Yield production,chemical composition,and functional properties of emulsifier H28 synthesized by <Emphasis Type="Italic">Halomonas eurihalina </Emphasis>strain H-28 in media containing various hydrocarbons

Halomonas eurihalina strain H-28 is a moderately halophilic bacterium that produces an extracellular polysaccharide not only in media with glucose but also in media supplemented with hydrocarbons (n-tetradecane, n-hexadecane, n-octane, xylene, mineral light oil, mineral heavy oil, petrol, or crude oil). In this study we investigated yield production, chemical composition, viscosity, and emulsifying activity of exopolysaccharides (EPS) extracted from the different media used. The largest amounts of biopolymer were synthesized in media with glucose and n-hexadecane. Chemical composition varied with culture conditions; thus EPS from cultures grown in the presence of hydrocarbons had lower contents of carbohydrates and proteins than EPS from media with glucose. However, the percentages of uronic acids, acetyls, and sulfates were always higher than glucose EPS. Crude oil was the substrate most effectively emulsified. All EPS were capable of emulsifying crude oil more efficiently than the three control surfactants tested (Tween 20, Tween 80, and Triton X-100). All polymers gave low viscosity solutions. EPS H28 could be attractive for application in the oil industry and/or in bioremediation processes, bearing in mind not only its functional properties, but also the capacity of producer strain H-28 to grow in the presence of high salt concentrations and oil substrates.     

Production of bioemulsifier by Bacillus subtilis, Alcaligenes faecalis and Enterobacter species in liquid culture

Three bacterial strains isolated from waste crude oil were selected due to their capacity of growing in the presence of hydrocarbons and production of bioemulsifier. The genetic identification (PCR of the 16S rDNA gene using fD1 and rD1 primers) of these strains showed their affiliation to Bacillus subtilis, Alcaligenes faecalis and Enterobacter sp. These strains were able to emulsify n-octane, toluene, xylene, mineral oils and crude oil, look promising for bioremediation application. Finally, chemical composition, emulsifying activity and surfactant activity of the biopolymers produced by the selected strains were studies under different culture conditions. Our results showed that chemical and functional properties of the bioemulsifiers were affected by the carbon source added to the growth media.     

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.     

Characteristics of bioemulsifiers synthesised in crude oil media by <Emphasis Type="Italic">Halomonas eurihalina</Emphasis> and their effectiveness in the isolation of bacteria able to grow in the presence of hydrocarbons

Halomonas eurihalina strains F2-7, H28, H96, H212 and H214 were capable of producing large amounts of exopolysaccharides (EPS) in MY medium with added crude oil. The biopolymers showed lower carbohydrate and protein content than those synthesised in control medium without oil. Nevertheless, the percentages of uronic acids, acetyls and sulphates were higher. The emulsifying activity of biopolymers was measured; crude oil was the substrate most efficiently emulsified. Furthermore, all the EPS tested emulsified higher volumes of crude oil than the commercial surfactants used as controls. We have also proved the effectiveness of both Halomonas eurihalina strains and their EPS to select indigenous bacteria able to grow in the presence of polycyclic aromatic hydrocarbons (naphthalene, phenanthrene and pyrene) from waste crude oil. The majority of isolated strains belonged to the genus Bacillus.     

Production and characterization of a new bioemulsifier from Pseudomonas putida ML2

AIMS: To characterize the bioemulsifier produced by a nonfluorescent strain of Pseudomonas putida isolated from a polluted sediment and to determine the influence of pH, temperature, media composition, and carbon and nitrogen source on growth and emulsifying activity. METHODS AND RESULTS: Different indexes were employed to determine the emulsifying properties of culture supernatants of P. putida ML2 in defined and complex media. Surface tension of cell-free supernatants was measured. Purification and chemical analysis of the emulsifier was performed. Confirmed results indicate that a polysaccharide with hexasaccharide repeating units is responsible for the emulsifying activity in a mineral medium with glucose as sole carbon source. Moreover, an emulsifier is produced when growing on naphthalene. CONCLUSIONS: Culture media composition influences the amount and the properties of the emulsifier produced by this P. putida strain. Under nitrogen limiting conditions, a polysaccharide is responsible for the emulsifying activity in defined mineral media. In complex nitrogen rich medium, a different kind of emulsifier is produced. The exopolymer may contribute to hydrocarbons solubilization. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first exopolysaccharide with emulsifying properties produced by a Pseudomonas strain reported to the present. Also chemical composition is significantly different from previous reports. This strain has potential use in bioremediation and the purified polysaccharide may be used in food and cosmetic industry. Moreover, the production of the exopolymer may play a role on biofilm formation.     

Characterization of a novel bioemulsifier from <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis>

This study describes a novel and efficient alasan-like bioemulsifier produced by Pseudomonas stutzeri NJtech 11-1, which was isolated from the Shengli Oilfield. The strain was found to produce a new and interesting emulsion stabilizer. The crude bioemulsifier showed super stability with 50% salinity and broad pH 3–10. The emulsion index (EI₂₄) was increased to 100% after heating from 45 to 95 °C and the emulsion could be stable for at least 30 days. The yield of Ps-bioemulsifier (pure bioemulsifier) was 0.68?±?0.05 mg mL^?1. The Ps-bioemulsifier was composed of carbohydrates (80?±?2.6%) and proteins (9.5?±?0.5%). A low concentration (0.2 mg mL^?1) of the Ps-bioemulsifier was obtained maximum emulsifying activity at pH 7.1 and its emulsifying activity strengthened by suitable salinity. Furthermore, Ps-bioemulsifier could also emulsify cyclohexane, hexadecane, kerosene, xylene hydrocarbons efficiently. Therefore, the Ps-bioemulsifier showed emulsifying characteristics which make it a good candidate for potential applications in bioremediation and microbial enhanced oil recovery.     

Curdlan-like exopolysaccharide production by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> UNP3 during growth on hydrocarbon substrates

Cellulomonas flavigena UNP3, a natural isolate from vegetable oil contaminated soil sample has been studied for growth associated exopolysaccharide (EPS) production during growth on glucose, groundnut oil and naphthalene. The EPS showed matrix formation surrounding the cells during scanning electron microscopy. Cell surface hydrophobicity and emulsifying activity studies confirmed the role of EPS as bioemulsifier. Emulsifying activity was found to increase with time (0.2 U/mg for 10 min to 0.27 U/mg for 30 min). Emulsification index, E₂₄ value increased with the increase in EPS concentration. Degradation of polyaromatic hydrocarbons was confirmed using gas chromatography analysis. FTIR analysis showed presence of characteristic absorbance at 895.10 cm⁻¹ for β-configuration of glucan. NMR studies also revealed EPS produced by C. flavigena UNP3 as a linear β-1, 3-d-glucan, and a curdlan like polysaccharide.     

烃降解菌株T7-2产生的生物乳化剂及其理化性质研究

从石油污染海域海底泥中筛选到的1株低温石油烃降解菌, 经鉴定为红平红球菌(Rhodococcus erythropolis), 命名为T7-2。该菌株能以十六烷为碳源代谢产生一种对柴油等烃类具良好乳化作用的生物乳化剂。研究表明, 该乳化剂主要由糖类、脂类和蛋白质组成, 其比例为55.43:31.24:12.65。进一步研究证实, 该乳化剂糖类的单糖组成为甘露糖和鼠李糖; 脂类由十碳、十二碳、十六碳及十八碳脂肪酸组成; 蛋白质由16种氨基酸构成。本文还对乳化剂的理化性质进行了分析, 发现它是一种性能稳定、乳化效率高、适应范围较为广泛的生物乳化剂, 对海洋污染生物修复及石油开采都具有潜在的应用价值。     

烃降解菌株T7-2产生的生物乳化剂及其理化性质研究

从石油污染海域海底泥中筛选到的1株低温石油烃降解菌,经鉴定为红平红球菌(Rhodococcus erythropolis),命名为T7-2.该菌株能以十六烷为碳源代谢产生一种对柴油等烃类具良好乳化作用的生物乳化剂.研究表明,该乳化剂主要由糖类、脂类和蛋白质组成,其比例为55.43:31.24:12.65.进一步研究证实,该乳化剂糖类的单糖组成为甘露糖和鼠李糖;脂类由十碳、十二碳、十六碳及十八碳脂肪酸组成;蛋白质由16种氨基酸构成.本文还对乳化剂的理化性质进行了分析,发现它是一种性能稳定、乳化效率高、适应范围较为广泛的生物乳化剂,对海洋污染生物修复及石油开采都具有潜在的应用价值.     

Screening and Evaluation of Biosurfactant-Producing Strains Isolated from Oilfield Wastewater

The six biosurfactant-producing strains, isolated from oilfield wastewater in Daqing oilfield, were screened. The production of biosurfactant was verified by measuring the diameter of the oil spreading, measuring the surface tension value and emulsifying capacity against xylene, n-pentane, kerosene and crude oil. The experimental result showed three strains (S2, S3, S6) had the better surface activity. Among the three strains, the best results were achieved when using S2 strain. The diameter of the oil spreading of the biosurfactant produced by S2 strain was 14 cm, its critical micelle concentration (CMC) was 21.8 mg/l and the interfacial tension between crude oil and biosurfactant solution produced by S2 strain reduced to 25.7 mN/m. The biosurfactant produced by S2 strain was capable of forming stable emulsions with various hydrocarbons, such as xylene, n-pentane, kerosene and crude oil. After S2 strain treatment, the reduction rate of oil viscosity was 51 % and oil freezing point reduced by 4 °C.     

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.     

Isolation and characterization of exopolysaccharide produced by Vibrio harveyi strain VB23

AIMS: The aim of the study was to isolate and characterize exopolysaccharide (EPS) produced by Vibrio harveyi strain VB23. METHODS AND RESULTS: Growth and EPS production by V. harveyi strain VB23, was studied in mineral salts medium supplemented with NaCl (1.5%) and glucose (0.2%). The rate of EPS production in batch cultures was highest during the late log phase of growth when compared with stationary growth phase. The exopolymer was recovered from the culture supernatant by using a cold ethanol precipitation-dialysis procedure. Chemical analyses of EPS revealed that it is primarily composed of neutral sugars, uronic acids, proteins and sulfates. The purified EPS revealed prominent functional reactive groups, such as hydroxyl, carboxylic and amides, which correspond to a typical heteropolymeric polysaccharide and the EPS, also possessed good emulsification activity. The gas chromatographic analysis of an alditol acetate-derivatized sample of EPS revealed that it is composed primarily of galactose and glucose. Minor components found were rhamnose, fucose, ribose, arabinose, xylose and mannose. CONCLUSIONS: The EPS produced by V. harveyi strain VB23 is a heteropolysaccharide possessing good emulsification activity. EPS was readily isolated from culture supernatants, which suggests that the EPS was a slime-like EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of EPS characterization in luminous V. harveyi bacteria, which describes the isolation and characterization of an EPS expressed by V. harveyi. The results of the study contributes significantly towards an understanding of the chemical composition and applications of the EPS in environmental biotechnology and bioremediation.     

Isolation and characterization of mucous exopolysaccharide (EPS) produced by Vibrio furnissii strain VB0S3

Marine bacterial strains were isolated from coastal regions of Goa and screened for the strains that produce the highest amount of mucous exopolysaccharide (EPS). Our screening resulted in the identification of the strain Vibrio furnissii VB0S3 (hereafter called VB0S3), as it produced the highest EPS in batch cultures during the late logarithmic growth phase. The isolate was identified as VB0S3 based on morphological and biochemical properties. Growth and EPS production were studied in mineral salts medium supplemented with NaCl (1.5%) and glucose (0.2%). The exopolymer was recovered from the culture supernatant by using three volumes of cold ethanol precipitation and dialysis procedure. Chemical analyses of EPS revealed that it is primarily composed of neutral sugars, uronic acids, and proteins. Fourier-transform infrared (FT-IR) spectroscopy revealed the presence of carboxyl, hydroxyl, and amide groups, which correspond to a typical heteropolymeric polysaccharide, and the EPS also possessed good emulsification activity. The gas chromatographic analysis of an alditol-acetate derivatized sample of EPS revealed that it was mainly composed of galactose and glucose. Minor components found were mannose, rhamnose, fucose, ribose, arabinose, and xylose. EPS was readily isolated from culture supernatants, which suggests that the EPS was a slime-like exopolysaccharide. This is the first report of exopolysaccharide characterization that describes the isolation and characterization of an EPS expressed by Vibrio furnissii strain VB0S3. The results of the study contribute significantly and go a long way towards an understanding of the correlation between growth and EPS production, chemical composition, and industrial applications of the exopolysaccharide in environmental biotechnology and bioremediation.     

Biotechnological Potential of Bacillus salmalaya 139SI: A Novel Strain for Remediating Water Polluted with Crude Oil Waste

Environmental contamination by petroleum hydrocarbons, mainly crude oil waste from refineries, is becoming prevalent worldwide. This study investigates the bioremediation of water contaminated with crude oil waste. Bacillus salamalaya 139SI, a bacterium isolated from a private farm soil in the Kuala Selangor in Malaysia, was found to be a potential degrader of crude oil waste. When a microbial population of 10⁸ CFU ml^-1 was used, the 139SI strain degraded 79% and 88% of the total petroleum hydrocarbons after 42 days of incubation in mineral salt media containing 2% and 1% of crude oil waste, respectively, under optimum conditions. In the uninoculated medium containing 1% crude oil waste, 6% was degraded. Relative to the control, the degradation was significantly greater when a bacteria count of 99 × 10⁸ CFU ml^-1 was added to the treatments polluted with 1% oil. Thus, this isolated strain is useful for enhancing the biotreatment of oil in wastewater.     

Production and properties of a bioemulsifier synthesized by phenanthrene-degrading Penicillium sp.

In this study, we describe the production and properties of the bioemulsifier synthesized by Penicillium sp. grown in a medium supplemented with 300 mg L⁻¹ of phenanthrene, where bioemulsifier production was not growth-associated. The maximum emulsifier production (2 ± 0.3 g L⁻¹) and emulsifying activity in cell-free culture medium (E₂₄ = 60 ± 4%) were recorded on the 4th and 5th days, respectively. Of the various hydrocarbons tested, the best emulsifying activity was observed for kerosene, diesel, and xylene. The emulsifying agent maintained its properties over a wide range of pH (3–9), at high salinity (20% NaCl), and during exposure to elevated temperatures (93 °C). The fungal bioemulsifier was effective at these extreme environmental conditions and was able to emulsify the tested pure aromatic and aliphatic hydrocarbons and mixtures of the hydrocarbons. The bioemulsifier was composed by lipids (67%), carbohydrates (11%), and protein (7%). Myristic, stearic, and oleic were the major acids detected in the lipidic fraction.     

Bioconversion of biodiesel refinery waste in the bioemulsifier by Trichosporon mycotoxinivorans CLA2

ABSTRACT: BACKGROUND: The microbial bioemulsifiers was surface active compounds, are more effective in stabilizing oil-in-water emulsions. The yeasts have been isolated to produce bioemulsifiers from vegetable oils and industrial wastes. RESULTS: Trichosporon mycotoxinivorans CLA2 is bioemulsifier-producing yeast strain isolated from effluents of the dairy industry, with ability to emulsify different hydrophobic substrates. Bioemulsifier production (mg/L) and the emulsifying activity (E24) of this strain were optimized by response surface methodology using mineral minimal medium containing refinery waste as the carbon source, which consisted of diatomaceous earth impregnated with esters from filters used in biodiesel purification. The highest bioemulsifier production occurred in mineral minimal medium containing 75 g/L biodiesel residue and 5 g/L ammonium sulfate. The highest emulsifying activity was obtained in medium containing 58 g/L biodiesel refinery residue and 4.6 g/L ammonium sulfate, and under these conditions, the model estimated an emulsifying activity of 85%. Gas chromatography and mass spectrometry analysis suggested a bioemulsifier molecule consisting of monosaccharides, predominantly xylose and mannose, and a long chain aliphatic groups composed of octadecanoic acid and hexadecanoic acid at concentrations of 48.01% and 43.16%, respectively. The carbohydrate composition as determined by GC-MS of their alditol acetate derivatives showed a larger ratio of xylose (49.27%), mannose (39.91%), and glucose (10.81%). 1 H NMR spectra confirmed by COSY suggested high molecular weight, polymeric pattern, presence of monosaccharide's and long chain aliphatic groups in the bioemulsifier molecule. CONCLUSIONS: The biodiesel residue is an economical substrate, therefore seems to be very promising for the low-cost production of active emulsifiers in the emulsification of aromatics, aliphatic hydrocarbons, and kerosene.     

Identification and characterization of the<Emphasis Type="Italic"> carAB</Emphasis> genes responsible for encoding carbamoylphosphate synthetase in<Emphasis Type="Italic"> Halomonas eurihalina</Emphasis>

Halomonas eurihalina is a moderately halophilic bacterium which produces exopolysaccharides potentially of great use in many fields of industry and ecology. Strain F2-7 of H. eurihalina synthesizes an anionic exopolysaccharide known as polymer V2-7, which not only has emulsifying activity but also becomes viscous under acidic conditions, and therefore we consider it worthwhile making a detailed study of the genetics of this strain. By insertional mutagenesis using the mini-Tn 5 Km2 transposon we isolated and characterized a mutant strain, S36 K, which requires both arginine and uracil for growth and does not excrete EPS. S36 K carries a mutation within the carB gene that encodes the synthesis of the large subunit of the carbamoylphosphate synthetase enzyme, which in turn catalyzes the synthesis of carbamoylphosphate, an important precursor of arginine and pyrimidines. We describe here the cloning and characterization of the carAB genes, which encode carbamoylphosphate synthetase in Halomonas eurihalina, and discuss this enzyme's possible role in the pathways for the synthesis of exopolysaccharides in strain F2-7.     

Characterization and emulsifying property of a novel bioemulsifier by Aeribacillus pallidus YM-1

Aims: Biosurfactants and bioemulsifiers commonly have the advantages of biodegradability, low toxicity, selectivity and biocompatibility over chemically synthesized surfactants. The goal of the study is to present a novel bioemulsifier with great application potential. Methods and Results: Aeribacillus pallidus YM‐1, isolated from crude oil contaminated soil, was found to produce a novel high molecular bioemulsifier with an emulsification index of 60 ± 1% without remarkable surface tension reduction (45·7 ± 0·1 mN m^?1). The number‐average molecular weight was determined as 526 369 Da by gel permeation chromatography analysis. Bioemulsifier was subjected to FT‐IR and a complex of carbohydrates (41·1%), lipids (47·6%) and proteins (11·3%) was determined. Conclusions: The bioemulsifier of A. pallidus YM‐1 was isolated from the glucose‐based culture medium and characterized with the help of chemical analytical techniques. The bioemulsifier exhibited a promising emulsifying property for biotechnology application potential in bioremediation and microbial enhanced oil recovery. Significance and Impact of the Study: This is the first report of the bioemulsifier production by A. pallidus. The potential emulsifying activity of the bioemulsifier in the present study may be explored in various biotechnological and industrial applications.     

Evaluation of bioemulsifier mediated Microbial Enhanced Oil Recovery using sand pack column

Bacillus licheniformis K125, isolated from an oil reservoir, produces an effective bioemulsifier. The crude bioemulsifier showed 66% emulsification activity (E(24)) and reduced the surface tension of water from 72 to 34 mN/m. It contains substantial amount of polysaccharide, protein and lipid. This bioemulsifier is pseudoplastic non-Newtonian in nature. It forms oil in water emulsion which remains stable at wide range of pH, temperature and salinity. It gave 43+/-3.3% additional oil recovery upon application to a sand pack column designed to simulate an oil reservoir. This is 13.7% higher than that obtained from crude lipopeptide biosurfactants produced by the standard strain, Bacillus mojavensis JF2 and 8.5% higher than hot water spring isolate, Bacillus licheniformis TT42. The increased oil recovery obtained by using the crude bioemulsifier can be attributed to its combined surface and emulsification activity. Its mechanism of oil recovery must be similar to the mechanism exhibited by surfactant-polymer flooding process of chemical enhanced oil recovery.     

Utilization of phenol by hydrocarbon assimilating yeasts

77 Ascomycetous, basidiomycetous as well as imperfect yeast strains of 46 different species and 20 genera were tested for growth with the substrates n-octane, n-hexadecane, and phenol. Of 59 yeast strains with ascomycetous cell wall structure 33 grew on hydrocarbons and 32 on phenol. No yeast strain out of 26 which are unable to use n-alkanes as a source of carbon and energy grew on phenol. In comparison with the latter 32 out of 33 n-hexadecane assimilating yeasts were also capable of using phenol. All n-octane utilizing yeasts of this group also assimilate phenol as a carbon source for growth.The correlation of the hydrocarbon assimilation with the phenol assimilation seems to be not so strong in the basidiomycetous yeasts. 7 out of 18 strains from this group grew on n-hexadecane and 13 on phenol.Furthermore, it could be shown that the use of hydrocarbons and phenol (as well as methanol) is strongly correlated with the coenzyme Q structure of the respective yeast strain.The results are discussed with respect to the particular chemical properties of the substrates used and the fact that coenzyme Q structure is considered to be an important marker of evolutionary relationships among yeasts.