The effect of temperature on eicosane substrate uptake modes by a marine bacterium Pseudomonas nautica strain 617: relationship with the biochemical content of cells and supernatants

Three hydrocarbon uptake modes (adherence, emulsification and solubilization) were identified and quantified in cells and supernatants of a mesophilic marine bacterium Pseudomonas nautica strain 617 grown on eicosane. The adherence capacity was related to the enrichment of cells with wax esters and glycolipids. The emulsifying activity was related to the presence of extracellular biosurfactants composed of proteins, carbohydrates and lipids (35:63:2). The intensity of substrate uptake modes was sensitive to temperatures currently found in the original environment of P. nautica (16°C, 20°C and 32°C). When temperature decreased, a significant increase in adherence and emulsifying activity was observed in relation to biochemical changes, whereas solubilizing activity decreased. The marine bacterium was able to degrade 53–59% eicosane at the end of exponential growth after 13, 5 and 3 days incubation at 16°C, 20°C and 32°C respectively.     

Ectoine as the predominant osmolyte in the marine bacterium Marinobacter hydrocarbonoclasticus grown on eicosane at high salinities

L. FERNANDEZ-LINARES, R. FAURE, J.-C. BERTRAND AND M. GAUTHIER. 1996. Ectoine was detected by ¹³C-nuclear magnetic resonance spectroscopy in extracts of the halophilic marine bacterium Marinobacter hydrocarbonoclasticus grown at different NaCl concentrations (0.6, 1.5, 2.5 mol l^-1) in synthetic seawater on eicosane as the sole source of carbon and energy. Ectoine was synthesized as the principal osmolyte in response to stress. However, when the medium was supplemented with glycine betaine, this osmolyte was transported into the cells and replaced ectoine.     

Preparation and characterization of monolayer and multilayer Langmuir-Blodgett films of a series of 6-O-alkylcelluloses

Monolayer and multilayer Langmuir-Blodgett (LB) films of 6-O-alkylcelluloses with various chain lengths were prepared and studied. The surface pressure (pi)-area (A) isotherms of 6-O-alkylcelluloses exhibited characteristic behaviors depending on the length of the alkyl chain and temperature. 6-O-Stearylcellulose on the subphase formed a homogeneous monolayer at 10 mN m(-1). By transfer ratio, FT-IR, and contact angle measurements, it was proved that the monolayer of 6-O-stearylcellulose on the water surface was transferred successfully onto a substrate by a vertical dipping method to form a Z-type LB film. The transmission and reflection absorption IR spectrum indicated that the hydrocarbon chains had all-trans rotamers and were oriented nearly perpendicular to the surface in the film. AFM section analysis revealed that the thickness per layer was calculated to be 2.35 nm. These results suggested that the hydrocarbon chains were inclined at an angle of about 25.3 degrees to have high packing density in the alkyl region.     

铜绿假单胞菌对长链烷烃的摄取模式

研究了一株铜绿假单胞菌(CGMCC 1.1785)摄取长链烷烃的模式。铜绿假单胞菌1．1785能够以固态的长链烷烃为唯一碳源生长,在培养过程中产生表面活性代谢物。烃与水相的界面面积是细菌生长重要的影响因素,说明传质限制的存在。由于该菌不能够利用鼠李糖脂增溶的烃作为碳源,因此添加鼠李糖脂能够强化烃摄取的主要原因是烃界面的扩大。细胞表面疏水性从开始的急剧升高到后来的不断下降,说明在不同生长阶段细胞对烃的摄取模式是不同的。由此认为,铜绿假单胞菌1.1785既没有通过表面活性剂介导模式获取烃,也并非完全通过直接接触模式获取烃。据此提出该菌采用了一种运动接触的烃摄取模式,其趋化运动能力在这种摄取过程中起到重要作用。     

Streptavidin-based containment systems for genetically engineered microorganisms

The use of genetically modified microorganisms for environmental remediation continues to be debated. Conditional lethal systems with tightly regulated gene expression can be used to contain released microorganisms and ameliorate some of the concerns about horizontal gene transfer. We have described streptavidin-based suicide systems to address these concerns and evaluated their function in Pseudomonas putida containing the TOL plasmid for aromatic hydrocarbon metabolism. Tight regulation of expression of a truncated streptavidin gene was required to avoid premature production of the toxic protein. Streptavidin expression was induced by the absence of 3-methyl benzoate (hydrocarbon substrate) which resulted in the elimination of 99.9% of the bacterial culture within eight hours. Low mutant escape rates at 10(-7) per cell per generation were also realized.     

The mode of interaction between the substrate and cell surface of the hydrocarbon-utilizing yeast Candida tropicalis

A method for the measurement of the affinity of the cell surface to hydrocarbons is described. The affinity was basically unaffected by different pH values and temperature as well as by the chain length of the substrate. The contact time required for saturation of the cell surface with substrate was 30 sec. Cells grown on glucose showed a 25% lower adsorption capacity compared to those grown on n-alkane. The glucose grown cells showed also a more distinguished dependence of the amount of adsorbed hydrocarbon on the quality of the emulsion. The interaction between the substrate and cell surface turned out to be an adsorption that did not involve an enzymatic reaction. These results led to the conclusion that a lipopolysaccharide moiety present at the cell surface is responsible for the affinity.     

Molecular dynamics simulations of the aggregation behaviour of overlapped graphene sheets in linear aliphatic hydrocarbons

Molecular dynamics simulations were used to investigate the aggregation of two partially overlapped graphene sheets in hexane, dodecane and eicosane. When partially overlapped graphene sheets are adjacent to one another, they will expel the adsorbed layers of the solvent molecules on the graphene surface, and the amount of overlap will increase. When the overlapped regions of the graphene sheets are separated by solvent molecules, they cannot expel the adsorption layers between them, and so the sheets remain separated. The driving force for aggregation is the van der Waals interaction between the two graphene sheets, while the van der Waals interaction between the graphene sheets and the solvent molecules inhibits graphene aggregation. The diffusion rate of the hydrocarbon molecules with shorter chain lengths is higher. Thus, they diffuse faster during graphene aggregation, which leads to a higher rate of graphene overlapping in the shorter hydrocarbons. This work provides useful insights into graphene aggregation in linear hydrocarbon solvents of varying lengths at the nanoscale.     

Cell Surface Measurements in Hydrocarbon and Carbohydrate Fermentations

Acinetobacter calcoaceticus was grown in 11-liter batch fermentations with hexadecane or sodium citrate as the sole source of carbon. Surface and interfacial tension measurements of the microbial broth indicated that surface-active compounds were being produced only during growth on the hydrocarbon substrate. Contact angle measurements of an aqueous drop on a smooth lawn of cells in a hexadecane bath indicated a highly hydrophobic surface of the cells in the initial stages of the hydrocarbon fermentation (120° contact angle). At this stage, the entire cell population was bound to the hydrocarbon-aqueous interface. The contact angle dropped rapidly to approximately 45° after 14 h into the fermentation. This coincided with a shift of the cell population to the aqueous phase. Thus, the cells demonstrated more hydrophilic characteristics in the later stages of the fermentation. Contact angles on cells grown on sodium citrate ranged from 18 to 24° throughout the fermentation. The cells appear to be highly hydrophilic during growth on a soluble substrate. From the contact angle and aqueous-hydrocarbon interfacial tension, the surface free energy of the cells was calculated along with the cell-aqueous and cell-hydrocarbon interfacial tension. The results of these measurements were useful in quantitatively evaluating the hydrophobic nature of the cell surface during growth on hydrocarbons and comparing it with the hydrophilic nature of the cell surface during growth on a soluble substrate.     

Yeast and bacteria cell hydrophobicity and hydrocarbon biodegradation in the presence of natural surfactants: rhamnolipides and saponins

This study concerns the relation between hydrocarbon biodegradation in the presence of natural surfactants and cell hydrophobicity resulting from the use of these surfactants. The relative capabilities of two bacterial strains (Pseudomonas aeruginosa and Bacillus subtilis) and two yeast strains (Candida maltosa, Yarrowia lipolytica) were investigated. The selected microorganisms were tested separately and in combination in order to achieve the optimal degrading performance. The surface cell hydrophobicity of microorganisms and the degree of hydrocarbon biodegradation were measured. The microbial adhesion to the hydrocarbon (MATH) test was used to denote the surface cell hydrophobicity of the microbial species. The results indicate the correlation between the modification of the surface cell and the degree of hydrocarbon biodegradation; however results for bacteria differ from that obtained for yeast strains. Saponins, as the surfactant, was more effective than rhamnolipides during hydrocarbon biodegradation, though the concentration of this surfactant has no significant influence on the surface cell hydrophobicity.     

The effect of medium composition on the structure and physical state of sophorolipids produced by Candida bombicola ATCC 22214

Candida bombicola was grown using a variety of lipophilic carbon substrates. Most of the hydrocarbon and carboxylic acid substrates resulted in a mixture of sophorolipids consisting of free acids and the more desirable lactones. The ratio of diacylated lactone to free acid in these mixtures was a maximum when produced using hexadecane and heptadecane. All of the other lipophilic substrates resulted in significant amounts of free acids being produced. These lactone products were unique in that they precipitated as crystals, which were easily separated from the culture medium. All of the other products were isolated as oils as is usually reported in the literature. Finally, the amounts of these crystals recovered were significantly higher than those observed for any of the oily products. It was possible to determine the degree of direct incorporation of the lipophilic substrates into the sophorolipids for a homologous series of alkanes. The amount of direct incorporation increased with increasing chain length to a maximum for pentadecane, hexadecane and heptadecane. As the length of the alkane substrate increased further, the amount of direct incorporation then decreased until there was no apparent incorporation for eicosane.     

Diversity of bacterial strains degrading hexadecane in relation to the mode of substrate uptake

The relative distribution of the modes of hydrocarbon uptake, used by bacteria of the environment for the degradation of long-chain alkanes, has been evaluated. The first mode of uptake, direct interfacial accession, involves contact of cells with hydrocarbon droplets. In the second mode, biosurfactant-mediated transfer, cell contact takes place with hydrocarbons emulsified or solubilized by biosurfactants. Sixty-one strains growing on hexadecane were isolated from polluted and non-polluted soils and identified. The majority (61%) belonged to the Corynebacterium-Mycobacterium-Nocardia group. Criteria selected for characterizing hexadecane uptake were cell hydrophobicity, interfacial and surface tensions and production of glycolipidic extracellular biosurfactants. These properties were determined in flask cultures on an insoluble (hexadecane) and on a soluble (glycerol or succinate) carbon source for a subset of 23 representative strains. Exclusive direct interfacial uptake was utilized by 47% of studied strains. A large proportion of strains (53%) produced biosurfactants. The data on cellular hydrophobicity suggested the existence of two distinct alkane transfer mechanisms in this group. Accordingly, tentative assignments of biosurfactant-mediated micellar transfer were made for 11% of the isolated strains, and of biosurfactant-enhanced interfacial uptake for 42%.     

Hexadecane mineralization activity in ornithogenic soil from Seabee Hook,Cape Hallett,Antarctica

Ornithogenic soils that form in penguin rookeries contain high levels of organic carbon and nitrogen. On Seabee Hook, Cape Hallett, Antartica, ornithogenic soil was contaminated with hydrocarbons following establishment of a scientific research station. In these soils hydrocarbon biodegradation could be supported by available soil nitrogen. Hexadecane mineralization activity was detected in vitro in ornithogenic soil when incubated at 5 or 15°C. At 5°C the extent of hexadecane mineralization was higher in hydrocarbon-contaminated soil than in uncontaminated soil. Alkane-degrading bacteria isolated from Seabee Hook soil were identified as Rhodococcus or Gordonia spp. or an unclassified Corynebacterineae. The alkane degraders grew on n-alkanes from heptane (C8) to eicosane (C20) and pristane, and utilized uric acid or ammonium nitrate as nitrogen source. All of the isolates possessed urease activity. Results of this study indicate biodegradation of hydrocarbons may contribute to the natural attenuation of oil spills in ornithogenic surface soils in summer.     

Gas-chromatography and electroantennogram analysis of saturated hydrocarbons of cruciferous host plants and host larval body extracts of Plutella xylostella for behavioural manipulation of Cotesia plutellae

Saturated hydrocarbons (SHC) of five cruciferous host plants viz., cabbage, cauliflower, broccoli, knol khol and Brussels sprout and the larvae of diamondback moth (DBM), Plutella xylostella reared on these host plants were identified through gas-chromatography. The hydrocarbon profile of host plants and larval body extract of DBM reared on respective host plants revealed a wide variation in quantity as well as quality. Long chain hydrocarbons C26-C30 were detected in all the extracts. In electroantennogram (EAG) studies, SHCs at 10(-3) g dose elicited differential EAG response in the antennal receptors of gravid Cotesia plutellae females. Tricosane (C23) and hexacosane (C26) elicited 10-fold increased EAG response compared to control stimulus. Long chain hydrocarbons C27, C28 and C29 elicited, 6-7 fold increased responses. The sensitivity of antenna was 4-5 folds for C25, C14, C24, C15 and C30, while the short chain hydrocarbons elicited 2-3 fold increased EAG responses. Dual choice flight orientation experiments in a wind tunnel revealed that the gravid C. plutellae females preferred the odour of C16, C26, C29, C15, C21, C23, C30, C27, C24 and C22 as 60-70% females oriented and landed on SHC treated substrate compared to control odour, while the odour of eicosane (C20), pentacosane (C25) and octacosane (C28) were not preferred by the females.     

Pseudomonas oleovorans as a tool in bioconversions of hydrocarbons: growth,morphology and conversion characteristics in different two-phase systems

The bioconversion of hydrocarbons by Pseudomonas oleovorans has been studied in two-phase systems. In these systems, the hydrocarbon substrate is present in sufficient amounts to form the bulk apolar phase. High cell densities (up to 20 mg dry mass per ml water phase) are reached when the apolar phase consists of n-octane, 1-octene or 1-decene. There is considerable cell damage after incubation for 50–70 h. Loss of cell viability and membrane damage as observed by freeze-fracture electron microscopy correlate with a loss of hydrocarbon oxidation, measured as the conversion of 1-octene to 1,2-epoxyoctane. The final yield of oxidized hydrocarbon in the apolar substrate phase can be increased substantially by replacing the damaged cells with freshly grown cells. Yields up to 150 mg 1,2-epoxyoctane per ml 1-octene and up to 20–25 mg 1,2-epoxyoctane per ml culture were obtained with four cycles of the cell renewal procedure. Several other substrates in addition to octene were tested in the optimized two-phase system. Of these, 1-decene was converted into (R)-1,2-epoxydecane with an optical purity of 60%, while allylbenzene was converted into chiral 1,2-epoxy-3-phenylpropane. Some of the future applications of the conversion products are discussed.     

A Petroleum-Degrading Freshwater Alga Phormidium foveolarum Gom

The freshwater blue-green alga Phormidium foveolarum Gem. was grown on medium containing hydrocarbons. Measurements of O2 consumption during degradation of the N-tetradecane by Phormidium foveolarum Gom. and residues of the hydrocarbon substrate were made by Warburg manometry and gas-chromatography. The results show that: (1) Phormidium foveolarum Gom. has ability to absorb and degrade the N-tetradecane and then the concentration of N-tetradecane decreased. (2) Under the dark condition the O2 consumption increased with the reaction time for hydrocarbon degradation. (3) Notable changes in cell contents of Phormidium foveolarum Gom. have been observed after it has continuously grown on the substrate containing hydrocarbon. The oil droplets appeared and more large granules have been found in the cells.     

Enhanced hydrocarbon biodegradation by a newly isolated Bacillus subtilis strain

The relation between hydrocarbon degradation and biosurfactant (rhamnolipid) production by a new Bacillus subtilis 22BN strain was investigated. The strain was isolated for its capacity to utilize n-hexadecane and naphthalene and at the same time to produce surface-active compound at high concentrations (1.5 - 2.0 g l(-1)). Biosurfactant production was detected by surface tension lowering and emulsifying activity. The strain is a good degrader of both hydrocarbons used with degradability of 98.3 +/- 1% and 75 +/- 2% for n-hexadecane and naphthalene, respectively. Measurement of cell hydrophobicity showed that the combination of slightly soluble substrate and rhamnolipid developed higher hydrophobicity correlated with increased utilization of both hydrocarbon substrates. To our knowledge, this is the first report of Bacillus subtilis strain that degrades hydrophobic compounds and at the same time produces rhamnolipid biosurfactant.     

茶蚜体表漂洗物对天敌的引诱活性及组分分析

行为生测和触角电位反应都证明茶蚜Toxoptera aurantii体表的正己烷或乙醚漂洗物对茶蚜重要天敌中华草蛉Chysopa sinica、蚜茧蜂Aphidius sp.和七星瓢虫Coccinella septempunctata具有显著的引诱效应,正己烷漂洗物的活性稍强。GC、GC-MS分析表明正己烷漂洗物中主要组分是苯甲醛、十一烷、2, 5-己二酮、2,5-二氢噻吩、芳樟醇、萘、4-甲基-辛烷、1, 2-苯甲酸-双-（二丁基-邻苯二甲酸甲酯）、二丁基-邻苯二甲酸甲酯和二十烷,其中苯甲醛、2, 5-己二酮和芳樟醇含量稍大。乙醚漂洗物中主要组分为反-2-己烯酸、正十七烷、2,6,10,14-四甲基十五烷、二十烷、四甲基四十烷、二丁基-邻苯二甲酸酯和十九烷,前2种组分含量较大。     

Surface-tethered DNA complexes for enhanced gene delivery

Overcoming the barriers to efficient gene transfer is a fundamental goal of biotechnology. A versatile approach to enhance the delivery of nonviral DNA involves complexation with cationic polymers, which can be designed to overcome the barriers to effective gene transfer. More recently, DNA release from a polymer substrate or scaffold has been shown to enhance gene transfer, likely by increasing DNA concentrations in the cell microenvironment. We propose a novel approach that combines these two strategies in which cationic polymer/DNA complexes are tethered to a substrate that supports cell adhesion. The cationic polymers package the DNA for efficient internalization and the surface tethering functions to maintain elevated concentrations in the cell microenvironment for cells adhered to the substrate. The cationic polymer polylysine (degree of polymerization equal to 19 or 150) was modified with biotin groups, which was confirmed by mass spectrometry and biochemical analysis. Complex formation of DNA with biotinylated-polylysine, or mixtures of biotinylated and nonbiotinylated polylysines, was confirmed by gel electrophoresis. Plasmid DNA encoding for the reporter gene beta-galactosidase was complexed with different mixtures of biotinylated and nonbiotinylated polylysine and incubated on neutravidin (nonglycosylated avidin)-coated surfaces. DNA surface densities ranging from 0.1 to 4.3 microg/cm2 were observed and found to be a function of the number of biotin groups, the molecular weight of the polylysine, and the amount of DNA. HEK293T or NIH/3T3 cells were then seeded onto the DNA-modified surfaces, and transfection was quantified at 48 and 96 h. Transfection by the DNA surfaces was observed with both cell lines, and expression levels up to 100 fold greater than bulk delivery of the complexes was obtained. Transfection was found to be a function of the surface DNA quantities and the number of tethers on the complex. Transfected cells were observed only in the region in which DNA complexes were tethered, suggesting that the location of transfected cells can be specifically controlled. Surface tethering of DNA represents a promising approach to enhancing gene transfer and spatially controlling gene delivery, which may have applications to a multitude of fields ranging from tissue engineering to functional genomics.     

Different modes of hydrocarbon uptake by two Pseudomonas species

The mode of n-hexadecane uptake by two organisms-Pseudomonas M 1 and Pseudomonas N 1-was studied. During the growth of Pseudomonas M 1 on n-hexadecane, no extracellular biosurfactant/bioemulsifier was produced, no significant n-hexadecane pseudosolubilization was observed, and the reduction of surface and interfacial tensions in the cell-free culture broth was negligible. EDTA, a known inhibitor of hydrocarbon pseudosolubilization, did not inhibit the growth of the organism on n-hexadecane. Normal hexadecane-grown cells showed strong surface-active properties and capacity to adhere firmly to hydrocarbon phase. It was concluded that in this organism, surface-active properties of the cells facilitate attachment of cells to the hydrocarbon-water interface generated by agitation, and promote substrate uptake and growth; no hydrocarbon pseudosolubilization or extracellular mediator is involved in the substrate uptake. Pseudomonas N 1 grew on n-hexadecane much faster than Pseudomonas M 1. Growth of this organism on n-hexadecane was associated with the extracellular production of biosurfactant-bioemulsifier and n-hexadecane pseudosolubilizing factor; the growth was strongly inhibited by 5 mM EDTA, indicating that hydrocarbon pseudosolubilization was the dominant factor in substrate uptake. The rate of n-hexadecane pseudosolubilization was high enough to account for the substrate up take rate. Hydrocarbon emulsifying and n-hexadecane pseudosolubilizing factors were isolated and tentatively characterized as lipoprotein and glycoprotein, respectively. Both factors act in a synergistic manner to provide enhanced hydrocarbon transport to cells through pseudosolubilization. It is proposed that this facility of mediated hydrocarbon transport is the basis for the relatively fast rate of growth of Pseudomonas N 1 on hydrocarbon.     

一株石油烃降解菌的细胞疏水性及其乳化性质

【目的】从新疆油田石油污染土壤中分离到一株在25 °C条件下利用烃类产生生物表面活性剂的菌株红球菌(Rhodococcus sp.) HL-6, 对其菌体细胞疏水性及所产表面活性剂进行研究。【方法】通过细胞粘附性、表面张力及乳化活性测定对菌株所产表面活性剂进行性质研究。【结果】菌株HL-6在亲水性和疏水性基质中均能产生生物表面活性剂, 在疏水性基质中可以将培养液表面张力由初始的62.487 mN/m降到30.667 mN/m, 培养液在pH 6?9及NaCl浓度1%?5%范围内乳化效果良好, 在4 °C到55 °C范围内乳化效果均为100%, 菌株对柴油的耐受能力很高, 在30%柴油浓度下依然生长良好并且有44%的乳化活性。【结论】HL-6菌株的细胞表面具有很强的疏水性, 这有助于菌体细胞对烃类的摄取。该菌株能够利用烃类基质生产生物表面活性剂, 可以明显降低培养液表面张力并且对石油烃具有良好的乳化作用。说明菌株HL-6能够适应海洋滩涂石油污染的环境, 并可用于严重石油污染区域的生物修复。