一株海洋柴油降解菌的分离筛选鉴定及其生物表面活性剂特性分析

从中国浙江省舟山渔场油污染的海水和海洋沉积物中分离筛选产生物表面活性剂的柴油降解菌株。经富集培养、形态观察、测定单菌噬油斑、柴油降解率大小初筛到3株柴油降解菌。然后对初筛到的3株菌进行液滴坍塌实验、发酵液的表面张力、排油圈和乳化稳定性的大小测定进一步复筛,最终筛选出1株产生物表面活性剂的柴油降解菌,经18s rRNA鉴定为海洋解脂耶罗威亚酵母（Yarrowia lipolytica）。其柴油降解率为80%,发酵液液体表面张力可从73.4 mN/m降至23.56 mN/m,乳化效率E₂₄为60%。通过薄层色谱、傅里叶变换红外光谱、GC/MS鉴定,其产生的表面活性剂是由C₁₄、C₁₅β-羟基脂肪酸组成的脂肽。     

一株产生生物表面活性剂的海洋细菌培养条件优化与产物特性研究

微生物代谢产生的生物表面活性剂在海洋环境污染的生物处理方面具有较大应用潜力。从青岛近岸海水中分离到一株生物表面活性剂产生菌株S-22,鉴定为球型节杆菌（Arthrobacter globiformis）,通过摇瓶实验对其生长和产生生物表面活性剂的培养条件进行优化,最佳培养条件下6 h表面张力可降低至30.5 mN/m。该菌株产生生物表面活性剂的速度快,有利于大规模工业生产。综合薄层层析分析、傅立叶变换红外光谱分析和GC-MS分析,结果表明该生物表面活性剂是一种由海藻糖和两种脂肪酸（十六烷酸和9-双键十八烷酸）组成的海藻糖脂。该海藻糖脂表面活性高,临界胶束浓度为48.5 mg/L,具有良好的乳化能力,且耐盐和耐热能力较强,具有良好的应用前景。     

低能离子诱变产表面活性剂的烃降解菌研究

为有效治理石油污染土壤,从长期遭受石油污染的土壤中筛选出一株烃降解菌8-11作为出发菌,利用低能N+注入烃降解菌进行诱变,在能量为20 keV、剂量为90×2.6×1013ions/cm2条件下筛选出一株高效烃降解菌——诱变菌23。原油摇瓶发酵实验表明诱变菌对原油的降解率达到74%;降解后原油的全烃气相色谱图显示,经过7 d的作用,原油中的正构烷烃完全降解。诱变菌23能够产生大量的生物表面活性物质,傅里叶红外光谱分析表明其产生的生物活性物质为糖脂类化合物,该糖脂类生物表面活性剂能使发酵液的表面张力从空白对照的56.1 mN/m降低为29.3 mN/m。研究表明诱变菌23具有较高的烃降解能力,能有效降低表面张力,具有较大的应用潜力。     

生物表面活性剂产生菌的筛选及产剂性能研究

从长期被汽油污染的土壤中,筛选出了一株表面活性剂产生菌BSZ-07,初步确定其为铜绿假单胞菌。通过正交试验,优化出了BSZ-07的最优产剂条件。BSZ-07在48h内可使发酵液的表面张力降至34.2mN/m,且乳化性能稳定,是一种较优良的生物表面活性剂产生菌。经FTIR分析及元素分析,初步确定其所产表面活性剂为鼠李糖脂。     

铜绿假单胞菌S6分泌的生物表面活性剂特性

研究了1株铜绿假单胞菌S6(Pseudomonas aeruginosa S6)分泌的生物表面活性剂的理化性质.该生物表面活性剂的临界胶束浓度为50mg·L-1,此时其表面张力为29.3mN·m-1.pH值对S6产表面活性剂有一定影响,在中性及弱碱性条件下,S6长势较好且表面活性剂表面张力较低.该生物表面活性剂对菲具有非常明显的增溶效应,使水中菲的溶解度增加了约23倍.原油的加入有利于S6产表面活性物质.与原油的相互作用说明该生物表面活性剂能够乳化原油且维持乳化液稳定性于80%以上;原油浓度为6%～8%时,能达到最佳乳化效果.HPLC-ESI-MS分析检出该生物表面活性剂含有13种鼠李糖脂同系物.     

产表面活性剂解烃菌的筛选及其降解条件研究

以原油为唯一碳源和能源,从新疆克拉玛依油田土壤中筛选出1株能产生物表面活性剂的高效解烃菌XJBM,经形态观察、生理生化特征和Biolog分析,初步鉴定该菌为铜绿假单胞菌(Pseudomonas Aeruginosa)。薄层色谱分析结果表明,XJBM产糖脂类生物表面活性剂,在最适发酵条件下,生物表面活性剂的产量可达2.25 g/L,可将发酵液表面张力从68.20 m N/m降低到32.50 m N/m,乳化指数(E24)达到81.8%。采用单因素试验对影响XJBM降解率的因素进行了研究,得出最适降解条件为p H 7.5,温度30℃,盐浓度5 g/L,接种量10%。在此条件下,菌株对1%石油烃的7d降解率为63.78%。     

Pseudomonas aeruginosa W_3产鼠李糖脂的结构表征及理化性质

利用液相色谱/质谱联用仪(HPLC-ESI-MS)分析了石油降解菌Pseudomonas aeruginosa W3以甘露醇为碳源所产鼠李糖脂生物表面活性剂的组成.结果表明,所产鼠李糖脂共检出6种主要的鼠李糖脂同系物,均由1～2个鼠李糖分子和1～2个含β羟基的碳链长度为8～12的饱和或不饱和脂肪酸分子构成,其主要组分的m/z为649.6和621.5,对应的结构是RhaRhaC10C10和RhaRhaC8C10,分别占总检出物质量的57%和15.5%.该鼠李糖脂混合物中双鼠李糖脂的含量达到90%,是目前报道的双鼠李糖脂含量较高的菌株之一.该糖脂类生物表面活性剂可将水的表面张力从71.4mN.m-1降到30.5mN.m-1,临界胶束浓度为48mg·L-1,在高温、高盐度及高pH等极端环境下,仍能保持较高的表面活性和乳化能力,在生物修复中具有潜在应用价值.     

堆肥过程中产生生物表面活性剂的细菌的筛选

从不同温度的堆肥过程中 ( 45℃和 5 5℃ ) ,筛选了产生生物表面活性剂的菌种 ,对产生的生物表面活性剂进行了定性和定量的检验 .实验结果表明 ,Bacillussubtilis是堆肥过程中产生生物表面活性剂的主要菌种之一 ,并验证了其产生的生物表面活性剂为莎梵婷等脂肽类物质 .还对筛选出的优势菌种BacillussubtilisB产剂条件进行了优化 ,强化了它的产剂能力 ,结果表明碳源和氮源对产剂影响较大 ,而Fe2 浓度影响较小 .最后对筛选的菌种及其产生的生物表面活性剂在城市生活垃圾堆肥中的应用作了展望     

绿色荧光蛋白标记在生物修复中的应用

通过对铜绿假单胞菌进行增强型绿色荧光蛋白(EGFP)标记,获得带有EGFP遗传标记的铜绿假单胞菌.该菌株绿色荧光标记性能稳定,通过转化子基因组DNAPCR和斑点杂交检测,证明外源EGFP基因存在于转化子染色体上.转化子30h内将溶液的表面张力由70mN/m降至35mN/m,产生的生物表面活性剂性能良好.     

脂肽类生物表面活性剂产生菌的分离及特性研究

从石油污染土壤中分离筛选获得一株产生生物表面活性剂菌株Y8A,经生理生化实验、16S rDNA序列分析等将其鉴定为芽孢杆菌属（Bacillus sp.）.Y8A能在22h内将发酵液的表面张力从68.3mN·m-1降到23.5 mN·m-1.经TLC和傅立叶红外光谱分析, 菌株Y8A产生的生物表面活性剂为脂肽类.20mg·L-1 Ca2+和Fe2+能显著促进其生长和表面活性剂的产生;菌株Y8A在20～30℃,pH 5～12范围内产生表面活性剂的能力较强;LB培养基中添加1%乳糖对生长的影响不大,但能够明显促进Y8A产生生物表面活性剂,而葡萄糖、蔗糖抑制表面活性剂的产生.Y8A能够促进石油降解菌Y1D和F11对石油的降解和功夫菊酯降解菌ZZH对功夫菊酯的生物降解.     

Lipopeptide als natürliche Adjuvantien für Impfstoffe aus Gram-negativen Bakterien

Bacterial cell wall components such as lipopolysaccharide, a variety of membrane proteins, murein, and lipoprotein can act as immunoadjuvants for bacterial vaccines, thus enhancing protection from bacterial infections. Synthetically prepared N-terminal parts of the lipoprotein from Enterobacteria carrying three fatty acid residues or lipopeptide analogs containing one to four aminoacids bound to S-glycerylcysteine act as potent immunoadjuvants in vivo in combination with or covalently linked to antiges. Here we demonstrate that the supplementation ofSalmonella vaccines with these synthetic lipopeptides significantly enhances their vaccine efficiency in mice. Variations in the native lipopeptide structure regarding chain length and amino acid sequence of the peptide moiety, as well as modifications of the lipoamino acid, lead to reduction or even complete loss of the adjuvant activity. The immunoadjuvant properties of the lipopeptides as described here are mediated by an enhancement of the humoral immune response.     

Isolation of antifungal bacteria from Japanese fermented soybeans, natto

An inhibitory effect of a traditional Japanese fermented food, natto, was found against plant pathogens such as Rhizoctonia solani and Fusarium oxysporum, and the bacteria which showed inhibition were isolated from the natto. Among isolated bacteria, BC-1 and GAc exhibited a strong antagonistic effect in vitro against plant pathogens on an agar medium. The supernatant of bacterial culture also showed strong activity against R. solani, which meant the antimicrobial substances were produced and secreted into the medium. Both of the bacteria were estimated as Bacillus amyloliquefaciens from a partial sequence of the 16s rRNA gene. High performance liquid chromatography analysis clearly showed the production of the lipopeptide antibiotic iturin A by BC-1 and GAc.     

Isolation of antifungal bacteria from Japanese fermented soybeans,natto

An inhibitory effect of a traditional Japanese fermented food, natto, was found against plant pathogens such as Rhizoctonia solani and Fusarium oxysporum, and the bacteria which showed inhibition were isolated from the natto. Among isolated bacteria, BC-1 and GAc exhibited a strong antagonistic effect in vitro against plant pathogens on an agar medium. The supernatant of bacterial culture also showed strong activity against R. solani, which meant the antimicrobial substances were produced and secreted into the medium. Both of the bacteria were estimated as Bacillus amyloliquefaciens from a partial sequence of the 16s rRNA gene. High performance liquid chromatography analysis clearly showed the production of the lipopeptide antibiotic iturin A by BC-1 and GAc.     

Elektronen-Energieverlust-Spektroskopie (FELS) als Methode zur Lokalisierung von Antigenen und anderen Substanzen in Zellen und Geweben

The localization of antigens and other substances in cells and tissues by electron microscopy is usually performed by immunohistochemical techniques employing labelled conventional or monoclonal antibodies. For the ultrastructural localization of the antibodies, they are coupled to electron-dense labels like gold or ferritin. Here, we demonstrate a novel method to localize antigens in cells, tissues, and on other supports. By electron energy loss spectroscopy (EELS) it is possible to directly analyze the distribution of antigens, metabolites or other substances without the use of labelled antibodies: as an example we demonstrate the distribution of the immunomodulator lipopeptide in B lymphocytes and macrophages. EELS represents a novel, sensitive, and generally applicable method for the detection and localization of antigens and other substances in biology and medicine.     

Improvement of production of lipopeptide antibiotic iturin A using fish protein

To enhance the production of lipopeptide antibiotic iturin A, nutrient contents of the culture mediums were investigated in both submerged and biofilm fermentations. As a carbon source maltose and as nitrogen source, fish protein was used. In submerged fermentation maltose uptake was found lower (12%) compared to biofilm fermentation (15%) that was associated with higher cellular growth in biofilm. However, requirement of nitrogen (fish protein) concentration was found similar in both submerged and biofilm fermentations. Production of iturin A in submerged fermentation with 12% maltose and 5% fish protein was 4450 mg/L, and in biofilm fermentation it was 5050 mg/L when 15% maltose and 5% fish protein was used.     

Improvement of production of lipopeptide antibiotic iturin A using fish protein

To enhance the production of lipopeptide antibiotic iturin A, nutrient contents of the culture mediums were investigated in both submerged and biofilm fermentations. As a carbon source maltose and as nitrogen source, fish protein was used. In submerged fermentation maltose uptake was found lower (12%) compared to biofilm fermentation (15%) that was associated with higher cellular growth in biofilm. However, requirement of nitrogen (fish protein) concentration was found similar in both submerged and biofilm fermentations. Production of iturin A in submerged fermentation with 12% maltose and 5% fish protein was 4450 mg/L, and in biofilm fermentation it was 5050 mg/L when 15% maltose and 5% fish protein was used.     

Production of iturin A homologues under different culture conditions

Iturin A is a cyclic lipopeptide antibiotic and eight different kinds of iturin A have been reported based on its alkyl side chains. As iturin A is a promising biocontrol agent, total production of iturin A was tried to enhance and comparative production of its homologues was investigated by using different nitrogen and carbon sources. When Polypepton S and defatted soybean meal were used, total production as well as the ratio of the iturin A homologues were similar. However, production of iturin A was relatively lower and also the ratio of the iturin A homologues was different when Polypepton was used, where A2 was decreased and A4 was increased. Production ratio of the iturin A homologues was similar for the carbon sources like maltose, mannitol, sucrose and starch but relative production of iturin A2 was much enhanced compared to A3 when lactose or galactose was used. Interestingly production ratio of A4 was increased and A2 and A3 were decreased when no additional carbon source was used, and similar tendency was observed in the homologue ratio with glucose and fructose. Production of iturin A homologue A6 was significantly increased whereas A2 and A3 were decreased when defatted rapeseed cake was used. Utilization of different amino acids did not show significant differences in their production of the iturin A homologues. Oxygen supply found to be the factor affecting the production of iturin A homologues when it was investigated in a varied culture volume size and shaking speed. A2 found to be increased with increased oxygen supply where the production of A3 was affected inversely.     

Production characteristics of lipopeptide antibiotics in biofilm fermentation of Bacillus subtilis

In biofilm fermentation, from the very early moments, surfactin was produced along with the biofilm development in the lipopeptide antibiotic production medium by using Bacillus subtilis. However, almost no iturin A was produced in its first 24 hours of cultivation and the production of iturin A began much later. Volumes of the nutrient medium and available surface area of the biofilm reactors were found to be important with the relative production of these two antibiotics. Production of iturin A was increased from 12 mg to about 50 mg per reactor when the culture size was increased from 5 mL to 20 mL, as the depth of the medium was increased. The production level was saturated thereafter with larger volumes. On the other hand, surfactin production was remained similar, which was about 10 mg per reactor, from all the 5 mL to 80 mL of biofilm culture. Optimized temperature for iturin A and surfactin production was observed at 25 and 37°C, respectively. In the biofilm fermentation, production of surfactin was increased when the incubation temperature was increased within the temperature range of 25 to 37°C, on the other hand, iturin A production was gradually decreased with the increase of the incubation temperature.     

Alcaligenes sp.S-XJ-1利用废弃柴油合成生物破乳剂的研究

生物破乳剂是一种用于油水分离的新型破乳剂.采用废弃柴油培养生物破乳剂产生菌Alcaligenes sp.S-XJ-1,培养7 d,菌株干重最高可达2.0 g/L,10 g/L的菌株细胞悬液能够将水表面张力从72.0 mN/m降低到29.7 mN/m.生物破乳剂产量为0.3 g/L,其CMC为150 mg/L,表面活性优于化学表面活性剂SDS,且对W/O模型乳状液的破乳效果在70%以上.废弃柴油GC-MS测试结果表明,S-XJ-1菌株能够利用废弃柴油中的C14～C20正构烷烃,且C20正构烷烃几乎被完全利用,其利用率高达99%.S-XJ-1菌株对不同碳链长度正构烷烃复配碳源的利用率及破乳性能随着碳链长度的延长而逐渐增加.与其他正构烷烃复配碳源相比,S-XJ-1菌株对C20正构烷烃利用率最高,合成破乳剂的性能最好,且与废弃柴油研究结果最为接近.TLC和FTIR分析表明S-XJ-1菌株利用废弃柴油合成的生物破乳剂为脂肽类物质.     

Biofilm formation and lipopeptide antibiotic iturin A production in different peptone media

Biofilm fermentation is a newly developed promising technique in fermentation technology. In this study no.3 and no.3S media have been used for the lipopeptide antibiotic iturin A production by Bacillus subtilis RB14. The main component of no.3 and no.3S media is Polypepton and Polypepton S, respectively. B. subtilis RB14 produces thick stable biofilm and high amount of iturin A in no.3S medium. Whereas, impaired biofilm formation and lower iturin A production was observed in no.3 medium. From the analytical information it was observed that the amounts of metal ions, such as K⁺, Ca²⁺ and Mn²⁺, cysteine and cellulose are lower in Polypepton compared to the Polypepton S. To investigate their effect on biofilm formation and iturin A production cysteine, cellulose, K⁺, Ca²⁺ and Mn²⁺ were added respectively into the no.3 medium at similar amount that Polypepton S contains. It was observed that individual addition of K⁺, Ca²⁺, cysteine and cellulose had no effect on biofilm formation, cellular growth induction or iturin A production. However, when Mn²⁺ was supplemented in no.3 medium, biofilm development was restored with an improved production of iturin A. Finally, combined addition of investigated substances into the no.3 medium resulted with highly folded, thick biofilm with high cellular growth and iturin A production compared to the original no.3 medium.