Membrane potential based characterization by flow cytometry of physiological states in an aerobic anoxygenic phototrophic bacterium

A simplified protocol for determination of bacterial membrane potential (MP) by flow cytometry with 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)(3)) as the MP probe was established, and was applied to an aerobic anoxygenic phototrophic bacterium (AAPB strain, Roseobacter clade) with an Escherichia coli strain as a reference. It was shown to be capable of precise indication of the physiological states of the cells as well as cellular responses to environmental factors. Based on the experimental results, a MP curve model was proposed for characterization of species-specific features, which offers parameters including the maximum MP of the curve (MPmax), the time to reach the MPmax (T-max), the slope of the exponential phase of the MP curve (K-MP), and the minimum (threshold) concentration of a particular substrate required for the organism to reach its MPmax (S-max). These parameters are functions of substrates and incubation conditions for a single species, and can be used for comparison between different species under the same conditions. The MPmax, T-max, and K-MP of the AAPB strain were quite different form those of the E. coli strain, and the AAPB strain was shown to be less sensitive to temperature and pH changes than the E. coli strain

珠穆朗玛峰北坡6000m以上主要生境细菌群落特征

流式细胞技术及构建的细菌16S rRNA基因文库揭示了珠穆朗玛峰北坡6600~8000m表层雪中细菌的数量,以及6000m冰塔林、6350m冰川融水和6600m表层雪中细菌的群落特征.珠峰北坡表层雪中细菌数量高于南极地区,但与其他高山雪中相似.表层雪中细菌的数量有随海拔升高而增多的趋势,但与离子的浓度相关性不太.珠峰北坡冰冻环境中的细菌16S rRNA序列与土壤、湖泊和河流、动植物体及其他冷冻环境中细菌的相似.冰塔林、冰川融水和表层雪中细菌具不同的群落特征,Cytophaga-Flavobacterium-Bacteroides(CFB)类群细菌在冰川融水中占绝对优势,冰塔林中属于β-Proteobacteria和CFB类的细菌为优势种,而表层雪中细菌则以β-Proteobacteria和Actinobacteria类的细菌为主,不同生境中细菌群落的不同可能是由于细菌的沉积后变化引起.国家重点基础研究发展计划项目(批准号:2005CB422004);; 国家自然科学基金项目(批准号:40121101,40401054);; 中国科学院创新项目(批准号:KZCX3-SW-339);; “百人计划”项目及国际社会公益项目(批准号:2005DIA3J106

珠穆朗玛峰地区东绒布冰川冰雪微生物群落及其季节变化

通过流式细胞计数和构建环境样品16S rRNA基因的克隆文库,分析了珠穆朗玛峰地区东绒布冰川冰雪微生物数量和菌群结构特征及其与季节变化的关系．珠峰地区冰雪微生物的16S rRNA基因序列分属于α,β,γ-变形菌纲,放线菌门,厚壁菌门,CFB,蓝细菌,真核质体,待定门TM7共9大类,以γ-变形菌纲为主要类群,其中Acinetobacter和Leclercia属是整个菌群中的优势属．微生物的数量和菌群结构具有明显的季节特征,夏季微生物的数量高于冬季;菌群结构具有一些共有属种的同时,在夏、冬季雪中具有各自特有的属种,共有属种推测是青藏高原局地源的微生物,序列同源性分析结果表明,夏季较多细菌属种与海洋环境相关,冬季细菌则具有更强的耐冷性．微生物明显的季节变化可能是受珠峰地区夏季和冬季不同水汽来源的影响

中国海及邻近区域碳库与通量综合分析

中国海总面积约470万平方公里,纵跨热带、亚热带、温带、北温带等多个气候带.其中,南海北依\"世界第三极\"青藏高原、南邻\"全球气候引擎\"西太平洋暖池,东海拥有全球最宽的陆架之一,跨陆架物质运输显著,黄海是冷暖流交汇区域,渤海则是受人类活动高度影响的内湾浅海.中国海内有长江、黄河、珠江等大河输入,外邻全球两大西边界流之一的黑潮.这些鲜明的特色赋予了中国海碳储库和通量研究的典型代表意义.文章从不同海区（渤海、黄海、东海、南海）、不同界面（陆-海、海-气、水柱-沉积物、边缘海-大洋等）,以及不同生态系统（红树林、盐沼湿地、海草床、海藻养殖、珊瑚礁、水柱生态系统等）多层面对海洋碳库与通量进行了较系统地综合分析,初步估算了各个碳库的储量与不同碳库间的通量.就海气通量而言,渤海向大气中释放CO2约0.22Tg Ca-1,黄海吸收CO2约1.15Tg Ca-1,东海吸收CO2约6.92～23.30Tg Ca-1,南海释放CO2约13.86～33.60Tg Ca-1.如果仅考虑海-气界面的CO2交换,中国海总体上是大气CO2的\"源\",净释放量约6.01～9.33Tg Ca-1.这主要是由于河流输入以及邻近大洋输入所致.河流输入渤黄海、东海、南海的溶解无机碳（DIC）分别为5.04、14.60和40.14Tg Ca-1,而邻近大洋输入DIC更是高达144.81Tg Ca-1,远超中国海向大气释放的碳量.渤海、黄海、东海、南海的沉积有机碳通量分别为2.00、3.60、7.40、7.49Tg Ca-1.东海和南海向邻近大洋输送有机碳通量分别为15.25～36.70和43.39Tg Ca-1.就生态系统而言,中国沿海红树林、盐沼湿地、海草床有机碳埋藏通量为0.36Tg Ca-1,海草床溶解有机碳（DOC）输出通量为0.59Tg Ca-1;中国近海海藻养殖移出碳通量0.68Tg Ca-1,沉积和DOC释放通量分别为0.14和0.82Tg Ca-1.总计,中国海有机碳年输出通量为81.72～103.17Tg Ca-1.中国海的有机碳输出以DOC形式为主,东海向邻近大洋输出的DOC通量约15.00～35.00Tg Ca-1,南海输出约31.39Tg Ca-1.综上,尽管从海-气通量看中国海是大气CO2的\"源\",但考虑了河流、大洋输入、沉积输出以及微型生物碳泵（DOC转化输出）作用后,中国海是重要的储碳区.需要指出的是,文章数据是基于中国海各海区碳循环研究报道,鉴于不同研究方法上的差异,所得数据难免有一定的误差范围,亟待将来统一方法标准下的更多深入研究和分析.国家重点研发计划项目(编号:2016YFA0601400);;国家自然科学基金项目(批准号:91751207、91428308、41722603、41606153、41422603);;中央高校基础研究项目(编号:20720170107);;中海油项目(编号:CNOOC-KJ125FZDXM00TJ001-2014、CNOOCKJ125FZDXM00ZJ001-2014)资

Microbial production of recalcitrant dissolved organic matter: long-term carbon storage in the global ocean

The biological pump is a process whereby CO2 in the upper ocean is fixed by primary producers and transported to the deep ocean as sinking biogenic particles or as dissolved organic matter. The fate of most of this exported material is remineralization to CO2, which accumulates in deep waters until it is eventually ventilated again at the sea surface. However, a proportion of the fixed carbon is not mineralized but is instead stored for millennia as recalcitrant dissolved organic matter. The processes and mechanisms involved in the generation of this large carbon reservoir are poorly understood. Here, we propose the microbial carbon pump as a conceptual framework to address this important, multifaceted biogeochemical problem.National Basic Research Program of China [2007CB815904]; National Natural Science Foundation of China [40632013/40841023]; SOA project [201105021/DY1150243]; Gordon and Betty Moore Foundation ; US National Science Foundation [648116, 0752972, 0851113, MCB-0453993]; French Science Ministry [ANR07 BLAN 016]; Netherlands Organisation for Scientific Research-Earth and Life Science

Monitoring the Cadmium in the Ocean by Using MT as the Marker

以菲律宾花蛤Ruditapes philippin Adams et Reave 为实验动物,分别提取其外套膜和内脏团等组织的金属硫蛋白,经Sephadex G-75葡聚糖柱层析纯化后,用DV-600紫外可见分光光度计测定每个样品金属硫蛋白的吸收值,以研究菲律宾花蛤体内的金属硫蛋白浓度与镉污染之间的关系.结果表明:诱导实验能使检测菲律宾花蛤的内脏团和外套膜的金属硫蛋白含量均明显增加;金属硫蛋白的吸收值与菲律宾花蛤重量、壳长、壳宽均有较大的相关性.从这些关系中,认为利用金属硫蛋白作为菲律宾花蛤镉污染的检测指标是可行的.Ruditapes philippin Adams et Reave were used as the animal in the experiment. And Sephadex G-75 was used as the equipment of filtration. First, Metallothionein (MT) was extracted from the membrane of overcoat and internal organs separately. Then, MT was purified by filtration. The abstraction of MT was measured by DV-600 ultraviolet absorption spectrum. The relations between the density of MT and the pollution of Cadmium were studied. The results showed that the value of MT in the internal organs and the membrane of overcoat in the R. philippin would increase dramatically by inducing experiments. And there were relations between the density of MT and the weight, length and width of R. philippin. Using MT as the marker to monitor the pollution of Cadmium in the R. philippin is feasible

The Application of Flow Imaging Technology in Harmful Algae Bloom Monitoring

赤潮是我国近海常见的重要灾害.建立赤潮生物现场监测技术,是赤潮预警预报和防治控制的基础.将流式细胞分析技术与显微成像分析技术结合起来,建立了赤潮生物流式图像监测仪器,建立赤潮生物专家识别数据库,对于目标赤潮生物的识别准确率达到82.52%~99.02%,定量准确率达到80%以上.统计分析结果显示,赤潮生物流式图像监测仪器与显微镜方法具有相似的精密度,90.00%以上的分析结果具有一致性.与显微镜方法相比,赤潮生物流式图像监测仪器具有自动化程度高,分析速度快,无需人为干预等优点,为赤潮生物的监测提供了新的技术手段.Harmful algae bloom(HAB)is one of common environmental problems in coastal oceans of China.How to establish red tide monitoring technology is the basis for HAB prediction and prevention.In this study,we developed a flow imaging system that was combined with flow cytometry and microscopy,and established a HAB′s expert identification database.With the database,the flow imaging system could distinguish the target algae with the accuracy of 82.52%-99.02%,and the accuracy of automatic quantification was up to 80%.Statistical analysis showed that the flow imaging system had precision similar to the microscopy,and more than 90.00% of the analytical results were consistent.Compared with microscopy,the flow imaging system had higher automation,faster analysis speed,without human intervention,and etc.Therefore,the flow imaging system would provide a new tool for HAB′s monitoring in the field.国家高技术研究发展计划(863)项目(2012AA092002;2007AA092001-9); 上海市科学技术委员会科研计划项目(11dz1204703

Algorithm for Automatic Recognition of Red Tide Algal Images Captured by Flow Cytometry

赤潮是常见海洋自然灾害之一。为了早期预测和预报赤潮的发生,开发了基于流式图像的赤潮监测仪器,它将流式细胞、显微成像以及图像处理技术结合起来,采用基于背景差的方法快速准确地分割出藻类图像;为了克服藻类细胞处于不同生长周期和环境带来的形态和个体差异的影响,提取具有平移、旋转和尺度不变性的几何形状特征与基于灰度共生矩阵的纹理特征,采用一对一的多分类支持向量机进行分类识别。实验结果表明,该算法实现对了海洋原甲藻、红色裸甲藻、拟菱形藻和中肋骨条藻的自动分类,平均识别准确率高达94.37%。The red tide is a global marine natural disaster.In order to predict and forecast the occurrence of red tide,with the technology of flow cytometry,micro-image and image processing,a real-time harmful algae monitoring system was developed.A method based on background subtraction was used to quickly and accurately segment the algae images.In order to overcome the influence of algal cells in morphology and individual difference brought by the different growth period and environment,geometry features which are invariant to translation,rotation and scale and texture features based on GLCM were extracted.And finally,one-to-one multi-class support vector machine was adopted for identification.The experimental results show that the average recognition accuracy rate is as high as 94.37%.国家863计划(2012AA092002)资

Natural and anthropogenic forcing on the dynamics of virloplankton in the Yangtze river estuary

Seasonal investigation Of virus dynamics by flow cytometry was conducted in the Yangtze river estuarine area in April, August, November 2002 and February 2003, and a supplemental Investigation in the inner estuary and downstream of the river was conducted in October 2005. The majority of the total viral abundance was bacteriophage and only 5.4% of the total was algal virus. Total viral abundance varied with season and location, ranging from 6.75x10(-5)-1.68x10(7) particles/ml, and the virus:bacterium ratio (VBR) ranged from 1.52 to 72.02 with a mean of 8.7. In the present study, viral abundance peaked in both the summer and the winter, unlike the typical seasonal pattern reported in the literature, in which viral abundance peaks in the summer when bacterial hosts are also at their most abundant. However, the driving forces for the two peaks reported here were totally different, the summer viral abundance peak coupled With the development of bacterial hosts which were controlled largely by temperature year-round and by trophic state occasionally while the winter one seemed to be multi-factor controlled. The host-phage interaction was no longer predominant in control of the winter viral abundance as bacterial abundance was lowest in this season. The winter low temperature would help maintain a high vital abundance as high temperatures might increase viral inactivation and viral decay; the VBR peak values actually Occurred In the winter. More importantly, the high virus-containing freshwater discharge in winter due to a higher proportion Of anthropogenic sewage relative to low natural flooding in Winter run-off, turned Out to be the first Factor contributing to the high winter Viral abundance and VBR values. Ill addition, the variation of intrusion of warm and relatively oligotrophic water from oceanic Currents played a role alternating the distribution patterns of temperature, salinity and trophic conditions and consequently the distribution patterns of virus and bacteria seasonally and spatially. Dynamics of virus in the Yangtze river estuarine. rea is thus characterized by distinct seasonal and spatial variations due to natural forcing and by, and by pronounced alternation of the regular patterns due to anthropogenic Impacts

Red Tide Algae Recognition Based SVDD and SVM

提出了一种基于支持向量机(SVM)和支持向量域描述(SVdd)的赤潮藻类分类系统.该系统是赤潮藻类流式监测系统的子系统.设计这套系统的主要难点在于:1)同一种藻类的形态由于个体差异和生长期不同而不同;2)藻类图像是任意位置三维目标在成像平面的投影,投影存在任意性并可能产生局部遮挡;3)藻类图像包含非目标藻类和杂质.在特征提取算法的基础上,首先对输入的藻类采用SVdd进行拒识或接受处理,最后针对接受的藻类再利用基于超平面分割的SVM分类器进行分类判决.实验证明:基于SVM和SVdd的赤潮藻类分类系统分类精度更高并具有较好的拒识性能,是一种较好的藻类自动分类方法.This paper presents a real-time alga classifier designed for flow-cytometry-based marine alga monitoring systems.The difficulties of such classification includes:1)the shape of the same algae category is deformable,and largely variant due to the individual differences and mature stage,2)the image of algae may vary due to different 3D positions to the imaging plane and partial occlusion, 3)the images also contain unknown algae and contaminations.In the proposed method,several shape features were developed,support vector data description(SVDD) was trained to reject the contaminative objects and unknown algae and a support vector machine (SVM) was used to classify the algae to taxonomic categories.Our approach achieved greater than 90%accuracy on a collection of algal images.The test on contaminated algal image set(contains unknown algae and non-algae objects,such as sands) also demonstrated promising results.国家高技术研究发展计划(863)项目(2003AA635160