鼠李糖脂的表面化学和生物合成及其在垃圾堆肥中的应用展望

鼠李糖脂是生物表面活性剂中一类非常重要而应用广泛的微生物发酵产物,在环境污染修复中需求量越来越多。针对近十年来国内外对鼠李糖脂生物表面活性剂的研究,较系统地总结了其化学结构、性质、生物合成机理及产量调节方法,及大规模生产鼠李糖脂的基础研究工作,并对其在城市生活垃圾堆肥中的应用做了展望。     

一株生物表面活性剂产生菌的分离及其特性研究

模炼油厂污泥中分离得到1株生物表面活性剂产生菌C-3, 根据其生理生化特性和16S rDNA序列相似性分析, 将其鉴定为铜绿假单胞菌(Pseudomonas aeruginosa)。初步研究了其产生物表面活性剂的最适条件, 在以植物油为碳源、30°C、初始pH 8、Ca2+浓度20 mg/L、250 mL三角瓶中装75 mL发酵液的条件下, 最利于菌株的生长和生物表面活性剂产生。它的成分为糖脂类物质, 临界胶束浓度(CMC)为50 mg/L, 具有很好的增溶效果。     

铜绿假单胞菌高产鼠李糖脂菌株的筛选

从多种来源筛选高产鼠李糖脂的菌株,并研究菌种发酵特性和鼠李糖脂产物的理化性质。采用CTAB平板初步筛选鼠李糖脂合成菌株,通过分析菌株的16S r RNA基因序列确定细菌种属,采用薄层色谱、红外光谱分析产物性质。结果显示,利用CTAB平板初筛获得163株阳性菌株,初步发酵确定10株高产细菌鼠李糖脂的产量为12.2-17.7 g/L,10株细菌均鉴定为铜绿假单胞菌。挑选产量最高的菌株B12,分别以甘油、菜籽油、花生饼粉或葵花籽饼粉为碳源进行发酵,发现菜籽油为合成鼠李糖脂的最佳碳源。进一步对比在35℃、37℃和40℃的发酵水平,发现37℃条件下鼠李糖脂产量最高,为26.8 g/L。最后,对鼠李糖脂发酵产物进行了初步纯化,并进行了薄层色谱和红外光谱分析。菌株B12能够合成较高水平的鼠李糖脂,可能成为工业生产的候选菌株。     

1株耐高温生物表面活性剂产生菌的特性研究

研究了耐高温生物表面活性剂产生菌ZY-3的生理生化特性,并通过测定发酵液的菌体密度、表面张力和乳化活性等指标,研究不同碳源和初始pH对菌株ZY-3生长和产生物表面活性剂的影响,同时对其所产生物表面活性剂进行了初步分离和性质分析。菌株ZY-3被初步鉴定为芽胞杆菌属(Bacillus),具有产酸、不产H_2S、还原硝酸盐等特性。在以淀粉为碳源、初始pH 6.0的培养基中发酵,产生物表面活性剂多且稳定;在种子培养基和发酵培养基中都有淀粉的条件下,菌体生长较多,降低表面张力和乳化的作用均较强,所产生物表面活性剂可以使发酵液的表面张力从72.1 mN/m降到53.1 mN/m,乳化活性从0升高到24%。初步判断产物为糖脂类阴离子表面活性剂。     

一株产鼠李糖脂菌株的筛选、鉴定及其产物特性分析

鼠李糖脂是最常见,研究最深入,应用最广泛的一类生物表面活性剂。从油田附近、沼气池旁的土壤中分离得到了25株菌,通过硫酸-苯酚反应,乳化实验,排油性实验,薄层色谱实验筛选产鼠李糖脂的菌株并表征产生的鼠李糖脂,通过16S r DNA序列确定细菌的种属。硫酸-苯酚反应显示有2株菌可能产鼠李糖脂;5株菌的发酵液具有明显的乳化效果,命名为"其红"这株菌的乳化指数可达58.97%;1株菌的发酵液上清稀释10倍后排油圈直径仍可达3.53 cm;薄层色谱实验也显示"其红"菌产鼠李糖脂。在四个实验中"其红"菌都检测到了鼠李糖脂,故确认"其红"菌可以产鼠李糖脂。16S r DNA序列分析表明"其红"菌属于希瓦氏菌属(Shewanella)并与腐败希瓦菌(S.putrefaciens LMG 26268(T))相似度最高。     

基因工程微生物合成鼠李糖脂表面活性剂的研究进展

鼠李糖脂是一类重要的生物表面活性剂。相比于化学合成的表面活性剂,其具有更优秀的理化性质及环境友好等特点,被广泛应用于微生物采油、环境污染修复等工程中。目前,鼠李糖脂的工业生产主要采用铜绿假单胞菌这一具有致病性的天然合成菌株,与此同时,受菌株遗传背景的限制,优化发酵过程等方法在产量提升方面遇到了一些瓶颈问题。利用基因工程方法对菌株进行改良有望进一步提高鼠李糖脂生产的安全性、产量、产物性能等多项指标,因此受到了越来越广泛的关注。本文综述了近年来利用基因工程方法优化鼠李糖脂生物合成的最新进展,讨论了异源合成、代谢通路改造、基因表达优化、蛋白质工程、底盘工程等多种策略的应用,并展望了一系列可行的研究方向。     

一株产生脂肽的枯草芽孢杆菌的分离鉴定及脂肽对原油的作用

从大庆油田地层水中分离到一组能高效产生生物表面活性剂的菌株,采用sfp基因PCR鉴定的方法从中分离到一株芽孢杆菌ZW-3,该菌株能够产生大量表面活性物质,采用细菌生理生化鉴定结合16S rDNA序列的系统发育学分析确定该菌株为枯草芽孢杆菌(Bacillus subtilis),通过薄层层析色谱(TLC)、高效液相色谱(HPLC)分析其代谢产物,初步鉴定为脂肽(Lipopeptide);该脂肽生物表面活性剂理化性质显示它能使培养基的表面张力从68.92mN/m降低25.19mN/m、原油/水的界面张力从23.53mN/m降低到4.57mN/m,与1.8%的NaOH溶液复配可以将油水界面张力降低到1.2×10-3 mN/m,其临界胶束浓度为33.3mg/L(3.24×10-5 mol/L),并具有较好的乳化活性和发泡性能,说明该菌株代谢的脂肽生物表面活性剂在提高石油采收率中具有广泛的应用前景.     

鼠李糖脂生物表面活性剂的研究进展

鼠李糖脂是一种重要的生物表面活性剂。综述了鼠李糖脂生物表面活性剂的化学结构、特性、生理学功能及其发酵生产,特别讨论了利用廉价原料——工农业的废物,如植物油废渣等来生产鼠李糖脂,其不仅可降低生产成本,还能减少工农业废渣对环境的污染,降低其处理费用。     

表面张力曲线法测定生物表面活性剂的浓度

介绍一种利用表面张力曲线测定生物表面活性剂发酵液浓度的方法,通过测定不同浓度鼠李糖脂标准溶液的表面张力,制作浓度和表面张力的x-y散点图,再根据希斯科夫斯基经验公式利用Origin软件做x-y散点图的拟合曲线,得到该公式的相关参数,然后利用该经验公式,通过测定稀释后待测发酵液的表面张力,可求得原发酵液的鼠李糖脂浓度。和常用的方法相比,该方法具有快速、简单、准确和测定成本低的优点。     

9株海洋来源铜绿假单胞菌合成鼠李糖脂的比较分析

目的:从海洋来源的铜绿假单胞菌中筛选多株具有鼠李糖脂合成能力的菌株。方法:以9株分离自不同海洋环境的铜绿假单胞菌为研究对象,考察并比较其发酵合成鼠李糖脂生物表面活性剂的表面活性、产量和产物成分的差异,扩增并比对合成途径中的关键基因。结果:9株菌的发酵产物均具有表面活性,其中菌株1A01151发酵液的表面活性最强,表面张力值可降低至28 m N/m;9株菌的基因组中均含有鼠李糖脂合成途径中关键基因rhl AB和rhl C,都具有合成单、双鼠李糖脂的能力;菌株1A01151和1A00364的发酵产量最高(2.69 g/L),产物经LC-MS/MS检测,所合成的鼠李糖脂同系物组分不同,双糖双脂的含量最高(1A01151:75.96%;1A00364:61.01%)。结论:海洋来源的铜绿假单胞菌是具有鼠李糖脂高产潜力的菌株,可用于合成性能不同、组成多样的鼠李糖脂生物表面活性剂。     

Rhamnolipid stimulates uptake of hydrophobic compounds by Pseudomonas aeruginosa

The biodegradation of hexadecane by five biosurfactant-producing bacterial strains (Pseudomonas aeruginosa UG2, Acinetobacter calcoaceticus RAG1, Rhodococcus erythropolis DSM 43066, R. erythropolis ATCC 19558, and strain BCG112) was determined in the presence and absence of exogenously added biosurfactants. The degradation of hexadecane by P. aeruginosa was stimulated only by the rhamnolipid biosurfactant produced by the same organism. This rhamnolipid did not stimulate the biodegradation of hexadecane by the four other strains to the same extent, nor was degradation of hexadecane by these strains stimulated by addition of their own biosurfactants. This suggests that P. aeruginosa has a mode of hexadecane uptake different from those of the other organisms. Rhamnolipid also enhanced the rate of epoxidation of the aliphatic hydrocarbon alpha,omega-tetradecadiene by a cell suspension of P. aeruginosa. Furthermore, the uptake of the hydrophobic probe 1-naphthylphenylamine by cells of P. aeruginosa was enhanced by rhamnolipid, as indicated by stopped-flow fluorescence experiments. Rhamnolipid did not stimulate the uptake rate of this probe in de-energized cells. These results indicate that an energy-dependent system is present in P. aeruginosa strain UG2 that mediates fast uptake of hydrophobic compounds in the presence of rhamnolipid.     

Rhamnolipid production by Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene

The potential of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene (vgb) for rhamnolipid production was studied. P. aeruginosa (NRRL B-771) and its transposon mediated vgb transferred recombinant strain, PaJC, were used in the research. The optimization of rhamnolipid production was carried out in the different conditions of cultivation (agitation rate, the composition of culture medium and temperature) in a time-course manner. The nutrient source, especially the carbon type, had a dramatic effect on rhamnolipid production. The PaJC strain and the wild type cells of P. aeruginosa started producing biosurfactant at the stationary phase and its concentration reached maximum at 24 h (838 mg/l(-1)) and at 72 h (751 mg l(-1)) of the incubation respectively. Rhamnolipid production was optimal in batch cultures when the temperature and agitation rate were controlled at 30 degrees C and 100 rpm. It reached 8373 mg l(-1) when the PaJC cells were grown in 1.0% glucose supplemented minimal media. Genetic engineering of biosurfactant producing strains with vgb may be an effective method to increase its production.     

Biosurfactant synthesis by Pseudomonas aeruginosa LBI isolated from a hydrocarbon-contaminated site

Aims: Pseudomonas aeruginosa LBI (Industrial Biotechnology Laboratory) was isolated from hydrocarbon-contaminated soil as a potential producer of biosurfactant and evaluated for hydrocarbon biodegradation. The emulsifying power and stability of the product was assessed in the laboratory, simulating water contamination with benzene, toluene, kerosene, diesel oil and crude oil at various concentrations. Methods and Results: Bacteria were grown at 30°C and shaken at 200 rpm for 168 h, with three repetitions. Surface tension, pH and biosurfactant stability were observed in the cell-free broth after 168 h of incubation. The strain was able to produce biosurfactant and grow in all the carbon sources under study, except benzene and toluene. When cultivated in 30% (w/v) diesel oil, the strain produced the highest quantities (9·9 g l⁻¹) of biosurfactant. The biosurfactant was capable of emulsifying all the hydrocarbons tested. Conclusion: The results from the present study demonstrate that Ps. aeruginosa LBI can grow in diesel oil, kerosene, crude oil and oil sludge and the biosurfactant produced has potential applications in the bioremediation of hydrocarbon-contaminated sites. Significance and Impact of the Study: Pseudomonas aeruginosa LBI or the biosurfactant it produces can be used in the bioremediation of environmental pollution induced by industrial discharge or accidental hydrocarbon spills.     

Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials

This study was aimed at the development of economical methods for higher yields of biosurfactant by suggesting the use of low-cost raw materials. Two oil-degrading strains, Pseudomonas aeruginosa GS9-119 and DS10-129, were used to optimize a substrate for maximum rhamnolipid production. Among the two strains, the latter produced maxima of 4.31, 2.98, and 1.77 g/L rhamnolipid biosurfactant using soybean oil, safflower oil, and glycerol, respectively. The yield of biosurfactant steadily increased even after the bacterial cultures reached the stationary phase of growth. Characterization of rhamnolipids using mass spectrometry revealed the presence of dirhamnolipids (Rha-Rha-C(10)-C(10)). Emulsification activity of the rhamnolipid biosurfactant produced by P. aeruginosa DS10-129 was greater than 70% using all the hydrocarbons tested, including xylene, benzene, hexane, crude oil, kerosene, gasoline, and diesel. P. aeruginosa GS9-119 emulsified only hexane and kerosene to that level.     

In Situ Rhamnolipid Production at an Abandoned Petroleum Refinery

A simple screening method was developed to detect in situ biosurfactant production by exploiting the relationship between surface tension (ST) and surfactant concentration. Filtered groundwater from contaminated wells with ST values of 60 to 70 dynes/cm decreased to 29 dynes/cm after being concentrated 10 to 15 times in a rotary evaporator, indicating that biosurfactants in the sample reached the critical micelle concentration (CMC). Samples from uncon-taminated groundwater concentrated 25 times showed no decrease in ST below 72 dynes/cm, suggesting that biosurfactants were not present. Microorganisms from soil cores were cultured on diesel fuel and identified using fatty acid methyl ester (FAME) analysis. Pseudomonas aeruginosa was found at very low numbers in uncontami-nated soil but was the dominant species in contaminated soil, indicating that hydrocarbon release impacted microbial diversity significantly. High-performance liquid chromatography (HPLC) was used to quantify rhamnolipids, biosurfactants produced by P. aeruginosa, in concentrated ground-water samples. Rhamnolipid concentrations in samples from contaminated soil were observed equal to their CMC (50 mg/L), but were not detected in samples from un-contaminated wells. We conclude that biosurfactant production may be an indicator of intrinsic bioremediation.     

一种脂肽类生物表面活性剂产生菌的筛选

从油田地层水中筛选分离得到1株能够产生表面活性剂的细菌,经鉴定为枯草芽孢杆菌。分析了该菌株的生理形态和生长特性,以及该菌株代谢产生的生物表面活性剂的性质。薄层色谱与原位水解显色和红外光谱分析表明,培养后菌株代谢产生的生物表面活性为脂肽。它能使水的表面张力降低到26mN/m,其临界胶束浓度为0.025mg/mL。     

The Effect of Rhamnolipid Biosurfactant Produced by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> on Model Bacterial Strains and Isolates from Industrial Wastewater

In this study, the effect of rhamnolipid biosurfactant produced by Pseudomonas fluorescens on bacterial strains, laboratory strains, and isolates from industrial wastewater was investigated. It was shown that biosurfactant, depending on the concentration, has a neutral or detrimental effect on the growth and protein release of model Gram (+) strain Bacillus subtilis 168. The growth and protein release of model Gram (−) strain Pseudomonas aeruginosa 1390 was not influenced by the presence of biosurfactant in the medium. Rhamnolipid biosurfactant at the used concentrations supported the growth of some slow growing on hexadecane bacterial isolates, members of the microbial community. Changes in cell surface hydrophobicity and permeability of some Gram (+) and Gram (−) isolates in the presence of rhamnolipid biosurfactant were followed in experiments in vitro. It was found that bacterial cells treated with biosurfactant became more or less hydrophobic than untreated cells depending on individual characteristics and abilities of the strains. For all treated strains, an increase in the amount of released protein was observed with increasing the amount of biosurfactant, probably due to increased cell permeability as a result of changes in the organization of cell surface structures. The results obtained could contribute to clarify the relationships between members of the microbial community as well as suggest the efficiency of surface properties of rhamnolipid biosurfactant from Pseudomonas fluorescens making it potentially applicable in bioremediation of hydrocarbon-polluted environments.     

Biosurfactant production by Pseudomonas aeruginosa A41 using palm oil as carbon source

Biosurfactant production by Pseudomonas aeruginosa A41, a strain isolated from seawater in the gulf of Thailand, was examined when grown in defined medium containing 2% vegetable oil or fatty acid as a carbon source in the presence of vitamins, trace elements and 0.4% NH(4)NO(3), at pH 7 and 30 degrees C with 200 rpm-shaking for 7 days. The yield of biosurfactant steadily increased even after a stationary phase. Under such conditions the surface tension of the medium was lowered from 55-70 mN/m to 27.8-30 mN/m with every carbon source tested. However, types of carbon sources were found to affect biosurfactant yield. The yields of rhamnolipid biosurfactant were 6.58 g/L, 2.91 g/L and 2.93 g/L determined as rhamnose content when olive oil, palm oil and coconut oil, respectively, were used as a carbon source. Among them, biosurfactant obtained from palm oil was the best in lowering surface tension of the medium. Increase in biosurfactant activities in terms of oil displacement test and rhamnose content were observed to be higher with shorter chain fatty acids than that of the longer chains (C12>C14>C16). In addition, we found that C18:2, highly unsaturated fatty acid, showed higher oil displacement activity and rhamnose content than that of C18:1. The optimal oil displacement activity was found at pH 7-9 and in the presence of 0.5-3% NaCl. The oil displacement activity was stable to temperatures up to 100 degrees C for 15 h. Surface tension reduction activity was relatively stable at pH 2-12 and 0-5% of NaCl. Emusification activity tested with various types of hydrocarbons and vegetable oils showed similarity of up to 60% stability. The partially purified biosurfactant via TLC and silica gel column chromatography gave three main peaks on HPLC with mass spectra of 527, 272, and 661 m/z respectively, corresponding to sodium-monorhamnodecanoate, hydroxyhexadecanoic acid and an unknown compound, respectively.     

Anti-biofilm potential of a glycolipid surfactant produced by a tropical marine strain of Serratia marcescens

A tropical marine bacterium isolated from the hard coral, Symphyllia sp. was identified as Serratia marcescens on the basis of morphological, biochemical and 16S rDNA analysis. The bacterium showed antimicrobial activity towards the pathogens Candida albicans and Pseudomonas aeruginosa and the marine biofouling bacterium Bacillus pumilus. S. marcescens displayed biosurfactant activity as evidenced by drop collapse, blood hemolysis and surface tension reduction (52.0-27 mN m(-1)). The active compound was purified by solvent extraction and silicic acid chromatography. Characterization was by thin layer chromatography, gas chromatography mass spectroscopy (GC-MS), Fourier transform infrared (FTIR) spectroscopy and (1)H as well as (13)C nuclear magnetic resonance (NMR) analysis. The surfactant was found to be a glycolipid composed of glucose and palmitic acid. The glycolipid prevented adhesion of C. albicans BH, P. aeruginosa PAO1 and B. pumilus TiO1. The glycolipid also disrupted preformed biofilms of these cultures in microtitre plates. Confocal laser scanning microscopy and electron microscopy confirmed the effective removal of biofilms from glass surfaces. The glycolipid derived from S. marcescens could thus serve as a potential anti-biofilm agent.     

一株表面活性剂产生菌的分离及抑菌活性

【目的】研究分离得到的表面活性剂产生菌的产表面活性剂能力、分类地位和抑菌活性。【方法】采用血平板、油平板进行表面活性剂产生菌的分离,以排油圈法进行表面活性的测定;通过生理生化特性和16S rDNA序列相似性分析对BS1菌株进行初步鉴定;利用对峙培养法和菌丝生长、孢子囊形成、孢子萌发的抑制率测定研究其抑菌活性。【结果】从石油污染土壤中分离到的BS1菌株可产生表面活性剂,在分类学地位上属于假单胞菌属(Pseudomonas sp.)。BS1菌体、发酵上清液、挥发性物质对12种供试病原真菌均表现出一定的抑制作用。BS1菌体、发酵上清液对大豆疫霉菌(Phytophthora sojae)的抑制率最大,分别达到65.31%和95.93%。发酵上清液通过影响大豆疫霉菌菌丝生长、孢子囊形成、孢子萌发等方式抑制病原菌的正常生长,稀释20倍的发酵上清液依然具有明显的抑制作用。BS1菌株产生的挥发性物质对大豆菌核菌(Sclerotinia sclerotiorum)的抑菌效果最好,抑制率达到84.25%。【结论】BS1菌株在产生表面活性剂的同时,还具有生物防治作用潜力。