两株海藻内生真菌次生代谢产物及其生物活性研究

海洋微生物拥有丰富多样的次生代谢途径，其中海洋生物内生真菌次生代谢产物研究日益受到天然产物化学界的重视。本论文以菌丝体生物量、发酵产物重量、抗菌与细胞毒活性、薄层色谱分析结果以及高效液相色谱分析结果等为评价依据对采自青岛沿海的13株海藻内生真菌在四种液体培养基上的静置发酵产物进行了综合评价，并从中选择了黑曲霉Aspergillus niger EN-13（分离自褐藻囊藻Colpomenia sinuosa）和杂色曲霉A. versicolor EN-7（分离自褐藻鼠尾藻Sargassum thunbergii）两株真菌进行了30升规模发酵（分别采用GPYM培养基和PDB培养）和化学成分的研究，对分离得到的大部分化合物进行了初步的生物活性筛选。 发酵提取物采用常规的硅胶柱层析、反相硅胶柱层析，凝胶Sephadex LH-20柱层析、制备薄层层析、半制备高效液相色谱以及重结晶等分离手段，得到单体化合物。利用各种现代波谱技术(IR、UV、EI-MS、FAB-MS、HR-ESI-MS、1H-NMR、13C-NMR、DEPT、1H-1H COSY、HSQC、HMBC等)并结合化学方法从两种菌株发酵提取物中鉴定了55个化合物的结构。其中从菌株A. niger EN-13分离鉴定了31个化合物，发现9个新化合物，包括2个鞘酯类化合物（AN-1~2）、3个萘并-γ-吡喃酮类化合物（AN-3~5）、3个苯乙基取代的α-吡喃酮类化合物（AN-17, AN-19~20）和1个甾体Diels-Alder加成产物（AN-21），另有1个新的天然环二肽（AN-27）被分离鉴定；从菌株A. versicolor EN-7分离鉴定了24个化合物，发现2个新化合物，为蒽醌AV-12与AV-17，另外，从前一菌株（A. niger EN-13）中鉴定的2个新鞘酯类化合物（AN-1~2）在A. versicolor EN-7中也被再次分离到。 对大部分单体化合物进行了抗菌活性、DPPH自由基清除活性和细胞毒活性测试。结果显示新化合物AN-1、AN-5和AN-20具有弱或中等强度的抑制白色念珠菌生长的活性，AN-4、AN-5、AN-21显示了弱或中等强度的抑制黑曲霉生长的活性，AV-12、AV-17显示了弱的抑制大肠杆菌生长的活性。在DPPH自由基清除活性筛选中，AN-5显示了中等强度的活性，其EC50为109.3 mM，与阳性对照BHT相近（EC50为81.8 mM）。其它部分已知化合物在抗菌和DPPH自由基清除活性的筛选中也显示了弱或中等强度的活性。在针对人肝癌细胞株SMMC-7721和人肺腺癌细胞株A549的体外细胞毒活性筛选中，所测样品均未显示显著活性。

Accumulation of petroleum hydrocarbons and heavy metals in clams (Ruditapes philippinarum) in Jiaozhou Bay, China

Accumulation and distributions of aliphatic and polyaromatic hydrocarbons (PAHs) and heavy metals were measured in tissues of the clam Ruditapes philippinarum collected from 5 sites in Jiaozhou Bay, Qingdao, China. The concentrations of total aliphatic hydrocarbon and PAHs ranged from 570 to 2 574 ng/gdw (gram dry weight) and from 276 to 939 ng/gdw, in the most and least polluted sites, respectively. The bio-accumulation of hydrocarbons and PAHs in the clams appeared to be selective. Aliphatic hydrocarbons were predominantly represented by short chain (< nC(23)) n-alkanes, suggesting that petroleum hydrocarbons were likely the major contamination source. The selective uptake of 3 and 4 ring PAHs, such as naphthalene, fluorene, phenanthrene, fluoranthene and pyrene, by the clams was probably related to the physiological and bio-kinetic processes that were energetically favorable for uptake of compounds with fewer rings. Accumulation of the metals Cd, Cu, Zn, Pb, Cr, Hg, and As in the clam tissues also showed high variability, ranging from 0.043 to 87 A mu g/gdw. Among the 7 detected metals, Zn, Cd, Cu, and As had a particularly high potential of accumulation in R. philippinarum. In general, a positive correlation was found between the tissue concentrations and sediment concentrations of hydrocarbons and of some metals. Our study suggests that moderate contamination with polyaromatic hydrocarbons, and low to moderate contamination with metals, currently exists for clam R. philippinarum in Jiaozhou Bay, in comparison with other regional studies. A long-term monitoring program is certainly needed for assessment of the potential ecological influence and toxicity of these contaminants of R. philippinarum in Jiaozhou Bay.

One century record of contamination by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in core sediments from the southern Yellow Sea

Sixteen polycyclic aromatic hydrocarbons (PAHs) and 28 polychlorinated biphenyls (PCBs) were measured at a 2-cm interval in a core sample from the middle of the southern Yellow Sea for elucidating their historical variations in inflow and sources. The chronology was obtained using the Pb-210 method. PAHs concentrations decreased generally with depth and two climax values occurred in 14-16 cm and 20-22 cm layers, demonstrating that the production and usage of PAHs might reach peaks in the periods of 1956-1962 and 1938-1944. The booming economy and the navy battles of the Second World War might explain why the higher levels were detected in the two layers. The result of principal component analysis (PCA) revealed that PAHs were primarily owing to the combustion product. Down-cored variation of PCB concentrations was complex. Higher concentrations besides the two peaks being the same as PAHs were detected from 4 to 8 cm, depositing from 1980 to 1992, which probably resulted from the disposal of the out-dated PCB-containing equipment. The average Cl percentage of PCBs detected was similar to that of the mixture of Aroclor 1254 and 1242, suggesting they might origin from the dielectrical and heat-transfer fluid. The total organic carbon (TOC) content played a prevalent role in the adsorption of high molecular weight PAHs (>= 4-ring), while no obvious relationship among total PCBs, the concentration of congeners, and TOC was found.

Persistent organic pollutant residues in the sediments and mollusks from the Bohai Sea coastal areas, North China: An overview

The Bohai Sea costal area is one of the most developed zones of China and the sewage water from populous and developed cities, including Beijing, Tianjin. Qinhuangdao and Dalian is discharged into the Bohai Sea. Additionally, its semi-enclosed characteristic restricts water exchange, which leads to high accumulation of pollutants in the environment. This overview presents the residues of 6 classes of Persistent Organic Pollutants (POPs). including PAHs, DDTs. HCHs, PCBs and PCDD/Fs. in the sediments and mollusks of the Bohai Sea through analyzing previous literatures. In the sediments. the highest PAH concentrations were detected in the vicinities of Qinhuangdao, while the northeast corner of the Bohai Bay possessed the highest levels of DDTs and PCBs. The investigations on HCHs and PCDD/Fs distributions on the whole sea scale have not been reported. In mollusks, PAH concentrations were in the same order of magnitude in the whole Bohai Sea, so were DDTs. HCHs and PCBs, while the outlier maximum values of PCDDs and PCDFs occurred in Yingkou. In general, the POPs residues in mollusks collected from Shandong Province were higher than the other areas. The compositions of DDTs, HCHs and PCBs in sediments indicated their recent usage. By comparing POP concentrations in sediments with the recommended criterions, it was shown that some individual PAH compounds occasionally associated with adverse biological effects in the vicinities of the Liaodong Bay and Qinhuangdao, and the Liaohe River Estuary were heavily contaminated with DDTs, but PCBs were all below the thresholds. In order to reveal the transference and transformation of POPs in the environment, further studies concerning with their behavior, fate and bioaccumulation in the different trophic levels should be programmed. Moreover, laws and regulations should be enforced to ban the illegal usage of POPs-containing pesticides to guarantee health of the environment and human. (C) 2008 Elsevier Ltd. All rights reserved.

Antimicrobial screening and active compound isolation from marine bacterium NJ6-3-1 associated with the sponge Hymeniacidon perleve

Twenty-nine marine bacterial strains were isolated from the sponge Hymeniacidon perleve at Nanji island, and antimicrobial screening showed that eight strains inhibited the growth of terrestrial microorganisms. The strain NJ6-3-1 with wide antimicrobial spectrum was identified as Pseudoalteromonas piscicida based on its 16S rRNA sequence analysis. The major antimicrobial metabolite, isolated through bioassay-guide fractionation of TLC bioautography overlay assay, was identified as norharman (a beta-carboline alkaloid) by EI-MS and NMR.

Marine bacteria associated with marine macroorganisms: the potential antimicrobial resources

In order to explore marine microorganisms with medical potential, marine bacteria were isolated from seawater, sediment, marine invertebrates and seaweeds collected from different coastal areas of the China Sea. The antimicrobial activities of these bacteria were investigated. Ethyl acetate extracts of marine bacterial fermentation were screened for antimicrobial activities using the method of agar diffusion. The results showed that 42 strains of the isolates have antimicrobial activity. The proportion of active bacteria associated with marine invertebrates (20%) and seaweeds (11%) is higher than that isolated from seawater (7%) and sediment (5%). The active marine bacteria were assigned to the genera Alteromonas, Pseudomonas, Bacillus and Flavobacterium. The TLC autobiographic overlay assay implied that the antimicrobial metabolites produced by four strains with wide antimicrobial spectrum were different. Due to a competitive role for space and nutrient, the marine bacteria associated with marine macroorganisms (invertebrates and seaweeds) could produce more antibiotic substances. These marine bacteria were expected to be potential resources of natural antibiotic products.

Distribution pattern of marine flagellate and its controlling factors in the central and north part of the Huanghai Sea in early summer

A survey was carried out in the central and north part of the Huanghai Sea (34.5degrees similar to 37.0degreesN, 120.5degrees similar to124.0degreesE) during June 12 similar to 27, 2000. It was found that the abundance of marine flagellate ranged from 45 to 1278 cell/ml, 479 cell/ml in average. Flagellate was more abundant in the central part than in the north part of Huanghai Sea, and the abundance decreased with the increasing distance from the coast, showing a similar distribution pattern with isotherm. Vertically, high density of flagellate was always presented in the bottom of thermocline, and formed a dense accumulation in the central area of the Huanghai Sea Cold Water Mass. The effects of physical and biological factors on the distribution of marine flagellate in early summer were discussed. Water temperature (especially the existence of thermocline) rather than salinity showed significant effect on the distribution pattern of marine flagellate in the Huanghai Sea in early summer. When comparing the abundance of marine flagellate with that of other microorganisms, it revealed a comparatively stable relationship among these organhisms, with a ratio of heterotrophic bacteria: cyanobacteria: flagellate: dinoflagellate: ciliate being 10(5) 10(3):10(2):10(1):10(0).

Sources and distribution of aliphatic and polyaromatic hydrocarbons in sediments of Jiaozhou Bay, Qingdao, China

The composition and distribution of aliphatic (n-alkanes) and polyaromatic hydrocarbons (PAHs) were measured for the surface sediments collected at 25 sites from Jiaozhou Bay, Qingdao, China. Total n-alkanes and PAH concentrations ranged from 0.5 to 8.2 mu g/gdw and 0.02 to 2.2 mu g/gdw, respectively, and the distribution of both n-alkanes and PAHs showed large spatial variations in the bay. The distribution of PAHs in the sediments was predominated by the three or more ring compounds. High hydrocarbon levels were generally found in the areas associated with high anthropogenic impact and port activities in the bay. The calculated hydrocarbon indexes suggest that petroleum contamination was the main source of n-alkanes, while both pyrolytic and petrogenic sources contributed PAHs to the surface sediments of Jiaozhou Bay. In comparison to other polluted coastal sediments, the level of contamination from both aliphatic hydrocarbons and PAHs in Jiaozhou Bay sediments is relatively low at the present time. (c) 2005 Elsevier Ltd. All rights reserved.

Bromophenol derivatives from the red alga Rhodomela confervoides

Eight new bromophenol derivatives, 2,3-dibromo-4,5-dihydroxybenzyl methyl sulfoxide (1), 4-(2,3-dibromo-4,5-dihydroxyphenyl)-3-butene-2-one (2), 2-(3-bromo-5-hydroxy-4-methoxyphenyl)-3-(2,3-dibromo-4,5-dihydroxyphenyl)propionic acid (3), 2-(3-bromo-5-hydroxy-4-methoxyphenyl)-3-(2,3-dibromo-4,5-dihydroxyphenyl)propionic acid methyl ester (4), 2-phenyl-3-(2,3-dibromo-4,5-dihydroxyphenyl)propionic acid (5), 4'-methoxy-2",3',3"-tribromo-4",5',5"-trihydroxydiphenylacetic acid (6), and 3-bromo-5-hydroxy-4-methoxyphenylacetic acid (7) and its methyl ester (8), together with a known bromophenol, 3-bromo-5-hydroxy4-methoxybenzoic acid (9), were isolated from the red alga Rhodomela confervoides. Their structures were elucidated by spectroscopic methods including IR, EIMS, FABMS, ESIMS, HRFABMS, HRESIMS, 1D and 2D NMR, and single-crystal X-ray structure analysis. Compounds 1-4, 8, and 9 were found inactive against several human cancer cell lines and microorganisms.

Bromophenols coupled with derivatives of amino acids and nucleosides from the red alga Rhodomela confervoides

Three new bromophenols coupled with pyroglutamic acid derivatives and one bromophenol coupled with deoxyguanosine were obtained from the red alga Rhodomela confervoides. By spectroscopic methods including 2D NMR and single-crystal X-ray structure analysis their structures were elucidated as N-(2,3-dibromo-4,5-dihydroxybenzyl)methyl pyroglutamate (1), N-(2,3-dibromo-4,5-dihydroxybenzyl)pyroglutamic acid (2), N-[3-bromo-2-(2,3-dibromo-4,5-dihydroxybenzyl)-4,5-dihydroxybenzyllmethyl pyroglutamate (3), and 2-N-(2,3-dibromo-4,5-dihydroxybenzylamino)deoxyguanosine (4), respectively. Compounds 1-4 were evaluated against several microorganisms and human cancer cell lines, but found inactive. To our knowledge this is the first report of bromophenols coupled with amino acid or nucleoside derivatives through the C-N bond.

Possible suppression of exogenous beta-1,3-glucanase gene gluc78 on rice transformation and growth

Three genes encoding for fungal cell wall degrading enzymes (CWDE), ech42, nag7O and gluc78 from the biocontrol fungus Trichoderma atroviride were transformed into rice mediated by Agrobacterium tumefaciens singly and in all possible combinations. A total of more than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. Our data indicated that gluc78 gene had negative effects on transformation frequency and plant growth. Some regenerated plants with gluc78 gene were stunted; spontaneously produced brown specks; could not tassel. The combination with either one of the two other genes (ech42, nag70) present in the same T-DNA region reduced the negative effect of gluc78 on plant growth. These results indicated that expression of several genes in one T-DNA region interfered with each other and expression of exogenous gene in recipient plant was a complex behavior. (c) 2007 Published by Elsevier Ireland Ltd.

The characteristics of nutrient removal and inhibitory effect of Ulva clathrata on Vibrio anguillarum 65

To investigate the ecological effect of macroalgae on de-eutrophication and depuration of mariculture seawater, the variation of dissolved inorganic nitrogen (DIN) and phosphate (DIP), the amount of Vibrio anguillarum, and total heterotrophic bacteria in Ulva clathrata culture, as well as on the algal surface, were investigated by artificially adding nutrients and V. anguillarum strain 65 from February to April 2006. The results indicated that U. clathrata not only had strong DIN and DIP removal capacities, but also showed a significant inhibitory effect on V. anguillarum, although not reducing the total heterotrophic bacteria. Vibrio anguillarum 65 dropped from 5 similar to 8 x 10(7) cfu mL(-1) to 10 cfu mL(-1) (clone-forming units per mL) in 10 g L-1 of fresh U. clathrata culture within 2 days; i.e., almost all of the Vibrios were efficiently eradicated from the algal culture system. Our results also showed that the inhibitory effect of U. clathrata on V. anguillarum strain 65 was both DIN- and DIP-dependent. Addition of DIN and DIP could enhance the inhibitory effects of the algae on the Vibrio, but did not reduce the total heterotrophic bacteria. Further studies showed that the culture suspension in which U. clathrata was pre-cultured for 24 h also had an inhibitory effect on V. anguillarum strain 65. Some unknown chemical substances, either released from U. clathrata or produced by the alga associated microorganisms, inhibited the proliferation of V. anguillarum 65.

Characteristics of sulfide corrosion products on 316L stainless steel surfaces in the presence of sulfate-reducing bacteria

It has been found that microbial communities play a significant role in the corrosion process of steels exposed in aquatic and soil environments. Biomineralization influenced by microorganisms is believed to be responsible for the formation of corrosion products via complicated pathways of electron transfer between microbial cells and the metal. In this study, sulfide corrosion products were investigated for 316L stainless steel exposed to media with sulfate-reducing bacteria media for 7 weeks. The species of inorganic and organic sulfides in the passive film on the stainless steel were observed by epifluorescence microscope, environmental scanning electron microscope combined with energy dispersive spectroscopy and X-ray photoelectron spectroscopy. The transformation from metal oxides to metal sulfides influenced by sulfate-reducing bacteria is emphasized in this paper. (c) 2005 Elsevier B.V. All rights reserved.

污染土壤的菌根修复

菌根是植物根系与菌根真菌形成的共生体,能有效促进污染物的降解和转化,使污染环境得以修复。阐述了多环芳烃（PAHs）、农药、重金属等污染土壤中菌根的作用,分析了菌根修复可能的机理,旨在说明利用菌根修复是生物修复的一个重要方面,具有广阔的发展前景,为进一步研究菌根的作用以及更好地运用菌根技术奠定基础。

宣龙式赤铁矿含矿岩系的成矿过程研究

Xuanlong-type Hematite Deposits, distributed in Xuanhua and Longguang area in Hebei province and hosted in the Changchengian Chuanlinggou Formation of Mesoproterozoic, is an oldest depositional iron deposit characterized by oolitic and stromatolitic hematite and siderite. This thesis made an systematic study of its sedimentary, sedimentology, geochemistry, mineralogy and sequence stratigraphy. Based on above, the mechanism and background of biomineralization are discussed. There are four types of hematite ores including stromatolite, algal oolite, algal pisolite and oncolite. Based on detailed study on ore texture, the authors think both algal oolite and algal pisolite ores are organic texture ores, and related to the role of microorganisms. The process of blue-green algae and bacteria in the Xuanlong basin absorbing, adsorbing and sticking iron to build up stromatolite is the formation process of Xuanlong-type hematite deposit. Researches on ore-bearing series and ore geochemistry show that the enrichment of elements is closely related to the microorganism activities. Fe_2O_3 is enriched in dark laminations of stromatolite with much organic matter and SiO_2 in light laminations with detrital matters. The trace elements, especially biogenic elements, including V, P, Mo are enriched in ores but relatively low in country rocks. The paper also demonstrates on the sequence stratigraphy of hematite deposits and five sequences and twelve systems are divided. The characteristics of sequence stratigraphy show that the deposit-forming location has obviously selectivity and always exists under a transgressive setting. The oxygen isotope in hematite is about -2.2～5.7‰, which is similar to that of Hamlys iron formation of Australia but more negative than that of volcanic or hydrothermal iron deposits characterized by high positive values. The calculation by the result of oxygen isotope analysis shows that the temperature of ancient sea water was 48.53℃. The negative value of carbon isotope from siderite indicates its biogenic carbon source. Meanwhile, the occurrence of seismite in the ore-beds, which indicates the formation of hematite deposits is associated with frequent shock caused by structural movement such as distal volcano or ocean-bottom earthquake etc, show the occurrence of hematite deposits is eventual, not gradual. In shorts, Xuanlong-type hematite deposits were the result of interaction among geological setting of semi-isolated Xuanglong basin, favorable hot and humid climate condition, abundant iron source, microorganism such as algae and bateria as well as the fluctuation of the sea level.