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     

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。     

发酵碳源对铜绿假单胞菌NY3所产鼠李糖脂结构及性能的影响

为研究发酵碳源对铜绿假单胞菌NY3所产鼠李糖脂结构及性能的影响,从鼠李糖脂的结构组分、性能和应用效果等方面展开研究。薄层实验证明两种鼠李糖脂均含有单糖脂和双糖脂。液质分析发现以橄榄油作碳源时,鼠李糖脂中双糖脂(Rha-Rha-C5-C6:1和Rha-Rha-C8-C8:2)比例更大,约为73.09%。而地沟油作碳源时,单糖脂(Rha-C10-C10和Rha-C16-C16:2)的比例更高,约为76.91%。橄榄油和地沟油为碳源的鼠李糖脂的临界胶束浓度(CMC)分别为55 mg/L和80mg/L。相同投加量时,前者乳化性和乳化稳定性均优于后者。NY3菌降解含油污泥时,投加双糖脂含量高的鼠李糖脂会使C16-C30直链烷烃的去除率更高。     

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。     

预水解发酵碳源对铜绿假单胞菌NY3产鼠李糖脂特性及应用效能的影响研究

发酵碳源对铜绿假单胞菌NY3(Pseudomonas aeruginosa NY3)产鼠李糖脂(Rhamnolipids,Rha)的特性影响较大。研究了利用废弃动物油作为发酵碳源时,其碱预水解和酶预水解对NY3菌发酵产鼠李糖脂产量、产物结构和性能的影响,从碳源水解酸值与水解产物、鼠李糖脂组分结构和实际应用效果进行了研究。碱、酶预水解实验发现,碳源酸值由初始的19.81 mg/g分别提高到72.04 mg/g和73.75 mg/g,气质联用(GC-MS)分析检测结果表明,碱、酶预水解后,碳源均释放7种C14-C18碳链的脂肪酸,鼠李糖脂产量由未预水解的8.28 g/L分别提高到15.35 g/L和17.63 g/L。液质联用(LCMS-IT-TOF)分析结果表明,用未预水解及碱、酶预水解碳源发酵时,NY3菌所产鼠李糖脂中单糖脂含量分别为62.07%、65.67%、87.32%。利用NY3菌在中试条件下处理高浓度石化企业油污泥,发现鼠李糖脂能促进NY3菌去除油污泥中的石油烃,且促进作用强弱顺序为未预水解产Rha碱预水解产Rha酶预水解产Rha。     

铜绿假单胞菌生物膜和浮游菌的毒力表达差异

目的探究铜绿假单胞菌生物膜和浮游菌状态下毒力因子的表达差异。方法使用铜绿假单胞菌标准菌株PAO1,分别在生物膜(静置)和浮游菌(摇床)状态下培养,收集上清液,检测总蛋白酶、LasA和LasB弹性蛋白酶、鼠李糖脂、绿脓素、溶血活性;通过荧光定量PCR检测群体感应(quorum sensing, QS)系统相关基因的表达;同时,通过活菌计数检测PAO1在生物膜和浮游菌状态下的生长曲线。结果生物膜状态下,铜绿假单胞菌PAO1的总蛋白酶、LasA、LasB弹性蛋白酶、鼠李糖脂、绿脓素表达均增高(均P0.05),溶血活性增高(P0.05),生物膜和浮游菌状态下细菌生长曲线差异无统计学意义,QS相关基因rhlI、rhlR、rhlA、lasI、lasR、pqsA、pqsR表达增高(均P0.05)。结论铜绿假单胞菌PAO1在生物膜状态下毒力因子表达较浮游菌状态下增高。     

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。     

铜绿假单胞菌中鼠李糖脂生物合成的研究进展*

鼠李糖脂因其具有环境友好和卓越的物理化学特性,而有望成为化学合成表面活性剂的替代物。近年来鼠李糖脂得到了广泛的研究,其目的是利用低价的可再生资源进行大规模生产,但目前的研究成果仍不足以选育出更具商业竞争力的鼠李糖脂过量合成菌株。为此,进一步理解鼠李糖脂生物合成的复杂基因调控网络,探索降低生产成本的发酵工艺势在必行。综述了铜绿假单胞菌中鼠李糖脂的生物合成途径、群体感应对主要基因的调控、鼠李糖脂在生物膜形成中所发挥的作用,以及发酵优化对鼠李糖脂产量的影响。有助于加深对鼠李糖脂生物合成的认识,为提高鼠李糖脂产量提供重要参考信息。     

铜绿假单胞菌XJ601产鼠李糖脂的优化培养及其稳定性

以1株从原油污染样品中分离获得的铜绿假单胞菌XJ601为研究对象,采用蒽酮比色法定量分析鼠李糖脂,优化其产鼠李糖脂的培养基组成。研究表明:疏水性底物优于亲水性底物,具有更高的鼠李糖脂产量,尤以菜籽油最佳;氮源中,硝酸盐、NH_4Cl能促进鼠李糖脂的合成,以菜籽油为碳源时,最佳氮源为NaNO_3;C/N比值在20时,鼠李糖脂产量最高;P元素的微量添加会影响鼠李糖脂的合成。摇瓶培养获得的鼠李糖脂对不同温度、pH及NaCl浓度都具有较好的稳定性,表明其在三次采油及原油污染生物治理等领域具有较好的应用前景。     

铜绿假单胞菌中鼠李糖脂生物合成的研究进展*

鼠李糖脂因其具有环境友好和卓越的物理化学特性,而有望成为化学合成表面活性剂的替代物。近年来鼠李糖脂得到了广泛的研究,其目的是利用低价的可再生资源进行大规模生产,但目前的研究成果仍不足以选育出更具商业竞争力的鼠李糖脂过量合成菌株。为此,进一步理解鼠李糖脂生物合成的复杂基因调控网络,探索降低生产成本的发酵工艺势在必行。综述了铜绿假单胞菌中鼠李糖脂的生物合成途径、群体感应对主要基因的调控、鼠李糖脂在生物膜形成中所发挥的作用,以及发酵优化对鼠李糖脂产量的影响。有助于加深对鼠李糖脂生物合成的认识,为提高鼠李糖脂产量提供重要参考信息。     

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%.     

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.     

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.     

Characterization of a novel long-chain n-alkane-degrading strain, Dietzia sp. E1

The newly isolated strain E1, identified as a Dietzia sp., proved to have an excellent ability to degrade n-C12 to n-C38 alkane components of crude oil. The preferred substrate was the very long-chain alkane n-eicosane at an optimal temperature of 37 degrees C and an optimal pH of 8 under aerobic conditions. The growth and substrate uptake kinetics were monitored during the n-alkane fermentation process, and Dietzia sp. E1 cells were found to possess three distinct levels of cell-surface hydrophobicity. Gas chromatographic/mass spectrometric analysis revealed that intracellular substrate mineralization occurred through the conversion of n-alkane to the corresponding n-alkanal. The monoterminal oxidation pathway was presumably initiated by AlkB and CYP153 terminal alkane hydroxylases, both of their partial coding sequences were successfully detected in the genome of strain E1, a novel member of the Dietzia genus.     

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.     

Hydrocarbon assimilation and biosurfactant production in Pseudomonas aeruginosa mutants.

We isolated transposon Tn5-GM-induced mutants of Pseudomonas aeruginosa PG201 that were unable to grow in minimal media containing hexadecane as a carbon source. Some of these mutants lacked extracellular rhamnolipids, as shown by measuring the surface and interfacial tensions of the cell culture supernatants. Furthermore, the concentrated culture media of the mutant strains were tested for the presence of rhamnolipids by thin-layer chromatography and for rhamnolipid activities, including hemolysis and growth inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the conditions tested, lacked the capacity to take up 14C-labeled hexadecane, and did not grow in media containing individual alkanes with chain lengths ranging from C12 to C19. However, growth on these alkanes and uptake of [14C]hexadecane were restored when small amounts of purified rhamnolipids were added to the cultures. Mutant 59C7 was unable to grow in media containing hexadecane, nor was it able to take up [14C]hexadecane. The addition of small amounts of rhamnolipids restored growth on alkanes and [14C]hexadecane uptake. In glucose-containing media, however, mutant 59C7 produced rhamnolipids at levels about twice as high as those of the wild-type strain. These results show that rhamnolipids play a major role in hexadecane uptake and utilization by P. aeruginosa.     

一株不动杆菌降解石油烃的特性及关键烷烃降解基因分析

【背景】Acinetobacter sp. Tust-DM21 (GenBank登录号KX390866)是本实验室前期从渤海湾海洋石油勘探船废油收集区采集的水油混合样中分离出的一株高效石油降解菌,其对短、中、长链烷烃均表现出很强的降解能力,有较好的应用前景。【目的】从应用层面探究其最佳降解条件,同时从生物信息层面探究其降解基因的作用。【方法】将其在不同温度、pH下培养144h,通过GC-MS内标法测定石油烃各组分的变化情况,计算出其最佳降解条件;同时,通过生物信息学手段确定基因组中的降解基因,每个基因分别选择7个同源基因,对它们的蛋白序列进行比较;最后对2个降解基因在0-144 h的表达情况进行了Real-time PCR分析。【结果】Acinetobacter sp. Tust-DM21最佳降解条件为35°C、pH 8.5,该条件下对石油降解率可达97.5%,其中,对长链烷烃降解率达98.5%,对环烃为81%,对芳香烃为87%;同时,研究发现基因组中含有常见烷烃降解基因alk B(GenBank登录号MH368539)和长链烷烃降解基因alm A (GenBank登录号MH357335),2个降解基因的蛋白经比较均与其同源蛋白表现出一定的相似性,同属菌的相似性最高;通过Real-timePCR发现这2个基因在0-144 h的相对表达量随时间逐步提高。【结论】Acinetobacter sp. Tust-DM21在最佳降解条件下对石油各组分都显示出了优良的降解能力,特别对长链烷烃的降解能力尤为突出;将2个降解基因的相对表达量结合该时间段的生长趋势,证明了菌株Acinetobacter sp. Tust-DM21的生长和降解与alk B和alm A基因的上调表达存在关联。     

Differential expression of the components of the two alkane hydroxylases from Pseudomonas aeruginosa

Oxidation of n-alkanes in bacteria is normally initiated by an enzyme system formed by a membrane-bound alkane hydroxylase and two soluble proteins, rubredoxin and rubredoxin reductase. Pseudomonas aeruginosa strains PAO1 and RR1 contain genes encoding two alkane hydroxylases (alkB1 and alkB2), two rubredoxins (alkG1 and alkG2), and a rubredoxin reductase (alkT). We have localized the promoters for these genes and analyzed their expression under different conditions. The alkB1 and alkB2 genes were preferentially expressed at different moments of the growth phase; expression of alkB2 was highest during the early exponential phase, while alkB1 was induced at the late exponential phase, when the growth rate decreased. Both genes were induced by C(10) to C(22)/C(24) alkanes but not by their oxidation derivatives. However, the alkG1, alkG2, and alkT genes were expressed at constant levels in both the absence and presence of alkanes.