环境工程论文翻译参考文献.doc

Biofouling Vol. 26, No. 3, April 2010, 301312 The uronic acids assay: a method for the determination of chemical activity on biolm EPS Kristina D.A. Mojica and Michael J. Cooney Department of Oceanography, School of Ocean and Earth Sciences and Technology, University of Hawaii, Honolulu, HI 96822, USA; bHawaii Natural Energy Institute, School of Ocean and Earth Sciences and Technology, University of Hawaii, Honolulu, HI 96822, USA (Received 7 June 2009; nal version received 20 November 2009) In this work, the uronic acids assay was evaluated for its potential to function as a bioassay to screen for antagonistic activity gainst the production of microbial biolm exopolysaccharide(EPS). The assay was rst applied to biolms produced in the presence of two universal disinfectants (sodium hypochlorite and sodium dodecyl sulfate) known o inhibit microbial growth and biolm formation. The performance of the assay was the characterized through statistical assessment of threshold concentrations for disinfection eciency and consistency relative to values reported in the literature. The assay was then evaluated for its utility in screening for enzymatic or chemical inhibitors of biolm formation (eg glycosidases, halogenated furanones, and semi-crude fractions extracted from minimally fouled marine plants) and its ability to distinguish between true anti-biolm activity and simple disinfection. Activity was characterized as (i) no eect, (ii) a true positive eect (ie increased biolm EPS), (iii) anti- bacterial activity (ie decreased biolm EPS and analogous decrease in planktonic growth), and (iv) anti-biolm EPS activity (ie decreased biolm EPS, without analogous decrease in planktonic growth). Results demonstrate that the uronic acids assay can augment existing biolm characterization methods by providing a quantitative measure of biolm EPS. Keywords: uronic acid; biolm; exopolysaccharide; assay; C. lytica Introduction 生物污染卷。 26日，第3期，2010年4月301-312糖醛酸的测定：用于化学活性生物膜每股收益克里斯蒂娜和Michael J.多巴胺Mojica库尼海洋学系，海洋和地球科学与技术学院测定方法夏威夷，檀香山，嗨96822，美国; bHawaii自然能源研究所，海洋与地球科学与技术，夏威夷，檀香山，嗨96822，美国大学法学院（2009年6月7日收稿;最终版本收到2009年11月20日）在这项工作中，糖醛酸的测定是评价其潜力，作为一个生物测定为拮抗微生物它多屏幕生物膜胞外多糖生产（EPS）的。该法首次应用到已知的两个普遍消毒剂（次氯酸钠和十二烷基硫酸钠）的存在产生生物膜抑制微生物的生长和生物膜的形成。本方法的性能通过阈值浓度的统计评估消毒效率和一致性相对于文献报道值的特点。该法是然后评估其效用，用于筛检生物膜形成（如糖苷酶，卤代呋喃酮，并从海洋植物中提取的微创犯规半粗分数）酶或化学抑制剂，它有能力区分真正的抗生物膜的活动和简单消毒。活动的特点为：（我）没有影响，（二）真正的正效应（即每股收益增加生物膜），（三）抗菌活性（即每股收益下降生物膜和浮游生长类似减少），（四）反生物膜每股收益的活动（即每股收益下降生物膜的生长无类似浮游减少）。结果表明，糖醛酸的检测提供了一个可以增加每股收益的生物膜生物膜现有的定量测量表征方法。关键词：糖醛酸;生物膜;胞外多糖;试验; Introduction It is now recognized that surface-associated bacteria are as important as planktonic free-living cells in the ecology of aquatic environments (Webb et al. 2003). Any surface with episodic or continuous exposure to water will quickly become colonized with bacteria, whose growth and activity support the formation of complex three-dimensional matrices of cells and excreted exopolysaccharides (EPSs) commonly termed biolms. The establishment of such structures aords bacteria with a measure of homeostasis, a primitive circulatory system, and a framework for the develop-ment of cooperative and specialized cell functions(Costerton et al.1994; Davey and OToole 2000; Stoodley and Stoodley 2005; Nobile and Hall- Mitchell 2007). Biolms also contribute signicantly to micro-bial productivity through improvementof nutrient andmetabolite exchange (eg enhancing nutrient availabil-ity and removal of potentially toxic metabolites) and by providing protection against a range of antagonists(eg antibiotics, biocides, immune systems, heavymetals, UV radiation, predators) (Dang and Lovell2000; Davey and OToole 2000;Parsekand Singh. 2003; Webb et al. 2003; Hall-Stoodley et al .2004).现在是承认，表面相关浮游细菌一样自由生活在水生环境生态学（韦伯等人。2003）细胞的重要。任何偶发或连续暴露表面的水将很快成为殖民统治与细菌，其生长和活动支持复杂的三维矩阵的细胞和分泌胞外多糖（EPSs）的形成通常被称为生物膜。戴维和奥图尔2000;这种结构能提供一个衡量的平衡，一种原始的循环系统，并为发展，合作和专门彪细胞功能（Costerton等al.1994框架细菌和斯托德利2005年建立斯托德利;诺比尔和霍尔米切尔2007年）。生物膜也极大地促进微bial生产力通过improvementof营养andmetabolite交换（如加强营养availabil性和具有潜在毒性代谢产物的清除），并通过提供针对拮抗剂（如抗生素，杀虫剂，免疫系统，heavymetals，紫外辐射范围的保护，食肉动物）（党和Lovell2000;戴维和奥图尔2000 Parsekand辛格2003年。韦伯等人2003;。霍尔斯托德利等.2004）。Biolms provide an important survival strategy for microbes in natural aquatic systems and have signicant inuences on aquatic ecology, the maintenance of industrial processes, and human health (Costerton 1995; Davey and OToole 2000; deSaravia and deMele 2003; Hall-Stoodley et al. 2004).生物膜为微生物的天然水生系统的一个重要的生存策略，而对水生生态信号，相重大的影响，工业进程的维护，以及人类健康（Costerton 1995;戴维和奥图尔2000 deSaravia和deMele 2003;霍尔斯托德利等人。2004年）。 Previously, Mojica et al. (2007)reported on the application of the uronic acids assay as a method to accurately assess the quantity of microbial EPS present using the component uronic acids as a proxy. In the present study,the method is further evaluated for its ecacy as a screening technique for the identication of compounds that inhibit the production of biolm EPS by Cytophaga lytica.The assay was evaluated using four dierent types of chemical activity,viz.disinfection, cellcell signaling inhibition,enzymatic degradation and unknown activity (Table 1).The ecacy of the assay was characterized by its ability to distinguish alterations in the quantity of biolm EPS due to true anti-biolm activity from simple disinfec-tion. The uronic acids assay,as presented here, is distinct from current biolm assays in that it measures a specic component of the biolm EPS,thus facili- tating a focus on the inhibition of biolm formation rather than the susceptibility of established biolms to various chemical treatments.此前，Mojica等。 （2007年）报告了该法糖醛酸作为一种方法来准确地评估目前使用的微生物数量每股收益作为代理组件糖醛酸的应用。在本研究中，该方法是进一步评价其作为的化合物抑制生物膜每股收益由Cytophaga lytica这法生产的评估采用四种化学活性，viz.disinfection，识别不同类型的细胞筛选技术效能细胞信号抑制，酶降解和未知的活动（见表1）。本方法的有效性是它是否有能力分辨出由于生物膜每股收益真正反生物膜从简单disinfec - tion活动量变化的特点。糖醛酸的测定，为这里提出，是从当前生物膜检测措施，它的每股收益的具体内容生物膜从而facili - tating对生物膜形成的抑制，而不是建立生物膜易感性的焦点，各种不同的化学处理。C. lytica was chosen as the model bacterium in this study as it provides a potential target for the chemical regulation of biofouling by macroalgae. In Hawaii,C.lytica biolms have been recognized as an important settlement and metamorphosis cue for Hydroides elegans, a dominant member of fouling communities(Huang and Hadeld 2003). Members of the genusCytophaga are well-known for their production of biolm EPS (Huang and Hadeld 2003) and, like other members of this genus,C. lytica is found worldwide and is a common member of the bacterial communities of coastal sea regions,especially on the surfaces marine benthic macroalgae and in tide pools (Johansen et al. 1999)C lytica被选为本研究模型细菌，因为它提供了一种海藻的生物污染的化学调控的潜在目标。在夏威夷，C.lytica生物膜已被确认为一项重要的结算及Hydroides线虫，有污染的社区（黄和哈德菲尔德2003年）的主要成员变态的线索。属Cytophaga大家所熟知他们的生物膜每股收益（黄和哈德菲尔德2003年）生产，该属植物一样，C.其他成员， lytica被发现，是一个世界范围内的细菌群落的沿海区域的共同成员，特别是对海洋底栖海藻的表面，在潮池，（约翰森等人。1999年）Materials and methods 材料和方法Bacterial strain and growth conditions 菌株和培养条件The C. lytica strain used in this study was isolated from a natural marine biolm from Pearl Harbor, HI(donated by Michael Hadeld,University of Hawaii, USA) (Huang and Hadeld 2003). Cultures were prepared by recovering the strain from bacterial stock solutions (1:1 ratio of growth culture and 20% glycerolin deionized (DI) water,-80 storage) by culturing them in seawater tryptone medium (ie nutrients added at half-strength of the original recipe) for 24 h at25and 160 rpm.Preparation of biolm inoculate应变的C. lytica用于此项研究从自然中分离从珍珠港，嗨（黄和哈德菲尔德2003）（由迈克尔哈德菲尔德，美国夏威夷大学的捐赠）海洋生物膜。文化是由股票恢复解决方案是由细菌株（1:1的比例增长20glycerolin文化和去离子片（DI）水，-80储存）编写的，以海水蛋白胨培养液（即营养补充他们在半场实力原配方24小时）在25和160 rpm.PreparationPreparation of biofilm inoculate生物膜的制备接种In order to induce the formation of a biolm, bacterial cells were harvested from liquid cultures by centrifugation(4000 rpm, 10 min) and resuspended in ltered sterile (F/S) seawater to a nal optical density of 1.000 + 0.005 at 600 nm (OD600). Ten milliters of the resulting standardized cell suspension, 1.25 108cells,were then used as the inoculum for biolm growth in all assays. 为了诱导形成生物膜，细菌细胞收获由离心液体培养（4000转，10分钟），并重新在过滤消毒（女/ S）的海水款，最后光密度1.000 + 0.005在600纳米（OD600值）。由此产生的十大标准化细胞悬液，1.25 108细胞，当时用作生物膜在所有检测的增长接种。Cultivation of biolms生物膜的培养Biolms were cultivated by taking 10 ml aliquots of well-mixed biolm inoculum into pre-treated (acid- washed on ice using HCl/HNO (3:1);sterilized by autoclaving) glass test tubes (50 ml) containing chemical treatment and incubating for 24 h at 25and 160 rpm. After incubation, the solution was decanted and the residual biolms in each tubewere rinsed three times with 2 ml aliquots of (F/S) deionized (DI) water. The test tubes were then air-dried (4560 min) under a sterile laminar ow hood and stored at -20until assayed for uronic acid content.Quadruplicate replicates were performed in all experiments. 生物膜培育的考虑到前处理（酸洗使用盐酸/硝酸（3:1）冰;通过高压灭菌消毒）10充分混合生物膜接种毫升等份玻璃试管（50毫升）含有化学治疗和孵化24小时在25和160转。孵育后，溶液倒出，并在每个tubewere剩余生物膜冲洗2给出了（F / S）的去离子（直接投资）毫升水等分三次。在试管中，然后风干（45-60分钟在无菌的层流罩于-20保存，直到为糖醛酸含量.在所有实验进行重复检测。Positive controls contained 10 ml of biolm inoculum and 1 ml of DI water.A second positive solvent controlwas created by combining 10 ml of biolm inoculum with 1 ml of the appropriate chemical treatment solvent.The solvent control wasused to determine whether the presenceof solvent hadany impact upon planktonic growth and/or uronic acid content.Negative controls consisted of 10 ml (F/S) seawater and 1 ml (F/S)DI water.All controls were incubated for the entire 24 h biolm growth period. 阳性对照含有10毫升的生物膜接种和1秒的直接投资water.A积极溶剂相结合，一适当的化学处理solvent.The溶剂对照毫升10毫升创建controlwas生物膜接种，以确定是否毫升wasused后浮游生长和/或糖醛酸content.Negative 10毫升（女/ S）的海水和1毫升（F / S模式）直接投资water.All对照组在整个生育期24小时培养生物膜包括控制presenceof溶剂hadany影响。 Planktonic CFU counts浮游菌落形成单位计数In order to determine whether alterations in thequantity of biolm EPS resulted from simple disinfection and,therefore,a lack of cells to participate in biolm formation,or were the consequence of true anti-biolm activity,crude plate counts were performed on the planktonic populations remaining after the biolm growthperiod.Specically,at the end of each growth period,the solution in each test tube was sampled to obtain 125 ml aliquots. These samples were subsequently subjected to a 1:10 serial dilution using(F/S) seawater. Twenty-ve microliters of each dilution of 10010-5were smeared onto seawater-tryptoneyeast extract (SWT) agar plates. The plates were then incubated at 25for 48 h, after which each plate that revealed distinct colony forming units (CFU)was enumerated.为了确定是否在生物膜每股收益thequantity改变，从简单的消毒造成的，因此，一个细胞缺乏参与生物膜形成，或者是真正的抗生物膜活动造成原油板计数剩余的浮游种群进行生物膜增长period.Specifically后在每个生育期结束后，在每个试管溶液取样，获得125毫升等份。这些样品随后遭受了1:10稀释使用（女/ S）的海水。二十五年的每100 - 10 - 5上半海水tryptoneyeast提取物（SWT）的琼脂平板稀释涂微升。板块，然后在25培养48小时，每盘之后，发现有明显的集落形成单位（CFU）的枚举。Chemical treatments化学处理 The purpose of the assay, as applied in this study, was to identify chemicals with potential activity against the production of biolm EPS.Successful chemical regulation of biolm growth requires that the chemical be present in sucient quantity upon initiation of biolm formation.Therefore, unless otherwise specied,all solutions tested for chemical activity on biolm EPS were added prior to the addition of biolm inoculum and thus were present during the entire biolm growth period.本方法的目的，在这项研究中的应用，是确定对生物膜与生物膜增长EPS.Successful潜在活性化学调控生产的化学品要求在启动后，生物膜的形成.目前数量充足，除非另有规定外，化学活性测试生物膜每股收益增加了所有解决方案之前，除了接种的生物膜，从而为在整个生长期生物膜的存在。Group 1: disinfectants第1组：消毒剂Concentrations of sodium hypochlorite(NaClO) and sodium dodecyl sulfate (SDS) were obtained through a 1:2 serial dilution of Clorox brand household bleach solution containing 6% NaClO and a 6% SDS solution (w/v) with DI water, respectively.次氯酸钠（次氯酸钠）和十二烷基硫酸钠（SDS）的浓度，获得通过1:2稀释的家用漂白Clorox公司品牌含6次氯酸钠溶液和6的SDS溶液与去离子水（瓦特/ V），分别为。Group 2: cellcell signaling inhibitors第2组：细胞间的信号抑制剂(5Z)- 4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone(donated by Stea Kjelleberg),termedhalogenated furanone compound 2 in the literature and hereafter in this article, was dissolved in dimethyl sulfoxide (DMSO) (0.87% nal test concentration) and tested at 18m and 180m concentrations.（5Z） - 4 -溴- 5 -（bromomethylene）- 3 -丁基- 2（5小时），呋喃（由Steffa Kjelleberg捐赠），termedhalogenated在文献和以后在这篇文章中呋喃酮化合物2 ，是溶于二甲基亚砜（DMSO）（0.87最终测试浓度）和18m和180m间，随后浓度测试。Group 3: glycosidases糖苷酶(Sigma as a potential antagonist of biolm EPS,the following concentrations were used:16.67 mg ml-1 of cellulase(Sigma C1184), 1 mg ml-1 of-amylase (Sigma A6211),and 5 mg ml-1 of -glucanase (Sigma 49106). All enzymes were dissolved in sodium carbo-natebicarbonate buer (0.1 M, pH 8).Glycosidases are enzymes that function by hydrolyzing glycosidic linkages of complex saccharides. Moreover, enzymes are sensitive to degradation,changes in pH,and lose activity over time. As a consequence, glycosidases might function more effectively when applied to established biolms. To ensure that any lack of effect resulting from the application of glycosidases in this study was not caused by the loss of activity of the enzymes, a second exposure period of 2 h (ie 22 h after biolm growth was initiated) was added to experiments in which glycosidases were employed.（西格玛作为生物膜的每股收益的潜在对手，以下浓度分别采用：16.67毫克，纤维素酶（西格玛C1184），1毫升1毫克毫升- 1-淀粉酶（Sigma公司A6211）和5毫克毫升- 1 -葡聚糖酶（西格玛49106）。所有的酶溶解在钠卡尔沃-内特，碳酸氢盐缓冲液（0.1米，pH值8）。糖苷酶是水解酶，糖苷键的复杂的糖类的功能。此外，酶是敏感的退化，pH值的变化，随着时间的推移而失去活性。因此，糖苷酶可以更有效地运作时，适用于建立生物膜。为了确保任何从本研究中的应用产生的效果糖苷酶缺乏不是由活性的酶造成的损失，为2小时（即22小时后开始生长生物膜）秒曝光时间，在其中加入糖苷酶被雇用实验。Biological extracts生物提取液Three macrophytes,Ulva reticulata, Acanthophora spicifera, and Gracilaria coronopifolia found previously to control settlement of H. elegans using pre-settlement processes (Walters et al. 1996), were targeted for collection from shallow water areas around the island of Oahu, HI. No less than 5 g of dry weight material were collected for each targeted species. Collected material was visually inspected and any macroscopic epifauna removed. The material was then ash-frozen in liquid nitrogen and freeze-dried under vacuum. Freezedried samples were crushed and extracted three times using 100% methanol for 1 h, 3 h, and overnight.The resulting crude extracts were then pooled and subsequently ltered through a cotton-tipped pipette followed by a 0.45 mm syringe lter. The solvent was then removed in a rotary evaporator at 110 mbar and 35三植物，石莼马丁，Acanthophora spicifera，和江蓠coronopifolia发现以前的H.控制使用预结算解决进程线虫（沃尔特斯等。1996年），被收集对象为来自各地的瓦胡岛，嗨岛浅水区。干重的材料不小于5克共收集各目标物种。收集的材料目视检查，任何宏观epifauna删除。当时的材料闪存液氮冷冻和冷冻真空干燥。 Freezedried样品粉碎，提取三次使用1小时，3小时100甲醇，粗提取物和overnight.The产生汇集，然后通过一个棉签用0.45毫米注射器过滤器过滤后吸管其后。然后删除该溶剂在旋转蒸发器在110毫巴和35Further renement of the crude extracts was accomplished using solid-phase extraction (SPE).Specically,crude extracts were loaded onto SPE columns containing silica gel 60 RP-18 in a 1:10 weight ratio of extract to stationary phase.Loaded columns were then dried for 2 h at 30. Columns were then eluted repeatedly while varying the polarity of the mobile phase (ie water/methanol/isopropanol) and, for each fraction, the solvent was removed by a rotaryEvaporator. 粗提物进一步细化为完成使用固相萃取（SPE）。具体来说，含粗提取物上硅胶固定的提取物的重量比为1:10 phase.Loaded列60的RP - 18固相萃取柱，然后装干燥2日30。列，然后反复而改变洗脱的流动相（即水/甲醇/异丙醇）极性，并为每个部分，溶剂是由一个旋转蒸发器除去。For growth of biolms in the presence of biological extracts,the extracts were dissolved in the extraction solvent and tested at a concentration of 200 mg ml-1 . 对于生物膜生长的生物提取物的存在，提取物溶解在溶剂萃取和浓度为200毫克毫升测试。 Uronic acids assay of biolms生物膜法糖醛酸 Analysis of the uronic acid content of dried biolms was executed as described previously (Mojica et al. 2007), except in those cases where sulfamate was used. In the latter case,sulfamate (sulfamic acid/potassium sulfamate) (4 M, pH 1.6) was added and the solution was vortexed (2.5 min) prior to addition of the sodium tetraborate solution (0.0125 M sodium tetratborate in concentrated sulfuric acid). Following incubation at 100 and subsequent cooling, the solutions were centrifuged (2000 rpm, 10 min) to pellet the precipitate before addition of the m-hydroxydiphenol to the supernatant. Thereafter, all steps remained the same as described previously by Mojica et al. (2007).干生物膜的糖醛酸含量的分析如前所述被处决（Mojica等。2007年），但在那些用氨基磺酸的案件。在后一种情况下，氨基磺酸（氨基磺酸/钾氨基磺酸）（4米，pH值 1.6）的溶液中加入了涡旋（2.5分）之前，四硼酸钠溶液（0.0125 M在浓硫酸钠tetratborate）的加入。在100以下的培养和随后的冷却，解决方案离心（2000转，10分钟），以颗粒之前的M - hydroxydiphenol除上清沉淀。此后，所有的步骤是一样的Mojica如前所述等。 （2007年）。Microscopy显微镜Sample preparation样品制备Pre-treated (acid-washed on ice using HCl/HNO3(3:1); sterilized by autoclaving) glass microscope slides suspended in a 50 ml Falcon tube containing biolm inoculum (30 ml) and the potential antagonistic solution (3 ml)(to be analyzed for its aect biolm EPS production) mixed together in the previously described ratio (10:1). After incubation and subsequent biolm formation, the slides were removed and rinsed three times with F/S DI water. Samples were then immediately stained and analyzed.前处理（酸冰洗使用HCl/HNO3（3:1），由高压灭菌消毒）玻璃镜在50毫升管中生物膜接种猎鹰（30毫升）和潜在的敌对溶液（3毫升）暂停幻灯片（要分析其影响生物膜EPS生产）在前面描述的混合比（10:1）在一起。经过孵化和随后形成生物膜，幻灯片被拆除，冲洗三与F / S的DI水倍。然后立即样品染色和分析。Lectin staining凝集素染色To visualize the biolm EPS matrix, the biolms were stained with uorescently labeled lectins comprising concanavalin A (Con A) and wheat germ agglutinin(WGA) (Invitrogen) conjugated to Alexa Fluor 488.Con A has a carbohydrate-binding specicity