Extracellular polysaccharides of Rhodococcus rhodochrous S-2 stimulate the degradation of aromatic components in crude oil by indigenous marine bacteria

Rhodococcus rhodochrous S-2 produces extracellular polysaccharides (S-2 EPS) containing D-glucose, D-galactose, D-mannose, D-glucuronic acid, and lipids, which is important to the tolerance of this strain to an aromatic fraction of (AF) Arabian light crude oil (N. Iwabuchi, N. Sunairi, H. Anzai, M. Nakajima, and S. Harayama, Appl. Environ. Microbiol. 66:5073-5077, 2000). In the present study, we examined the effects of S-2 EPS on the growth of indigenous marine bacteria on AF. Indigenous bacteria did not grow significantly in seawater containing AF even when nitrogen, phosphorus, and iron nutrients were supplemented. The addition of S-2 EPS to seawater containing nutrients and AF resulted in the emulsification of AF, promotion of the growth of indigenous bacteria, and enhancement of the degradation of AF by the bacteria. PCR-denaturing gradient gel electrophoresis analyses show that addition of S-2 EPS to the seawater containing nutrients and AF changed the composition of the bacterial populations in the seawater and that bacteria closely related to the genus Cycloclasticus became the major population. These results suggest that Cycloclasticus was responsible for the degradation of hydrocarbons in AF. The effects of 15 synthetic surfactants on the degradation of AF by indigenous marine bacteria were also examined, but enhancement of the degradation of AF was not significant. S-2 EPS was hence the most effective of the surfactants tested in promoting the biodegradation of AF and may thus be an attractive agent to use in the bioremediation of oil-contaminated marine environments.     

Structural elucidation of the EPS of slime producing Brevundimonas vesicularis sp. isolated from a paper machine

The slime forming bacteria Brevundimonas vesicularis sp. was isolated from a paper mill and its EPS was produced on laboratory scale. After production, the exopolysaccharide (EPS) was purified and analysed for its purity and homogeneity, HPSEC revealed one distinct population with a molecular mass of more than 2,000 kDa. The protein content was around 9 w/w%. The sample was analysed to determine its chemical structure. The EPS was found to consist of rhamnose, glucose, galacturonic acid and glucuronic acid. Due to the presence of uronic acids the molar ratio between the four sugars found varies from 3:5:2:4 by sugar composition analyses after methanolysis to 1:1:1:1 found by NMR. A repeating unit with a molecular mass of 678 Da was confirmed by MALDI-TOF mass spectrometry after mild acid treatment. 13C and 1H hetero- and homonuclear 2D NMR spectroscopy of the native and partial hydrolysed EPS revealed a repeating unit, no non-sugar substituents were present.     

Structural analysis of an acidic, fatty acid ester-bonded extracellular polysaccharide produced by a pristane-assimilating marine bacterium, Rhodococcus erythropolis PR4

Rhodococcus erythropolis PR4 is a marine bacterium that can degrade various alkanes including pristane, a C(19) branched alkane. This strain produces a large quantity of extracellular polysaccharides, which are assumed to play an important role in the hydrocarbon tolerance of this bacterium. The strain produced two acidic extracellular polysaccharides, FR1 and FR2, and the latter showed emulsifying activity toward clove oil, whereas the former did not. FR2 was composed of D-galactose, D-glucose, D-mannose, D-glucuronic acid, and pyruvic acid at a molar ratio of 1:1:1:1:1, and contained 2.9% (w/w) stearic acid and 4.3% (w/w) palmitic acid attached via ester bonds. Therefore, we designated FR2 as a PR4 fatty acid-containing extracellular polysaccharide or FACEPS. The chemical structure of the PR4 FACEPS polysaccharide chain was determined by 1D (1)H and (13)C NMR spectroscopies as well as by 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The sugar chain of PR4 FACEPS was shown to consist of tetrasaccharide repeating units having the following structure: [structure: see text].     

Structural analysis of an extracellular polysaccharide produced by a benzene tolerant bacterium, Rhodococcus sp. 33

Rhodococcus sp. 33 can tolerate and efficiently degrade various concentrations of benzene, one of the most toxic and prevailing environmental pollutants. This strain produces a large quantity of extracellular polysaccharide (33 EPS), which plays an important role in the benzene tolerance in Rhodococcus sp. 33, especially by helping the cells to survive an initial challenge with benzene. This EPS has been reported to be composed of D-galactose, D-glucose, D-mannose, D-glucuronic acid, and pyruvic acid at a molar ratio of 1:1:1:1:1. To understand the protective effect of 33 EPS, we determined its chemical structure by using 1H and 13C NMR spectroscopy including 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The polysaccharide was shown to consist of tetrasaccharide repeating units with the following structure: [structure: see text].     

Bacterial exopolysaccharides produced by newly discovered bacteria belonging to the genus Halomonas, isolated from hypersaline habitats in Morocco

We have studied the exopolysaccharides (EPS) from a new group of moderately halophilic bacteria belonging to the genus Halomonas. The quantity of EPS produced, its chemical composition and physical properties depend greatly upon the bacterial strain. The majority of the polymers produced viscous solutions and/or emulsified different hydrocarbon compounds. The most interesting strain, S-30, produced EPS at 2.8 g/l with a maximum viscosity of 23.5 Pa·5 and exhibited pseudoplastic behavior. This EPS emulsified five hydrocarbons more efficiently than did four control surfactants tested. Its monosaccharide composition was glucose:galactose:manose:glucuronic acid in equimolar ratio. Some two-thirds of the strains tested emulsified crude oil better than control surfactants did. There are many potential industrial applications for polysaccharides with these qualities. Journal of Industrial Microbiology & Biotechnology (2000)24, 374–378. Received 09 August 1999/ Accepted in revised form 23 March 2000     

黄土高原石油污染土壤微生物群落结构及其代谢特征

针对污染胁迫下土壤微生物群落变化和代谢变异等问题,基于平板稀释法和Biolog微平板分析方法,研究了陕北黄土高原石油污染土壤微生物群落结构、代谢特征及其功能多样性。结果表明,不同类群的土壤微生物对石油污染胁迫的响应不同,污染土壤细菌和真菌数量高出清洁土壤1个数量级,而污染土壤的放线菌数量极显著减少(P0.01);污染土壤和清洁土壤微生物对糖类和多聚物类碳源较易利用,污染土壤微生物总体上代谢碳源的种类和活性均低于清洁土壤。微生物群落主成分分析(PCA)表明,石油污染土壤和清洁土壤的微生物群落存在显著差异(P0.01),起分异作用的碳源主要为糖类,其次是羧酸类和氨基酸类;随着土壤石油含量增加,典型变量值变异(离散)增大,土壤微生物群落结构稳定性降低。微生物群落多样性分析表明,Shannon丰富度指数(H)、McIntosh均一度指数(U)和Simpson优势度指数(1/D)均达到极显著差异(P0.01),污染土壤微生物群落H和U低于清洁土壤,但是一定浓度的石油污染可以刺激土壤微生物群落中优势种群的生长,1/D增高。研究结果为陕北黄土高原石油污染区土壤微生物修复提供理论基础。     

Characteristics of bioemulsifier V2-7 synthesized in culture media added of hydrocarbons: chemical composition, emulsifying activity and rheological properties

The bioemulsifier V2-7 is an exopolysaccharide (EPS) synthesized by strain F2-7 of Halomonas eurihalina and it has the property of emulsifying a wide range of hydrocarbons i.e. n-tetradecane, n-hexadecane, n-octane, xylene mineral light and heavy oils, petrol and crude oil. Characteristics of exopolysaccharide V2-7 produced in media supplemented with various hydrocarbons (n-tetradecane, n-hexadecane, n-octane, xylene, mineral light oil, mineral heavy oil, petrol or crude oil) were studied. Yield production varied from 0.54 to 1.45 g L(-1) according to the hydrocarbon added, in the same way chemical composition, viscosity and emulsifying activity of EPS varied with the culture conditions. Respect to chemical composition, percentage of uronic acids found in exopolymers produced in hydrocarbon media was always higher than that described for V2-7 EPS (1.32%) obtained with glucose. This large amount of uronic acid present could be useful in biodetoxification and waste water treatment. On the other hand, the highest amount of biopolymer was synthesized with mineral light oil, while the most active emulsifiers were those obtained from media added with petrol and n-octane. Furthermore, all EPS were capable of emulsifying crude oil more efficiently than the three chemical surfactants tested as control (Tween 20, Tween 80 and Triton X-100). The capacity of strain F2-7 to grow and produce bioemulsifier in presence of oil hydrocarbons together with the high emulsifying activity and low viscosity power of the biopolymers synthesized in hydrocarbons media could be considered highly beneficial for application of both bioemulsifier and producing strain in bioremediation of oil pollutants.     

Microemulsion for topical delivery of fenoprofen calcium: in vitro and in vivo evaluation

The aim of this study was to investigate microemulsion (ME) based topical delivery system for fenoprofen calcium (FPCa) to eliminate its oral gastrointestinal adverse effects. ME was prepared by the water titration method using oleic acid as oil phase, tween 80 as a surfactant and propylene glycol as a cosurfactant. Oleic acid was selected as oil phase due to its good solubilizing capacity. ME existence region was determined using pseudo-ternary phase diagrams for preparing different formulations. Six different formulations were selected with various values of oil (25–68%), water (2–3%), and the mixture of surfactant and cosurfactant (1:1) (24–67%). The selected ME formulae were characterized for optical birefringence, transmission electron microscopy (TEM), pH, % transmittance, electronic conductivity, drug content, droplet size, rheological properties and stability evaluation. In vitro release study of FPCa from ME s through the synthetic membrane and hairless rat skin were evaluated. The optimized formula ME5 consisting of 5% w/w FPCa, 60% w/w oleic acid as oil phase, 3% w/w aqueous phase, and 32% w/w of surfactant phase containing Tween 80 and propylene glycol (1:?1) showed the highest transdermal flux and highest skin permeation rate. Finally, the % inhibition of carrageenan-induced rat paw edema of the optimized formula ME5 was highly significant (p?0.001) as compared to plain gel of FPCa. In conclusion, ME is a promising technique for topical delivery of FPCa.     

Production of fatty acid components of meadowfoam oil in somatic soybean embryos

The seed oil of meadowfoam (Limnanthes alba) and other Limnanthes spp. is enriched in the unusual fatty acid Delta(5)-eicosenoic acid (20:1Delta(5)). This fatty acid has physical and chemical properties that make the seed oil of these plants useful for a number of industrial applications. An expressed sequence tag approach was used to identify cDNAs for enzymes involved in the biosynthesis of 20:1Delta(5)). By random sequencing of a library prepared from developing Limnanthes douglasii seeds, a class of cDNAs was identified that encode a homolog of acyl-coenzyme A (CoA) desaturases found in animals, fungi, and cyanobacteria. Expression of a cDNA for the L. douglasii acyl-CoA desaturase homolog in somatic soybean (Glycine max) embryos behind a strong seed-specific promoter resulted in the accumulation of Delta(5)-hexadecenoic acid to amounts of 2% to 3% (w/w) of the total fatty acids of single embryos. Delta(5)-Octadecenoic acid and 20:1Delta(5) also composed <1% (w/w) each of the total fatty acids of these embryos. In addition, cDNAs were identified from the L. douglasii expressed sequence tags that encode a homolog of fatty acid elongase 1 (FAE1), a beta-ketoacyl-CoA synthase that catalyzes the initial step of very long-chain fatty acid synthesis. Expression of the L. douglassi FAE1 homolog in somatic soybean embryos was accompanied by the accumulation of C(20) and C(22) fatty acids, principally as eicosanoic acid, to amounts of 18% (w/w) of the total fatty acids of single embryos. To partially reconstruct the biosynthetic pathway of 20:1Delta(5) in transgenic plant tissues, cDNAs for the L. douglasii acyl-CoA desaturase and FAE1 were co-expressed in somatic soybean embryos. In the resulting transgenic embryos, 20:1Delta(5) and Delta(5)-docosenoic acid composed up to 12% of the total fatty acids.     

Chemical Composition of Two Exopolysaccharides from Bacillus thermoantarcticus

The thermophilic bacterium Bacillus thermoantarcticus produces two exocellular polysaccharides (EPS 1 and EPS 2), which can be obtained from the supernatant of liquid cultures by cold-ethanol precipitation, in yields as high as 400 mg liter(sup-1). The EPS fraction was produced with all substrates tested, although a higher yield was obtained with mannose as the carbon and energy source. The EPS content was proportional to the total biomass. On a weight basis, EPS 1 and EPS 2 represented about 27 and 71%, respectively, of the total carbohydrate fraction. EPS 1 is a sulfate heteropolysaccharide containing mannose and glucose in a relative molar proportion of 1.0 and 0.7, respectively. EPS 2 is a sulfate homopolysaccharide containing mannose as the major component. The absolute configurations of hexoses were shown to be d for both EPSs. Nuclear magnetic resonance spectra confirmed the presence of (alpha)-d-mannose and (beta)-d-glucose in EPS 1 and only (alpha)-d-mannose in EPS 2. In addition, (sup1)H nuclear magnetic resonance analysis and chemical analysis indicated the presence of pyruvic acid in EPS 2.     

Extracellular polysaccharides of Erwinia futululu,a bacterium associated with a fungal canker disease of Eucalyptus spp

Extracellular polysaccharides (EPSs) produced by an Erwinia spp. associated with a fungal canker disease of Eucalyptus were fractionated into two polysaccharides, one that was identified with that produced by Erwinia stewartii. The other has a similar structure, but with one terminal Glc residue replaced by pyruvic acid to give 4,6-O-[(R)-1-carboxyethylidene)-Galp. Their structures were determined using a combination of chemical and physical techniques including methylation analysis, periodate oxidation, low-pressure gel filtration and anion-exchange chromatographies, high-pH anion-exchange chromatography, mass spectrometry and 1D and 2D 1H NMR spectroscopy. The new polysaccharides, identified as EPS Futululu FF-1 and FF-2, have the following structures:The molecular weights of the polysaccharides range from 1.3-2.1x10(6) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions.     

Production of extracellular polysaccharide, EPS WN9, fromPaenibacillus sp. WN9 KCTC 8951P and its usefulness as a cement mortar admixture

The production of extracellular polysaccharide, EPS WN9, fromPaenibacillus sp. and its suitability as a viscosity modifying admixture for cement mortar mixing were investigated. After 48 h culture in an optimized medium, cell growth and EPS production were 1,2 g/L and 4.0 g/L, respectively. By adding EPS WN9 to mortar, it was possible to prepare a homogeneous mortar without material segregation and excess air entrapment. The optimal amount of EPS addition to mortar was found to be 0.02 to 0.05%(w/w) of the cement used. Increasing the dosage of EPS WN9 from 0 to 0.05%(w/w) resulted in a setting retardation of 0.14 h to 0.8 h and an increase in the compressive strength of mortar of 10 to 20%.     

Bioremediation of Oil-contaminated Soil Using Chicken Manure

In less developed countries, the prevalence of soil contaminated with used lubricating oil is high and the situation worsens with the economic advancement. The contamination has been shown to adversely affect the environment and human health. To mitigate, bioremediation could be adopted to tackle the problem of hydrocarbon-contaminated soil. Thus, this experimental research carried out the bioremediation using chicken manure in soils contaminated with 5%, 10% and 20% w/w used lubricating oil for a 42-day composting period. To compare, this research also experimented with the 5%, 10% and 20% oil-contaminated soils untreated with chicken manure. The results showed that the highest total petroleum hydrocarbons (TPHs) reduction efficiency of >60% was achieved in the 5% oil-contaminated compost remediated with chicken manure. The highest biodegradation rate of lubricating oil of 0.023–0.0025 day^?1 as measured by the first-order kinetics could also be achieved under the 5% oil contamination condition with the application of chicken manure. The findings highlight the prospect of chicken manure as a proper nutrient for enhanced remediation of hydrocarbon-contaminated soils, particularly of low contamination concentrations.     

Essential oil constituents derived from different organs of a relictual conifer Wollemia nobilis

The chemical composition of the essential oil of leaves (0.9%, w/v) and twigs (0.33%, w/v) of Wollemia nobilis (Araucariaceae) – a remnant species thought to have been extinct for 65 million years – was investigated by GC/MS. The main constituents of both leaf- and twig-derived oil samples were 16-kaurene (61.8% and 38.2%, respectively) and germacrene D (9.9% and 22%). The principal difference was a considerably more pronounced sesquiterpene presence in the twig-oil, amounting to 33.5%, than in its folial counterpart (23.4%). On the contrary, while remaining the dominant group in both oil samples under investigation, diterpenoids were relatively more abundant in leaf-derived oil constituting 65.3%, versus 41.7% detected in twigs. To our knowledge, this is the first report dealing with the essential oil composition of Wollemi pine twigs, as opposed to the leaf-derived volatiles.     

Discovery of the dual polysaccharide composition of emulsan and the isolation of the emulsion stabilizing component

Emulsan has been reported as an emulsion stabilizing amphipathic lipoheteropolysaccharide secreted by the oil-degrading bacterium Acinetobacter venetianus RAG-1. Previously, emulsan was regarded as a single polymer. As a result of developing a new purification process, we have discovered that emulsan is a complex of approximately 80% (w/w) lipopolysaccharide (LPS) and 20% (w/w) high molecular weight exopolysaccharide (EPS). The EPS was purified to 98% (w/w) using tangential flow filtration, Triton X-114 phase extraction, ammonium sulfate precipitation, and hydrophobic interaction chromatography. Several previously reported physical properties of emulsan can be attributed to the LPS fraction, such as charge, fatty acid profile, and solution behavior, while the EPS is responsible for the emulsion stabilization activity. The EPS is believed to be cationic in nature, thus providing an electrostatic binding mechanism for the formation of the emulsan complex.     

Quantitative analysis for the effect of plant oil and fatty acid on Tuber melanosporum culture by uniform design combined with partial least squares regression

Uniform design and partial least squares regression were adopted to quantitatively analyze the effects of plant oil and fatty acid as well as their addition amount and addition time on the performance of Tuber melanosporum submerged fermentation. The regression models showed the optimal scheme was the addition of 1.2 mL soybean oil on day 9, which was validated by experiment. The maximal biomass of 25.89 ± 1.01 g/L and extracellular polysaccharides (EPS) production of 6.51 ± 0.68 g/L were obtained, which were enhanced by 18.5% and 86%, respectively. Palmitic acid was identified to be the key component to stimulate the cell growth and EPS biosynthesis, and stearic acid was beneficial for the production of intracellular polysaccharides. Not only the biomass but also EPS production obtained in this work are the highest reported in the batch fermentation of truffle.     

一株高温产粘菌株的筛选及其产胞外聚合物分析

从油井采出水中分离到一株高温产胞外聚合物的细菌MS-1,经16S rDNA基因序列分析属于芽孢杆菌属(Bacillus sp.).该菌能在60℃生长并产生胞外聚合物,其中胞外多糖含量为48.3%～54.5%.主要由甘露糖、葡萄糖、半乳糖组成,摩尔比分别是2.04:1.00:0.89.胞外聚合物中蛋白含量为37.2%～42.4%,主要由甲硫氨酸、亮氨酸、天门冬氨酸、丙氨酸、组氨酸和丝氨酸组成.利用透射电镜和环境扫描电镜对胞外聚合物的形成进行了观察.该菌的分离和研究为高温油藏的微生物调剖和驱油奠定了生物学基础.     

Effectiveness of bioremediation for oil-polluted Antarctic seawater

The effectiveness of a specific fertiliser (INIPOL EAP 22) addition on bioremediation of oil-contaminated Antarctic coastal seawater was determined in the “Terre Adelie” area. Mesocosm studies were conducted to evaluate the effects of “Arabian light” crude oil contamination on coastal bacterioplanktonic communities. After oil addition, regular surveys of the bacterial changes of the oil-contaminated seawater were performed during 5-week periods during the austral summer of 1992/1993 and 1993/1994. All results (total, saprophytic and hydrocarbon-utilising bacterial abundance) clearly revealed a significant response of Antarctic bacterial communities to hydrocarbon contamination. A 1 order of magnitude increase of bacterial microflora occurred in seawater after crude oil contamination. A concomitant enrichment in oil-degrading bacteria was generally observed, from less than 0.001% of the community in uncontaminated samples to up to 50% after 3 weeks of contamination. Addition of fertiliser (INIPOL EAP 22) induced clear enhancement of both saprophytic and hydrocarbon-utilising microflora. Chemical analysis of the residual hydrocarbon fractions confirmed that fertiliser application increased the rate of oil biodegradation. Received: 27 March 1997 / Accepted: 20 October 1997