红树林放线菌分离鉴定及活性代谢产物研究

以采集自四个红树林地点的16份混合土壤为研究材料,选用7种选择性培养基,共分离获得330株放线菌。其中217株菌经16SrRNA基因序列分析,发现近75%菌株属于小单孢菌属(Micromonospora),其他还包括多形态孢菌属(Polymorphospora),疣孢菌属(Verrucosispora)等小单孢菌科的2个属和非小单孢菌科的9个属。采用美蓝酶标仪法对所分离到的放线菌进行抗菌活性检测,共50株菌表现出对金黄色葡萄球菌(Staphylococcus aureus ATCC 51650)、大肠杆菌(Escherichia coli ATCC 25922)和白色念珠菌(Candida albicans ATCC 10231)有不同程度抗性。然后利用高效液相色谱(HPLC)和液质联用技术(LC-MS)对有生物活性的菌株进行化学筛选,最后确定了5株可能产新颖化合物的小单孢菌。     

Isolation and Characterization of Novel Hydrocarbon-Degrading Euryhaline Consortia from Crude Oil and Mangrove Sediments

Two novel and versatile bacterial consortia were developed for the biodegradation of hydrocarbons. They were isolated from crude oil from the Cormorant Field in the North Sea (MPD-7) and from sediment associated with mangrove roots (MPD-M). The bacterial consortia were able to degrade both aliphatic and aromatic hydrocarbons in crude oils very effectively in seawater (35 g/L NaCl) and synthetic media containing 0 to 100 g/L NaCl (1.7 M). Salinities over twice that of normal seawater decreased the biodegradation rates. However, even at the highest salinity biodegradation was significant. Ratios of nC17 to pristane and nC18 to phytane were significantly lowered across the range of salinity. The lowest values were at 0 and 20 g/L (0.34 M). Phytane was degraded in preference to pristane. The degradation of these compounds was constant over the salinity range, with evidence of a slight increase for consortium MPD-M with increasing salinity. In general, the consortium isolated from mangrove root sediments was more efficient in metabolizing North Sea crude oil than the consortium isolated from Cormorant crude oil. The 5 strains that comprise MPD-M have been tentatively identified as species of the genera Marinobacter, Bacillus, and Erwinia. This is the first report of hydrocarbon-degrading consortia isolated from crude oil and mangrove sediments that are capable of treating oily wastes over such a wide range of salinity. Received June 30, 1999; accepted May 29, 2000.     

漳江口红树林沉积物真菌多样性分析及抗茶叶致病真菌生物活性筛选

以福建漳江口红树林国家级自然保护区红树林沉积物分离的真菌为对象,研究红树林沉积物真菌的多样性和筛选抗茶叶病原真菌活性的菌株。将分离纯化的135株真菌通过形态学和Ribosomal DNA-Internal Transcribed Spacer(rDNAITS)序列测定进行鉴定及多样性分析,归为40个种类型,分别属于17个属,其中青霉属(25%)为优势菌,木霉属(15%)、曲霉属(10%)和镰刀属(10%)次之,表明红树林沉积物真菌具有丰富的多样性;利用平板对峙法对真菌的发酵粗提物抗茶叶病原真菌生物活性研究,结果发现,共有17株(占42.5%)真菌具有抗茶叶病原真菌活性,其中15株能够抑制茶叶轮斑病(Pestalotiopsis theae)LH13,12株能够抑制茶叶炭疽病(Colletotrichum gloeosporioides)LH30,8株能够抑制茶叶溃疡病(Neofusicoccumsp.)LH107,有两株对这三种茶叶致病菌均有较强活性。这些活性菌株分布在6个属中,分别是青霉属(7株)、木霉属(3株)、镰孢属(2株)、枝孢属(2株)、白地霉属(2株)和球腔菌属(1株)。由此可见,红树林沉积物真菌抗菌活性菌株的种属分布具有多样性。     

1株抗根结线虫红树林放线菌的筛选与鉴定

从红树林底泥中分离筛选具有抗线虫活性的放线菌,并对活性菌株进行分类鉴定。采用稀释平板法分离放线菌,采用24孔板液体筛选模型筛选具有抗线虫活性的菌株,并对活性菌株进行形态学和生理生化特征研究,测定其16S rDNA序列并进行系统发育分析。筛选得到1株具有杀线虫活性的菌株,编号为HA11090,该菌株发酵液在稀释20倍和40倍后,抗根结线虫校正死亡率分别为70.5%和65.0%。菌株HA11090的16S rDNA序列与Saccharopolyspora hirsuta subsp.kobensis和Saccharopolyspora jiangxiensis的相似性最高,分别为99.35%和99.27%,三者在系统发育树上聚为一个分支,形态和生理生化特征分析显示,菌株HA11090与Saccharopolyspora jiangxiensis基本一致,而与S.hirsuta subsp.kobensis差异较大。鉴定菌株HA11090为Saccharopolyspora jiangxiensis,其发酵液具有较强的抗根结线虫活性,值得进一步研究。     

Anti‐Adhesion Activity of Tannins Isolated from the Mangrove Laguncularia racemosa

In the search of new compounds with biofilm‐inhibiting properties, mangroves with their richness of secondary metabolites can be a valuable resource. Crude methanolic leaf extracts from the mangrove Laguncularia racemosa enriched in phenolic substances cause a reduction in initial cell adhesion of Candida glabrata and Candida albicans, but not on Escherichia coli. LC/MS‐guided fractionation of the phenolic compounds resulted in 19 fractions, of which ten were analyzed for their bioactivity against cell adhesion. Effects on cell adhesion and planktonic growth of Escherichia coli, Candida glabrata and Candida albicans were measured in 96‐well microtiter plates in the presence of 0.2 mg ml^?1 of the isolated fractions. Two fractions caused a reduction of cell adhesion of Candida albicans. These fractions containing bioactive compounds were analyzed by LC/MS and NMR spectroscopy. Casuarinin and digalloyl‐hexahydroxydiphenoyl‐glucose were identified in the active fractions, in addition to three signals of ellagitannins. These results indicate a specific mode of action of hydrolysable tannins against cell adhesion of Candida albicans, which needs to be further analyzed.     

Seasonal Variability of Extracellular Enzymes Involved in Carbon Mineralization in Sediment of a Subtropical Mangrove Wetland

This paper reports an investigation of microbially mediated Cr(VI) reduction in a hyperalkaline, chromium-contaminated soil-water system representative of the conditions at a chromite ore processing residue (COPR) site. Soil from the former surface layer that has been buried beneath a COPR tip for over 100 years was shown to have an active microbial population despite a pH value of 10.5. This microbial population was able to reduce nitrate using an electron donor(s) that was probably derived from the soil organic matter. With the addition of acetate, nitrate reduction was followed in turn by removal of aqueous Cr(VI) from solution, and then iron reduction. Removal of ～300 μM aqueous Cr(VI) from solution was microbially mediated, probably by reductive precipitation, and occured over a few months. Thus, in soil that has had time to acclimatize to the prevailing pH value and Cr(VI) concentration, microbially mediated Cr(VI) reduction can be stimulated at a pH of 10.5 on a time scale compatible with engineering intervention at COPR-contaminated sites.     

福建漳江口红树林湿地沉积物中四种重金属的空间分布特征

对漳江口红树林湿地沉积物中4种重金属(Pb、Cd、Ni、Fe)含量空间分布的研究表明,漳江口红树林湿地沉积物中Pb、Fe含量较高,Cd、Ni含量相对较低;林内沉积物的Pb、Cd含量显著高于林外沉积物;在垂岸方向上,表层沉积物中除Ni外其它三种元素含量均表现出从林外到林内增加的趋势;在垂直梯度上,四种元素含量均随着沉积物深度增加而逐渐降低。     

拉鲁湿地自然保护区放线菌组成分析及生物活性测定

探究拉鲁湿地自然保护区的放线菌组成及其抑菌和酶活性,为放线菌新药物先导化合物和高活性酶的筛选提供资源。从拉鲁湿地自然保护区不同土壤类型、不同优势植被采集25份土样。用分散差速离心法分离了拉鲁湿地中温放线菌和低温放线菌。从中温放线菌中选择15株代表菌株进行了初步分类鉴定。采用打孔法检测了其对2株细菌和4株病原真菌的抑菌活性。结果表明：（1）拉鲁湿地放线菌数量从水生环境向陆地生态系统递增,中温放线菌数量显著多于低温放线菌;（2）拉鲁湿地土壤中分离到链霉菌属、小单孢菌属、诺卡氏菌属、马杜拉菌属、小链孢菌属5个放线菌属。其中以链霉菌属和小单孢菌属为优势属。链霉菌属以金色类群、白孢类群和粉红孢类群为主,小单孢菌分离到黄橙类群和黑褐类群;（3）供试菌株分解纤维素能力较强,分解蛋白质活性较低,具有抗菌活性的菌株很少,且抗菌活性较弱;（4）供试菌株耐毒性物质的能力较强。这些菌可用于毒害有机物污染物的处理。     

灯盏花产黄酮内生放线菌筛选及产黄酮能力的初步研究

通过黄酮类物质特异颜色反应和可见分光光度法,筛选产黄酮的灯盏花内生放线菌,研究灯盏花产黄酮内生放线菌在PDA、高氏1号和淀粉3种培养基上的产黄酮能力。结果表明:59株灯盏花内生放线菌中,8株菌的镁粉+浓盐酸、氯化铝、浓氨水3种颜色反应均成阳性,能够产生黄酮。形态学观察初步鉴定这8株菌均为链霉菌属(Streptomyces)。3种培养基中,在PDA培养基上灯盏花产黄酮内生放线菌的菌丝体生物量、黄酮含量和产量较高。8株灯盏花产黄酮内生放线菌中,菌株RA′1-7生长较好,菌株ELA′3-2和RA2-1菌丝体黄酮含量较高,菌株ELA′3-2菌丝体黄酮产量较高。     

云南红豆杉内生放线菌TAR11活性代谢产物的初步研究

目的：初步研究从云南红豆杉植株根部筛选到的一株抗植物病害的内生放线菌TAR11的活性代谢产物性质。方法：以枯草芽孢杆菌为指示菌、以抑菌活性为指标,测定TAR11发酵液的最小抑菌浓度;用不同温度、pH值处理,了解活性物质的稳定性;用有机溶剂对活性物质萃取和溶解,并用纸层析对活性物质进行初步分类。结果：TAR11发酵液抗菌物质的最小抑菌浓度为0．78％,对温度敏感,在酸性和中性条件下稳定,可被三氯甲烷萃取,能溶于水、甲醇、乙醇、丙酮、乙醚。结论：低浓度的放线菌TAR11代谢产物能强烈抑制枯草芽孢杆菌活性,经纸层析实验初步鉴定为一类碱性抗生素。放线菌TAR11有望开发成为新一代生物药物。     

喜树内生放线菌多样性及抗菌活性评价

从采集于云南大学的喜树中分离到了90株内生放线菌,经16SrRNA基因序列分析鉴定,分布于6个科10个属。对所有分离到的菌株进行抗菌活性检测,发现33.4%的菌株有抗菌活性。通过PCR方法,检测了PKSⅠ、PKSⅡ和NRPS基因,阳性检出率分别为31.1%,48.9%和45.6%。     

海南红树林淡紫拟青霉胞外多糖提取条件的优化

为了探讨影响红树林淡紫拟青霉胞外多糖提取的因素,确定最佳提取方案,设置不同的发酵液浓缩倍数、三氯乙酸用量等因素,设计单因素实验测定多糖提取最佳条件。然后设计正交试验,检测多糖在不同条件下的提取率,以获得最佳提取工艺。结果发现,发酵液浓缩3~5倍时多糖提取率最高,10%的三氯乙酸对蛋白质脱除效果最好;正交试验表明影响多糖提取的因素依次为乙醇用量、沉淀时间和温度,最优方案为3倍95%乙醇、4 ℃沉淀24 h。该条件下,多糖提取率可达57.835%±1.206%。研究结果为红树林淡紫拟青霉胞外多糖的提取研究提供了参考。     

Mercury Concentration in Sediment Cores from Sundarban Mangrove Wetland,India

The work presents an extended database (n = 123) of total mercury (T_Hg) in fine-grained sediment fraction (<63 μm) of core samples in 10 sampling stations of the Sundarban mangrove wetland, India, formed at the estuarine realm of the Hugli (Ganga) River. Results revealed a wide range of spatial, seasonal, and intertidal flat variations of T_Hg (7.3 to 93.3 ng/g) with a definite enhancement level at the lower stretch of the estuary, which has extreme mangrove vegetation. An overall enrichment of T_Hg in surface/subsurface layers of the core is tentatively explained by remobilization and resuspension of the metal from deeper sediments (36–40 cm). A strong positive correlation was observed between the Hg and clay fraction content of the sediments, while correlations of Hg with organic carbon was poor. Based on the index of geoaccumulation (I_geo), enrichment factor (EF), and anthropogenic factor (AF) values, it is suggested that the sediments of Sundarban were found to be less polluted with respect to total mercury. The data reported are a useful baseline for T_Hg in Sundarban and would be of importance in future sediment quality studies.     

水霉拮抗放线菌的分离、筛选与鉴定

目的:以珍珠健康养殖水体底泥为材料分离放线菌,筛选对水产动物水霉病病原菌有抗菌活性的放线菌。方法:采用稀释涂布法,选用萘啶酮酸和放线菌酮双抗平板分离获得放线菌;以黄颡鱼和湘云鲫鱼卵水霉病原菌为靶标菌,采用琼脂块法测试所分离菌株抗水霉菌活性及其稳定性;对拮抗活性强的放线菌采用形态观察和16S r DNA序列分析进行分类鉴定。结果:从分离获得的27株放线菌中筛选出3株对水霉病原菌有拮抗活性的菌株QF1、DNC17和QHV2,其中QHV2抗菌活性与稳定性最好;形态学观察与16S r DNA序列分析结果表明QF1、DNC17和QHV2均属于链霉菌属(Streptomycete sp.),分别鉴定为Streptomyces diastatochromogenes、Streptomyces variabilis和Streptomyces collinus。结论:3株放线菌对水霉病原菌具有较好的拮抗活性,具有开发成抗水霉药物的潜在价值。     

南海半红树植物黄槿内源真菌GT20036029代谢产物研究

应用多种色谱技术从半红树药用植物黄槿内生真菌GT20036029中分离得到9个化合物,通过波谱学方法并与已知化合物数据作比较,鉴定它们分别为N-(2-羟基苯乙基)乙酰胺(1)、环(L-脯氨酸-D-异亮氨酸)(2)、环(L-亮氨酸-L-脯氨酸)(3)、环(D-亮氨酸-L-脯氨酸)(4)、环(亮氨酸-酪氨酸)(5)、环(苯丙氨酸-丝氨酸)(6)、脑苷脂B(7)、(25S)-纽替皂苷元-3-O-α-L-鼠李糖-(1→2)-β-D-葡萄糖苷(8)和(25S)-异纽替皂苷元-3-O-α-L-鼠李糖-(1→2)-β-D-葡萄糖苷(9)。化合物1为首次从海洋真菌代谢产物中分离得到。化合物8显示了较好的肿瘤细胞生长抑制活性。     

Aerobic Degradation of 3-Methylindole by Pseudomonas aeruginosa Gs Isolated from Mangrove Sediment

3-Methylindole (3MI), an N-heterocyclic aromatic compound also called skatole, is associated with animal waste and industrial processing. A pure culture of bacterium capable of using 3MI as the sole source of carbon and energy was isolated from mangrove sediment using an enrichment technique and identified as Pseudomonas aeruginosa Gs based on 16S rDNA sequence. Microbial degradation of 3MI was studied in batch culture experiments for several factors, including initial substrate concentrations, pH, and salinity. The optimum pH and salinity was 7.0 and 5‰, respectively. Degradation of 3MI by P. aeruginosa Gs was quantified by reversed-phase high-performance liquid chromatography. Two metabolites of 3MI degradation were detected and proposed to be indoline-3-carboxylic acid and indoline-3-ol based on data obtained from HPLC/MS. Our results suggest that 3MI can be rapidly degraded by indigenous microorganisms found in mangrove sediment.     

Effect of pH and Salinity on Sorption of Antimony (III and V) on Mangrove Sediment,Sundarban, India

The extent of toxic metalloid retention and bioavailability and mobility in the sediment is of interest for understanding their biogeochemical cycling and for accurate risk assessment in an ecosystem. Intensification of monsoon and rainfall, believed to be related to global warming, could drive future changes of temperature, salinity, and pH distribution pattern affecting antimony cycling in the Sundarbans. This study investigated sorption kinetics of antimony (Sb) (III and V) as a function of temperature, salinity, and pH following the Langmuir model, and demonstrated that clayey silt type mangrove sediment was an effective adsorbent with higher efficiency for Sb (V) than Sb (III). Background level of Sb in the sediment was 0.35–0.78% of the maximum adsorption capacity (Γ_m). Out of the two distinct type of sorption sites governing mobility and bioavailability of Sb in the sediment, site 1 (Humic acid) showed higher affinity for Sb than the site II (oxyhydroxide). Sb adsorption was strongly influenced by temperature, salinity, and pH, which may be altered by long-term changes in climate and rainfall pattern.     

Isolation and Characterization of Antimony-Reducing Bacteria from Sediments Collected in the Vicinity of an Antimony Factory

To date, there is very little information available on the oxidation and reduction of antimony by bacteria. An antimonate-respiring bacterium was isolated from sediment samples collected in the vicinity of an antimony oxide-producing factory in Korea. This bacterium was isolated by application of the Hungate standard anaerobic culture technique. Temporarily named strain JUK-1, it was found to be a rod-shaped bacterium and occurred individually or in pairs. Antimonate (pentavalent antimony) was reduced to antimonite (trivalent antimony) in the presence of JUK-1 in an anoxic minimal medium containing 5 mM antimonate and 10 mM acetate. The organism grew optimally at an initial pH of 7.7 and a temperature of 30°C. A part of the antimonite which was produced in medium precipitated as a bio-mineral containing approximately 50.0% antimony, 33.5% oxygen, 12.7% carbon, 2.0% sodium and 1.9% magnesium by weight. Based on the phylogenetics of 16S rRNA gene sequence and DNA G+C content, JUK-1 appears to be a new strain of the Sinorhizobium genus. The results suggest that bacteria may play a significant role in changing the redox state of antimony in the environment.     

Isolation and Characterization of Antidermatophytic Bioactive Molecules from Piper longum L. Leaves

Piper longum L. (Piperaceae) commonly known as “long pepper” is a well known medicinal plant in ayurveda. Different parts of this plant, such as root, seed, fruit, whole plant etc. are used traditionally in various ailments. Here we have investigated the antidermatophytic activity of sequentially extracted petroleum ether, chloroform, methanol and water extracts from P. longum leaf against Trichophytonmentagrophytes, T. rubrum, T. tonsurans, Microsporum fulvum and M. gypseum. Better activity of chloroform and methanol extracts was observed. The chloroform extract was selected for further study and the MIC value was recorded as 5.0 mg ml⁻¹ against the test organisms. In the chloroform extract, tannins and phenolic compounds were detected. Further activity-guided fractionation of chloroform extract by silica gel column chromatography yielded nine major fractions. Among these, fraction-1, 4, 5 and 7 showed higher antidermatophytic activity. Fraction-4 on further purification by repeated column chromatography yielded a potential antidermatophytic fraction showing MIC value of 0.625 mg ml⁻¹ against T. mentagrophytes and T. rubrum as determined by broth microdilution method. The major compounds were identified as 1,2-benzenedicarboxylic acid, bis(2-ethylhexyl) ester (C₂₄H₃₈O₄] (41.45 %), 2,2-dimethoxybutane (C₆H₁₄O₂] (13.6 %) and β-myrcene (C₁₀H₁₆) (6.75 %) based on GC–MS data.