The Development of a Marine Natural Product-based Antifouling Paint

Problems with tin and copper antifouling compounds have highlighted the need to develop new environmentally friendly antifouling coatings. Bacteria isolated from living surfaces in the marine environment are a promising source of natural antifouling compounds. Four isolates were used to produce extracts that were formulated into ten waterbased paints. All but one of the paints showed activity against a test panel of fouling bacteria. Five of the paints were further tested for their ability to inhibit the settlement of barnacle larvae, Balanus amphitrite, and algal spores of Ulva lactuca, and for their ability to inhibit the growth of U. lactuca. Two paints caused a significant decrease in the number of settled barnacles. One paint containing extract of Pseudomonas sp. strain NUDMB50-11, showed excellent activity in all assays. The antifouling chemicals responsible for the activity of the extract were isolated, using bioassay guided fractionation, and their chemical structures determined.

TIDAL DEPENDENCE OF PHOTOINHIBITION OF PHOTOSYNTHESIS IN MARINE MACROPHYTES OF THE SOUTH CHINA SEA

PHOTOSYSTEM-II; CHLOROPHYLL FLUORESCENCE; ULVA-ROTUNDATA; ELECTRON-TRANSPORT; FIELD EXPERIMENTS; O-2 EVOLUTION; QUANTUM YIELD; TEMPERATURE; MACROALGAE; RESPONSES

Cytoglobosins A-G, Cytochalasans from a Marine-Derived Endophytic Fungus, Chaetomium globosum QEN-14

Financial support from the Ministry of Science and Technology of China (2010CB833802 and 2007AA09Z446) and from the National Science Foundation of China (30910103914) is gratefully acknowledged.

Transforming kelp into a marine bioreactor

The past decade has seen the genetic engineering of various types of seaweed. To date, genetic transformation studies have been carried out in several seaweeds, including the red seaweeds Porphyra, Gracilaria, Grateloupia, Kappaphyclus and Ceramium and the green seaweed Ulva. A genetic transformation model system has been established in the most commonly cultivated seaweed, the brown seaweed Laminaria japonica (kelp), based on the transfer of technology used in land plant transformation and also by modulating the seaweed life cycle. This model showed the potential for application of transgenic kelp to the production of valuable products and an indoor cultivation system for transgenic kelp was proposed, taking into account necessary factors for bio-safety. In this review, the establishment at use of the kelp transformation model is introduced, highlighting the potential for transforming kelp into a marine bioreactor.

Molecular biotechnology of marine algae in China

Molecular biotechnology of marine algae is referred to as the biotechnology on the identification, modification, production and utilization of marine algal molecules. It involves not only the manipulation of macromolecules such as DNA, RNA and proteins, but also deals with low molecular weight compounds such as secondary metabolites. In the last decade, molecular systematic researches to investigate the relationship and to examine the evolutionary divergence among Chinese marine algae have been carried out by Chinese scientists. For example, RAPD has been widely used in several laboratories to elucidate genetic variations of the reds, such as Porphyra, Gracilaria, Grateloupia and the greens such as Ulva and Enteromorpha. Some important data have been obtained. The study on molecular genetic markers for strain improvement is now in progress. In 1990s, genetic engineering of economic seaweeds such as Laminaria, Undaria, Porphyra, Gracilaria and Grateloupia has been studied in China. For Laminaria japonica, the successfully cultivated kelp in China, a model transformation system has been set up based on the application of plant genetic techniques and knowledge of the algal life history. Progress has been made recently in incorporating a vaccine gene into kelp genome. Evidence has been provided showing the expression of gene products as detectable vaccines. In the present paper, the progress of molecular biotechnological studies of marine algae in China, especially researches on elucidating and manipulating nucleic acids of marine algae, are reviewed.

Three-stage transformation of chlorophyll transient fluorescence pattern under sustained dehydration and the discovery of critical water content in seaweeds

The chlorophyll fluorescence kinetics of marine red alga Grateloupia turutunt Yamada, green alga Ulva pertusa Kjellm and brown alga Laminaria japonica Aresch during natural sustained dehydration were monitored and investigated. The pulse amplified modulation (PAM) system was used to analyze the distinct fluorescence parameters during thallus dehydration. Results proved that the fluorescence kinetics of different seaweed all showed three patterns of transformation with sustained water loss. These were: 1) peak kinetic pattern (at the early stage of dehydration fluorescence enhanced and quenched subsequently, representing a normal physiological state). 2) plateau kinetic pattern (with sustained water loss fluorescence enhanced continuously but quenching became slower, finally reaching its maximum). 3) Platform kinetic pattern (fluorescence fell and the shape of kinetic curve was similar to plateau kinetic pattern). A critical water content (CWC) could be found and defined as the percentage of water content just prior to the fluorescence drop and to be a significant physiological index for evaluation of plant drought tolerance. Once thallus water content became lower than this value the normal peak pattern can not be recovered even through rehydration, indicating an irreversible damage to the thylakoid membrane. The CWC value corresponding to different marine species were varied and negatively correlated with their desiccation tolerance, for example. Laminaria japonica had the highest CWC value (around 90%) and the lowest dehydration tolerance of the three. In addition, a fluorescence "burst" was found only in red algae during rehydration. The different fluorescence parameters F-o, F-v and F-v, F-m were measured and compared during water loss. Both F-o and F-v increased in the first stage of dehydration but F-v/F-m. kept almost constant. So the immediate response of in vivo chlorophyll fluorescence to dehydration was an enhancement. Later with sustained dehydration F-o increased continuously while F-v decreased and tended to become smaller and smaller. The major changes in fluorescence (including fluorescence drop during dehydration and the burst during rehydration) were all attributed to the change in F-o instead of F-v This significance of F-o indicates that it is necessary to do more research on F-o as well as on its relationship with the state of thylakoid membrane.

MARINE ORGANISMS ATTACHED TO SEAWEED SURFACES IN JIAOZHOU BAY, CHINA

Scanning electron microscopy of the surfaces of the seaweeds Laminaria japonica, haploid Porphyra yezoensis, Ulva pertusa and the diploid conchocelis of P. yezoensis and P. haitanensis revealed Vibrio and Micrococcus to be abundant on the surfaces of U. pertusa and P. yezoensis. Vibrio, Flavobacterium, Pseudomonas, Staphylococcus, Bacillus, Corynebacterium and other genera were isolated from the surfaces of L. japonica.

Molecular phylogenetic analysis of attached Ulvaceae species and free-floating Enteromorpha from Qingdao coasts in 2007

Based on the sequence data of the nuclear ribosomal DNA internal transcribed spacer (ITS) 1, 5.8 S, and ITS 2, the molecular phylogeny was analyzed on Ulvaceae species collected from Qingdao coasts in summer of 2007, including 15 attached Ulva and Enteromorpha samples from 10 locations and 10 free-floating Enteromorpha samples from seven locations. The result supported the monophyly of all free-floating Enteromorpha samples, implying the unialgal composition of the free-floating Enteromorpha, and the attached Ulvaceae species from Qingdao coasts were grouped into other five clades, suggesting that they were not the biogeographic origin of the free-floating Enteromorpha in that season.

Temperature and light tolerance of representative brown, green and red algae in tumble culture revealed by chlorophyll fluorescence measurements

Laminaria japonica, Undaria pinnatifida, Ulva lactuca, Grateloupia turuturu and Palmaria palmata are Suitable species that fit the requirements of a seaweed-animal integrated aquaculture system in terms of their viable biomass, rapid growth and promising nutrient uptake rates. fit this investigation, the responses of the optimal chlorophyll fluorescence yield of the five algal species in tumble Culture were assessed at a temperature range of 10 similar to 30 degrees C. The results revealed that Ulva lactuca was the most resistant species to high temperature, withstanding 30 degrees C for 4 h without apparent decline in the optimal chlorophyll fluorescence yield. While the arctic alga Palmaria palmata was the most vulnerable one, showing significant decline in the optimal chlorophyll fluorescence yield at 25 degrees C for 2 h. The cold-water species Laminaria japonica, however, demonstrated strong ability to cope with higher temperature (24 similar to 26 degrees C) for shorter time (within 24 h) without significant decline in the optimal chlorophyll fluorescence yield. Grateloupia turuturu showed a general decrease in the optimal chlorophyll fluorescence yield with the rising temperature from 23 to 30 degrees C, similar to the temperate kelp Undaria pinnatifida. Changes of chlorophyll fluorescence yields of these algae were characterized differently indicating the existence of species-unique strategy to cope with high light. Measurements of the optimal chlorophyll fluorescence yield after short exposure to direct solar irradiance revealed how long these exposures could be without significant photoinhibition or with promising recovery in photosynthetic activities. Seasonal pattern of alternation of algal species in tank culture in the Northern Hemisphere at the latitude of 36 degrees N was proposed according to these basic measurements.

A comparative study of thermolysis characteristics and kinetics of seaweeds and fir wood

Thermal analysis and thermolysis kinetics of three kinds of seaweeds and fir wood (M. glyptostriboides Huet Cheng), a kind of typical land plant, had been conducted. The results showed that thermal stability follows the order of Grateloupia filicina < Ulva lactuca < Dictyopteris divaricata < fir wood. A notable difference on heat flow between seaweeds and fir wood during thermolysis was that the former were mainly connected with exothermic processes at relatively lower temperature regimes. while the latter was connected with an apparent endotherm at a relatively higher temperature regime followed by a maximum exothermic peak. This suggested that the heat coupling might be realized if co-thermolysis of seaweeds and fir wood were carried out. The main devolatilization phase of each seaweed could be described by Avrami-Erofeev equation, which indicated that thermolysis of seaweeds follows the mechanism of random nucleation and nuclei growth, whereas that of fir wood by Z-L-T equation and its thermolysis mechanism was three-dimensional diffusion. The activation energies calculated for both seaweeds and fir wood increase as conversion increases. However, those for the former have wider distribution. (c) 2006 Elsevier Ltd. All rights reserved.

大型藻类在工程治理海水富营养化和抑制病原微生物中的作用

沿海工农业生产的快速发展和人类活动对近海生态系统产生了很大影响，大量化肥的使用和工业污水、生活污水的排放导致近海环境污染，海水富营养化，赤潮频发。另外，由于近海养殖活动的迅猛发展以及养殖的不规范和不科学性导致近海生态系统结构和功能改变，一方面加重了海水富营养化，另一方面养殖动植物病害经常发生，严重影响了海产品的质量和效益。 大型海藻是海区重要的初级生产者，生命周期长、生长快，能通过光合作用吸收固定水体的C、N、P等营养物质来合成自身，同时增加水体溶解氧。因此，大型海藻被称为海洋环境中的生物过滤器。另外，由于大型藻类自身营养成分的复杂性和与藻共生的微生物多样性，大型海藻还可对生态系统中的浮游生物和微生物产生直接和间接影响。 在海洋环境，尤其是海水养殖水体环境存在着两个主要问题：海水富营养化和病源微生物控制，本文针对海洋环境中存在的这两个问题进行了探索研究。 以大型经济藻种长心卡帕藻（Kappaphycus alvarezzi）作为实验材料，分别在实验室内、室外藻类处理系统和海湾养殖现场三种条件下，进行藻类去除海水氮磷的一次性实验、半连续实验和连续实验，研究了其对海水中无机氮、无机磷的吸收速率和去除能力，初步评估了其生态价值。 构建了一种半封闭海域富营养化治理模式，以长心卡帕藻为实验材料，研究了其去除海水富营养化的能力，主要结果如下： （1）室内实验研究发现，长心卡帕藻对氮、磷的吸收速率随底物浓度升高而升高。在氮磷比为10:1，温度28℃条件下，氮浓度为50μmol • L-1时，藻对氮、磷的吸收速率达到最大，分别为0.93µmol • g-1（FW）• h-1和0.072µmol • g-1（FW）• h-1。 （2）人工修建的藻类养殖系统中进行的长心卡帕藻去除氮、磷的半连续实验，结果表明该藻具有连续去除海水DIN、DIP的能力。只要保持足够的底物浓度，长心卡帕藻对无机氮、无机磷的吸收速率达到最大，分别为0.3µmol • g-1（FW） • h-1和0.03µmol • g-1（FW）• h-1。但是对氮磷的吸收速率较室内实验有所降低。 （3）自然条件下，通过调查黎安海湾水质情况发现，长心卡帕藻具有较大的生态效益。在整个海湾大面积养殖卡帕藻，通过收获藻体，每年大约可以从海水中带走33吨氮素，7.5吨磷素。由于在海湾长心卡帕藻的作用，全年海湾水质保持在1-2级国家海水质量标准，产生了明显的生态效益。 另外，我们对大型藻类浒苔（Ulva clathrata）吸收氮磷和抑制鳗弧菌（Vibrio anguillarum）的效果进行了初步探索，结果表明：浒苔不仅对培养系统内无机氮和磷具有明显的去除作用，而且在异养细菌总量没有降低的情况下，对鳗弧菌有显著抑制作用，该抑制作用还受到水体中氮磷营养盐浓度的影响。在10g • L-1海藻的条件下，鳗弧菌以105－107 cfu • mL-1接入2天后，无论是否添加外源氮磷，鳗弧菌密度降到10 cfu • mL-1以下，鳗弧菌去除率几乎达到100%。实验数据还显示，添加氮磷营养盐可以增强浒苔对鳗弧菌的抑制作用，但没有降低其中的异养菌群数量，系统内异养细菌总量均维持在较高水平。进一步研究表明，培养浒苔24h后的海水，也对鳗弧菌65#产生抑制作用，这说明浒苔代谢释放到水体中某种化学成分或与藻共栖的微生物对鳗弧菌生长产生了抑制。

贝藻混养系统中贝类食物来源的定量分析

应用稳定碳、氮同位素比值法和脂肪酸标志分析法，较为系统地研究了贝藻混养系统中滤食性贝类的食物来源，评估了大型藻类对混养系统及滤食性贝类的物质贡献。主要研究结果如下： 1.综述了典型生态系统中大型藻类和滤食性贝类各自的生态学地位和作用，大型藻类与滤食性贝类不仅在水体营养盐方面存在互利关系，二者在物质循环与收支方面同样具有耦合性，大型藻类提供的颗粒态有机质可以为滤食性贝类提供饵料来源。 2.总结了稳定同位素比值法和脂肪酸标志法在海洋生态系统食物来源及食物网分析中的应用，并建立了两种方法的具体操作规程。 3.分析了栉孔扇贝Chlamys farreri和海带Laminaria japonica混养系统中海带碎屑形成及释放不同阶段的生态学特征，评估了碎屑对扇贝的饵料贡献。海带在6周内释放了自身约27％的碳；碎屑形成及释放过程中C:N比值显著下降，同时伴随着旺盛的细菌降解，碎屑中也发现有大量硅藻类和原生动物存在。稳定同位素分析证实海带碎屑是混养期间扇贝的主要食物来源。 4.查明了春季胶州湾潮间带自然分布的长牡蛎Crassostrea gigas、紫贻贝Mytilus galloprovincialis和湾内浅海筏式养殖栉孔扇贝的可能食物来源。湾内栉孔扇贝饵料组成中浮游硅藻类为最主要部分，同时混杂有陆源有机质和细菌类物质；潮间带自然生长的牡蛎和贻贝饵料组成中，浮游植物占86.2-89.0％，种类组成中除硅藻外还包括一定比例的金藻和甲藻类；潮间带繁盛的孔石莼Ulva pertusa藻床为两种贝类提供了8.7-11.0％的补充食物来源。 5.揭示了桑沟湾贝藻混养海区春、夏季栉孔扇贝饵料来源组成情况及其季节变化，评估了海带养殖区碎屑碳量季节变化及海带来源碳对扇贝组织碳的贡献。结果表明，湾内贝藻混养区碎屑碳量为75.52-265.19 μg l-1，其在水体总颗粒态有机碳中的比例为25.6-73.8％。海带来源碎屑碳对栉孔扇贝组织碳的贡献比例为14.1-42.8％，且与水体碎屑碳比例的季节变化存在极显著相关性(F=0.992, P=0.004)。5月份湾外海带养殖区水体碎屑碳量为110.12-144.71 μg l-1，显著高于湾内无海带区（75.52 μg l-1），湾外养殖的扇贝组织中海带来源碳比例为22.0-24.1％，显著高于湾内单养区扇贝（9.6％）。估算结果表明，桑沟湾每年收获的6967吨（总湿重）栉孔扇贝中，海带提供了约57.1吨碳，换算为海带干物质为219.6吨。脂肪酸标志分析结果表明，2月份至8月份硅藻类在扇贝饵料组成中比例逐渐下降，而细菌类比例逐渐升高。整个采样期间，EPA/DHA比例较低，说明扇贝饵料组成中可能包括高DHA含量的组分。

海藻表面附着弧菌多样性分析及方法研究

对四种大型海藻：江篱(Gracilaria textorii)、孔石莼（Ulva pertusa）、海带苗（Laminaria japonica）和多管藻（Polysiphonia urceolata）表面附着弧菌的多样性进行了分析，同时对多样性分析中采用的不同方法进行了比较。 利用TCBS培养基从四种海藻表面总共分离到12株细菌：G1、G2分离自江篱，U3分离自孔石莼，L4、L5、L6分离自海带苗，P7、P8、P9、P10、P11、P12分离自多管藻。菌株G1，G2属于盐单胞菌(Halomonas)，其余10株细菌都属于弧菌(Vibrio)。 对12株细菌的酶学活性、抗生素抗性进行了研究。发现除G1、G2外，其余菌株都可产生淀粉酶和明胶酶。菌株G1、G2和U3对氨苄青霉素、链霉素和四环素有很强抗性。 采用多种分子生物学方法（16S rDNA、gyrB、RFLP、DGGE ）分析了上述12株菌的系统发育关系，对不同方法获得的结果进行了比较。 12株菌的16S rDNA系统发生树可分为4个明显分支。G1、G2与细菌H. meridiana构成一个分支。U3与V. lentus构成一个分支。L4、L5、L6、P9、P10、P11、P12与V. tasmaniesis形成一个分支。P7、P8与V. splendidus形成一个分支。 以gyrB基因序列为基础构建的系统发生树将G1、G2以外的10株弧菌分成4个较大分支，L4、P9属于同一分支，L6、P8、P10、P11和P12属于一个大的分支， U3、L5各自构成一个分支。菌株G1、G2没有得到gyrB基因序列扩增带。表明试验设计的引物是弧菌特异性引物。相对16S rDNA，不同菌株间gyrB基因序列差异性更大。 RFLP分析结果显示，12株细菌HinfI酶切后得到6种带型，SmaI酶切后得到4种带型。结合16S rDNA、gyrB基因的分析结果可知，RFLP可以在种的水平上有效进行细菌多样性分析。 12株细菌的DGGE分析结果显示，G1、G2与其它菌株电泳图谱有明显不同。菌株U3独自构成一种带型，L5、L6构成一种带型。表明DGGE技术在属的水平上分析细菌多样性效果较好，在种的水平上分析细菌多样性有一定的局限性。

孔石莼主要化学组分的提取分析及多糖生物活性的研究

孔石莼（Ulva pertusa Kjellm）是一种大型经济海藻，除了可直接食用外在医药方面也有广泛的应用。在古代医书上便记载有多种药用活性，我国民间也流传用孔石莼治疗中暑、水肿和小便不利等病症。本论文比较全面地分析了孔石莼多糖和类脂等主要化学组合，探讨用红外和核磁光谱来解释多糖结构，并首次研究了孔石莼多糖的体外抗肿瘤的体内降血脂活性。为进一步开发孔石莼资源做了部分基础性的工作。通过对提取扎石莼多糖和类脂的方法研究，确定了两种主要组仇的提取方法，得到了多糖样品WPS、APS、DWPS、DAPS和类脂样品。用化学和仪器分析方法对多糖样品进行了水分、灰分、硫酸基含量、总糖含量、特性粘度、元素分析、单糖分析、氨基酸分析、红外和核磁光谱分析等。结果表明：用乙醇脱脂后的脱脂孔石莼水提物（DWPS）得率最高，含硫酸基量和总糖量最高，特性粘度最小；孔石莼水提物（WPS）得率和含硫酸基量次之；孔石莼碱提物（APS）因深解性不好未能按常规方法测定其总糖含量，且未检测出水解硫酸根。孔石莼多糖的单糖要有鼠李糖、葡萄糖、木糖、糖醛酸等，并检测到少量的甘露糖和阿拉伯糖。谷氨酸、天冬西氨酸是多糖样品蛋白质中的主要氨基酸，D－3－OH－半胱氨酸含量亦较高。IR谱图中出现了硫酸基的吸收峰：~H－NMR及~(13)C－NMR谱图中出现了鼠李糖和糖醛酸的特征峰。类脂样品中，非极性类脂与极性类脂的比例约为1：2；GC－MS 分析结果表明，不饱和脂质含量达61.37％，其中多不饱和脂类占44.17％，n-3系列高度不饱和脂类占39.90％。三个多糖样品WPS、DWPS、DAPS进行抗口腔上皮癌（KB）和肝癌（Bel）肿瘤细胞体外实验，结果表明在5μg/ml的剂量时三个样品抑制率均在10％以下，无明显活性表现。以烟酸肌醇为阳性对照药，蒸馏水为空白对照，对孔石莼水提多糖WPS进行了三个剂量的降血脂动物实验。实验结果表明：脂蛋白改善情况有明显的剂量依赖性；中高剂量组可显著降低胆固醇、甘油酸三脂，明显改善脂蛋白胆固醇的含量分布：高剂量样品组（500mg/kg·d~(-1)）的药用活笥最高，与相同剂量的阳性对照药烟酸肌醇结果相比，药效理佳。并对孔石莼多糖血脂的机理进行了讨论。

孔石莼多糖及其衍生物的研究

在中国，孔石莼应用于医药方面已有几千年的历史，其煎剂常被当地居民用来治疗中暑和泌尿方面的疾病。实验室以前所做的工作表明，孔石莼的水溶多糖具有很好的抗高血脂活性，本论文在此研究的基础上主要就多糖的长期毒性、一般药理、多糖的衍生化、不同分子量孔石莼多糖的抗氧化活性、衍生物的抗氧化活性和动物调血脂活性进行了研究。 水提醇沉制备多糖，多糖主要由糖醛酸、鼠李糖、木糖、葡萄糖和硫酸根组成，还含有微量的半乳糖、甘露糖和阿拉伯糖。多糖中主要的二糖重复单位为[β-D-Glcp A-(1->4)- α-L-Rhap 3S] 和 [α-L-Idop A-(1->4)- α-L-Rhap 3S]。急性毒性实验表明，KM小鼠对孔石莼多糖的最大耐受量(MTD)大于4000 mg/kg，其腹腔注射雌性小鼠的半致死量(LD50)为408.7 mg/kg，雄性动物为432.7 mg/kg。长期毒性（6个月）实验表明孔石莼多糖无毒反应剂量为1.2 g/kg。一般药理研究表明孔石莼多糖对小鼠中枢神经系统无明显影响，对麻醉犬心血管系统和呼吸系统均无明显影响。 采用过氧化氢降解的方法制备了不同分子量的孔石莼多糖，并且测定了体外抗氧化活性；制备了高硫酸根含量的孔石莼多糖、乙酰化和苯甲酰化孔石莼多糖衍生物，测定了体外抗氧化活性和动物调血脂试验。结果表明，不同分子量的孔石莼多糖其抗氧化活性是不同的，分子量低的孔石莼多糖表现出了较强的抗氧化活性。孔石莼多糖的衍生物其抗氧化活性要优于孔石莼多糖。调血脂动物试验表明，孔石莼多糖以及其衍生物都具有很好的调血脂效果。高硫酸根含量孔石莼多糖的中、低剂量组的调血脂活性要优于高剂量组（低剂量组降低低密度脂蛋白的能力要稍弱于原料），而且，中剂量组与原料组相比，小鼠血清TG明显降低（P＜0.05），LDL－C 明显降低（P＜0.01）。低剂量组乙酰化衍生物降低甘油三酯（TG）和低密度脂蛋白（LDL－C）的能力要优于中高剂量组。对于孔石莼多糖以及衍生物调血脂的机制还需进一步的研究。