phase viscosity resulted in a longer emulsification time required for approaching a minimum microsphere size. Microspheres could be formed with the proportion of dispersed phase approaching 30%. The yield of alginate microspheres was 70%, with losses attributed to incomplete recovery during washing and filtration operations. The yield of DNA encapsulation within the fraction of recovered microspheres, was 94%. The small loss was thought to occur by surface release during the washing of the microspheres. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 464-470, 1997.