烃的微生物摄取机制研究进展*

综述了近期在烃的微生物摄取机制方面的研究进展。对提高环境生物整治效果而言,微生物对非水溶性底物,尤其是烃的摄取机制是重要的课题。随着理论认识的深入和研究手段的丰富,在该领域已有了更多发现和结论。介绍了表面活性剂对烃摄取的影响,细胞表面性质的调整和烃的跨膜输送等方面的近期研究结果;同时,提供了细胞亚微结构分析,细胞趋化性等相关证据,指出了在该领域尚待解决的问题。     

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。     

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     

石油烃降解菌的研究进展

该文归纳了细菌、真菌和藻类对石油烃的降解作用;讨论了微生物降解石油烃的影响因素,包括微生物种类、石油烃种类、温度、pH、营养物质、电子受体等;总结了微生物固定化技术、生物表面活性剂和基因工程技术在微生物降解石油烃领域的应用;最后,提出今后生物降解石油烃的研究重点可能是开发具有高效降解能力的菌群联合体。     

低温微生物修复石油烃类污染土壤研究进展

耐冷菌、嗜冷菌等低温微生物广泛存在于极地、高山以及高纬度等土壤环境中,是石油烃类污染物在低温条件下降解与转化的重要微生物资源.利用低温微生物的独特优势,石油污染土壤的低温生物修复技术的研究成为当前热点领域.本文系统综述了低温石油烃降解菌的分类及冷适机制,低温微生物对不同类型石油烃组分的降解特征和降解机理,低温环境中接种降解菌、添加营养物质和表面活性剂等强化技术在石油污染土壤中生物修复的应用.以及微生物分子生物学技术在低温微生物降解石油烃的研究现状,为拓展我国石油污染土壤生物修复技术提供参考.     

MATH法表征环境微生物细胞表面疏水性的研究进展

环境微生物的细胞表面疏水性对其生长代谢过程以及在环境领域的应用效率具有重要影响。目前用于测试细胞表面疏水性最常用的方法是碳氢吸附能力(Microbial adhesion to hydrocarbons,MATH),该方法因具有操作简便、有一定的准确度等优点在环境、生物工程、医学、食品等领域应用广泛。本文综述了MATH法在环境微生物领域中的污泥絮体性能表征、有机物降解、膜污染和破乳方面的应用,同时介绍了MATH法在实验操作、计数方法和数据分析方面的优化。最后展望了该方法今后的研究方向。     

生物表面活性剂对微生物生长和代谢的影响

综述了生物表面活性剂在微生物生长和代谢过程中的影响。根据其分子结构特征 ,系统分析了生物表面活性剂通过与难溶底物和微生物细胞之间的相互作用促进烷烃摄取的机理 ,利用该机理可以合理解释生理现象。生物表面活性剂还在参与细胞代谢活动的过程中发挥特殊功能。     

长链烷烃降解菌的降解特性

对长链烷烃降解菌的降解能力和摄取模式进行了研究。评价14株烃降解菌利用中长链烃生长的能力,发现只有少数烃降解菌能够获得良好生长,其中Mycobacterium fortuitum514,Pseudomonas aeruginosa1785和Pseudomonas marginata766等3株菌能够高效降解C20到C33的长链烷烃。辛烷不能支持这些长链烷烃降解菌的生长,说明其烃氧化酶与Pseudomonas oleovorans的OCT质粒编码的单氧酶不同。此外,M.fortuitum不产胞外表面活性剂,而P.aeruginosa和P.marginata则是表面活性剂产生菌,然而三者在以烃为碳源生长时均显示出很高的细胞表面疏水性。根据生长现象分析3株菌采用了不同的烷烃摄取模式。     

微生物有机酸转运蛋白的研究进展

转运蛋白是一类膜蛋白,可介导生物膜内外化学物质的跨膜转运及信号交换。有机酸转运蛋白在微生物有机酸代谢的跨膜转运过程中发挥重要作用,根据转运蛋白有机酸转运的方向不同可以分为摄取转运蛋白和外排转运蛋白。在微生物代谢中,有些有机酸可以作为能源直接参与体内代谢,有些是能量转换过程中的重要中间产物;摄取转运蛋白的过表达,可以促进微生物细胞获取能源物质,高效的生产目标产物;有机酸摄取转运蛋白敲除或外排转运蛋白表达,有利于底盘细胞外排更多目标产物,进而促进有机酸的生物合成。研究有机酸转运蛋白的结构和功能,有助于解析微生物细胞有机酸生物合成及利用的机制,对于提高工业微生物对有机酸的利用及生物合成具有重要作用。本文综述了微生物有机酸转运蛋白分类和结构、转运方式和转运功能等方面,重点综述了转运蛋白在有机酸生产中的应用,为工业微生物有机酸的高效生物合成及未来发展提供参考。     

微生物细胞表面呈现技术研究进展

微生物细胞表面工程是近年来发展起来的,它利用细胞表面展示技术使外源蛋白固定化于细胞表面,从而生产微生物细胞表面蛋白。微生物细胞表面工程可用于细胞催化剂、细胞吸附剂、活疫苗、生物传感器的开发等。微生物细胞表面工程具有广阔的应用前景,但是国内对这一领域的研究刚起步。在介绍细胞表面工程的基础上,对微生物细胞表面工程技术进展进行了综述,展望了对该技术的发展。     

Characterization of bacterial isolates from industrial wastewater according to probable modes of hexadecane uptake

Bacterial isolates from industrial wastewater were characterized according to probable modes of hexadecane uptake based on data for cell surface hydrophobicity, emulsifying activity, glycoside content and surface tension of cell-free culture medium. The results obtained suggested that both modes of biosurfactant-enhanced hexadecane uptake by bacterial strains take place, direct uptake and alkane transfer. The increase in cell surface hydrophobicity and glycoside production by the strains suggested the existence of biosurfactant-enhanced interfacial uptake of the alkane. Such mechanism is probably predominant for three isolates, Staphylococcus sp. HW-2, Streptococcus sp. HW-9 and Bacillus sp. HW-4. Secreted biosurfactants enhanced mainly alkane emulsification for most hydrophobic isolate Arthrobacter sp. HW-8, and micellar transfer for most hydrophilic isolate Streptococcus sp. HW-5. For other strains (67%) both mechanisms of biosurfactant-enhanced hexadecane uptake probably take place in similar degree, interfacial uptake and alkane emulsification. The results obtained could contribute to clarifying the natural relationships between the members of water ecosystem studied as well as will reveal potential producers of surface active compounds.     

Role of rhamnolipid biosurfactants in the uptake and mineralization of hexadecane in Pseudomonas aeruginosa

A study was undertaken to investigate the mechanisms for biosurfactant-enhanced hexadecane uptake into Pseudomonas aeruginosa. Two strains of Ps. aeruginosa were studied, one producing rhamnolipids (PG201) and the other rhamnolipid deficient (UO299). Rhamnolipids produced by PG201 acted to increase the solubility of n-hexadecane in the culture medium (from 1.84 to 22.76 microg l(-1). Rates of(l4)C-n-hexadecane uptake and mineralization were higher in PG201 than in UO299. However, the degree of difference was lower than expected. Additional studies were carried out on the cell surface properties of the two strains. During growth on n-hexadecane, the cell surface hydrophobicity of both PG201 (50.5%) and UO299 (33.7%) increased compared with that observed in water-soluble growth substrates (7-8%). Studies were also carried out to ascertain any energy requirements for the transport of n-hexadecane into Ps. aeruginosa cells. The addition of CCCP (an inhibitor of cytochrome oxidase which thereby blocks oxidative phosphorylation) at a range of concentrations caused a marked decrease in n-hexadecane uptake, indicating that n-hexadecane uptake in Ps. aeruginosa is an energy-dependent process. These studies support the hypothesis of alkane transport into microbial cells by direct contact with larger alkane droplets and by pseudosolubilization. Also, it appears that both mechanisms occur simultaneously.     

Predominant role of hydrocarbon solubilization in the microbial uptake of hydrocarbons

Using EDTA and proteolytic enzymes to suppress hydrocarbon solubilization, direct evidence is presented in support of the mechanism of liquid hydrocarbon uptake by microbial cells predominantly from the solubilized or accommodated substrate. EDTA (2-5mM) strongly inhibited growth of three yeast species and one bacterial species on n-hexadecane and the inhibition was removed by surfactant-emulsified and surfactant-solubilized alkane and also by excess addition of Ca(2+). EDTA had no inhibitory effect on the growth of the organisms on soluble substrates such as sodium acetate and nutrient broth or on n-pentane, a volatile alkane which was primarily transported by diffusion from gas phase. EDTA was shown to have no significant effect on the adsorption of cells on alkane drops. EDTA inhibition of growth was considered to be due to suppression of alkane solubilization, brought about by the solubilizing factor(s) produced by cells. It was shown that this chelating agent did not inhibit the growth of yeast on solubilized alkane but strongly inhibited its growth on alkane drops. It was demonstrated that adherent capacity of microbial cell to oil phase was closely related to the state of hydrocarbon emulsification and had no relationship to the ability of organisms to grow on hydrocarbon. Certain proteolytic enzymes inhibited the growth of yeast on alkane, presumably by digesting the alkane solubilizing protein, but not on glucose, and the inhibition was removed by a supply of surfactant-emulsified and surfactant-solubilized alkane. Specific solubilization of various hydrocarbon types during growth of the prokaryotic bacterial strain was demonstrated. The specific solubilization of hydrocarbon was strongly inhibited strain was demonstrated. The specific solubilization of hydrocarbon was strongly inhibited by EDTA, and the inhibition was removed by excess Ca(2+). It was concluded that specific solubilization of hydrocarbons is an important mechanism in the microbial uptake of hydrocarbons.     

Mode of uptake of insoluble solid substrates by microorganisms. II: Uptake of solid n-alkanes by yeast and bacterial species

Using two species of yeast and one of bacterium, evidence has ben obtained which indicates that the microbial uptake of solid alkane powders occurs primarily through a substrate solubilization mechanism. EDTA, a strong inhibitor of hydrocarbon solubilization by the cells, inhibited the growth of these organisms on alkane powder; the inhibition could be removed vai a supply of artificially solubilized alkane. One of the yeast strians, which was a mutant incapable of growing on solid alkane powder and liquid alkane, could grow very well on artifically solubilized alkanes. It was demonstrated that the solid alkane solubilization rate during microbial growth could satisfactorily account for the maximal alkane uptake rate actully observed during growth. The specificity of solubilization for the solid alkane used as the growth substrate was demonstrated.     

The concentrations of hexadecane and inorganic nutrients modulate the production of extracellular membrane-bound vesicles, soluble protein, and bioemulsifier by Acinetobacter venetianus RAG-1 and Acinetobacter sp. strain HO1-N

In the present study, we addressed the possibility that the production of both bioemulsifiers and membrane-bound vesicles may be a common feature of the growth of Acinetobacter spp. on alkanes, and we determined the extent to which the release of extracellular products by these organisms is regulated by the concentrations of the alkane substrate and inorganic nutrients. To accomplish this objective, we grew Acinetobacter venetianus RAG-1 and Acinetobacter sp. strain HO1-N with different concentrations of nutrients and assayed for extracellular products. The results indicated that the release of vesicles, soluble protein, and bioemulsifier was promoted in various degrees by higher concentrations of hexadecane and inorganic nutrients, while the specific activities of the bioemulsifiers were enhanced with lower nutrient concentrations. Based on our findings, we propose that under conditions of nutrient excess, these strains produce membrane-bound vesicles to function in "luxury uptake" of the alkane substrate for delivery and storage in the form of inclusions. Under the same conditions, soluble bioemulsifier and protein may perform auxiliary roles in cell desorption and (or) alkane uptake. With low concentrations of nutrients, the decreased production of vesicles, protein, and bioemulsifier and the increased activity of the emulsifier may represent a mechanism for reducing biosynthetic demands and conserving cellular material.     

Effects of non-ionic surfactants on the uptake and hydrolysis of fluoresceindiacetate by alkane-oxidizing bacteria

Biological effects of non-ionic surfactants on alkane-oxidizing bacteria were studied by assessing their influence on the uptake of prefluorochrome fluoresceindiacetate (FDA) and its intracellular hydrolysis to fluorescein. Both decreasing and increasing rates of hydrolysis as a consequence of the presence of surfactants were observed. The surfactants influenced the uptake of FDA, but not its intracellular hydrolysis. The effects of the surfactants on the uptake rate depended strongly on the structure and physico-chemical properties of the surfactants. There was no qualitative or significant quantitative difference in surfactant susceptibility between induced (alkane grown) and non-induced bacteria (acetate grown), even though the induced cells possess greater cell surface hydrophobicity.     

Modulating the import of medium-chain alkanes in <Emphasis Type="Italic">E. coli</Emphasis> through tuned expression of FadL

Background

In recent years, there have been intensive efforts to develop synthetic microbial platforms for the production, biosensing and bio-remediation of fossil fuel constituents such as alkanes. Building predictable engineered systems for these applications will require the ability to tightly control and modulate the rate of import of alkanes into the host cell. The native components responsible for the import of alkanes within these systems have yet to be elucidated. To shed further insights on this, we used the AlkBGT alkane monooxygenase complex from Pseudomonas putida GPo1 as a reporter system for assessing alkane import in Escherichia coli. Two native E. coli transporters, FadL and OmpW, were evaluated for octane import given their proven functionality in the uptake of fatty acids along with their structural similarity to the P. putida GPo1 alkane importer, AlkL.

Results

Octane import was removed with deletion of fadL, but was restored by complementation with a fadL-encoding plasmid. Furthermore, tuned overexpression of FadL increased the rate of alkane import by up to 4.5- fold. A FadL deletion strain displayed a small but significant degree of tolerance toward hexane and octane relative to the wild type, while the responsiveness of the well-known alkane biosensor, AlkS, toward octane and decane was strongly reduced by 2.7- and 2.9-fold, respectively.

Conclusions

We unequivocally show for the first time that FadL serves as the major route for medium-chain alkane import in E. coli. The experimental approaches used within this study, which include an enzyme-based reporter system and a fluorescent alkane biosensor for quantification and real-time monitoring of alkane import, could be employed as part of an engineering toolkit for optimizing biological systems that depend on the uptake of alkanes. Thus, the findings will be particularly useful for biological applications such as bioremediation and biomanufacturing.

     

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%.