Stable-isotope probing of bacterial community for dissolved inorganic carbon utilization in Microcystis aeruginosa-dominated eutrophic water

Dissolved inorganic carbon (DIC) is an important source of carbon in aquatic ecosystems, especially under conditions of increased frequency of cyanobacterial bloom. However, the importance of bacteria in direct or indirect utilization of DIC has been widely overlooked in eutrophic freshwater. To identify the functional bacteria that can actively utilize DIC in eutrophic freshwater during cyanobacterial bloom, stable-isotope probing (SIP) experiments were conducted on eutrophic river water with or without inoculation with cyanobacteria (Microcystis aeruginosa). Our 16S rRNA sequencing results revealed the significance of Betaproteobacteria, with similar relative abundance as Alphaproteobacteria, in the active assimilation of H¹³CO₃^? into their DNA directly or indirectly, which include autotrophic genera Betaproteobacterial ammonia-oxidizing bacteria. Other bacterial groups containing autotrophic members, e.g. Planctomycetes and Nitrospira, also presented higher abundance among free-living bacteria in water without cyanobacteria. Microcystis aggregates showed a preference for some specific bacterial members that may utilize H¹³CO₃^? metabolized by Microcystis as organic matter, e.g. Bacteroidetes (Cytophagales, Sphingobacteriales), and microcystin-degrading bacteria Betaproteobacteria (Paucibacter/Burkholderiaceae). This study provides some valuable information regarding the functional bacteria that can actively utilize DIC in eutrophic freshwater.     

溶藻细菌DC21的分离、鉴定及其溶藻特性

从云南滇池中分离得到一株溶藻细菌DC21,经鉴定为葡萄球菌属的1种(Staphylococcussp.),可溶解铜绿微囊等9种藻.试验表明,经过滤、离心沉降及高温灭菌处理的滤液,均能强烈抑制铜绿微囊藻的生长,说明其溶藻作用的因子之一为该菌的胞外分泌物,且这种物质具有热稳定性.细菌DC21使藻的光合作用受到抑制;在试验的第1d,受试藻的超氧物歧化酶及过氧化氢酶活性显著增高,但在第7d,这两种抗氧化保护酶的活性显著下降;受试藻的丙二醛含量明显增高,说明该菌促使其发生了膜脂过氧化.     

Microbial spheres: a novel cyanobacterial–diatom symbiosis

Cyanobacteria, algae and bacteria are widespread inhabitants of North Sea microbial mats. Our studies of these populations showed uncommon modes of living and extraordinary structures, which have not been described before. The structures are spherical objects covering a community of cyanobacteria, diatoms and bacteria. The cultivation of these communities in the laboratory and intensive observations of their exceptional movement has led to some spectacular findings. The sphere formations go through different phases with variation in the dominance of different microorganisms. The role of the bacteria is the most important in the first phase, and can be increased by the addition of signal substances. Spheres surrounded by envelopes of unknown composition and permeability appear, with numerous bacteria and sporadic diatoms inside. Then the cyanobacteria penetrate the spheres and arrange themselves at the surface. The communities proliferate over some weeks and are finally released. Laboratory expositions of the microbial communities to different parameters pinpoint the limits of sphere formation. The metabolic products of the sphere communities are concentrated in the spheres and lead to a different kind of compound compared with the surrounding environment. In this way, the microbial communities strongly influence the structure of the sediments. Uncommon circular structures, which develop into spheres between 0.08 and 3 mm in size were found in subcultures of non-axenic filamentous cyanobacteria enrichments from North Sea microbial mats. These filamentous cyanobacteria (Phormidium sp.) together with associated benthic diatoms of the genus Navicula and associated heterotrophic bacteria were held as reproducible synergistic cultures. Phormidium sp. filaments tightly intertwined with each other, formed the surface of the spheres, trapping diatoms inside. The formation of "spheres" was the result of radial and synchronous movements of the cyanobacteria. In old cultures, the direction of the cyanobacterial movement has turned in the opposite direction, away from the sphere. The integrity of large "spheres" was influenced by chemotactic phenomena and maintained by some type of trichome-trichome interaction. This suggests the presence of metabolic secondary products, which attract cyanobacteria and influence their movement in a form of chemotactic response.     

Isolation, identification and characterization of an algicidal bacterium from Lake Taihu and preliminary studies on its algicidal compounds

In an effort to identify a bio-agent capable of controlling cyanobacterial blooms, we isolated a bacterial strain, A27, which exhibited strong algicidal activity against the dominant bloom-forming species of Microcystis aeruginosa in Lake Taihu. Based on 16S rRNA gene sequence analysis, this strain belongs to the genus Exiguobacterium. This is the first report of an algicidal bacterial strain belonging to the genus Exiguobacterium. Strain A27 exhibited algicidal activity against a broad range of cyanobacteria, but elicited little or no algicidal activity against the two green algal strains tested. The algicidal activity of strain A27 was shown to be dependent on the density of the bacteria and to have a threshold density of 1.5× 10⁶ CFU/mL. Our data also showed that the algicidal activity of strain A27 depended on different growth stages of Microcystis aeruginosa (exponential ≈ lag phase > early stationary) rather than that of the bacterium itself. Our results also suggested the algicidal activity of strain A27 occurred via the production of extracellular algicidal compounds. Investigation of the algicidal compounds revealed that there were at least two different algicidal compounds produced by strain A27. These results indicated that strain A27 has great potential for use in the control of outbreaks of cyanobacterial blooms in Lake Taihu.     

太湖不同湖区冬季沉积物细菌群落多样性

为了探究太湖不同营养水平湖区冬季沉积物细菌群落多样性,利用高通量测序对太湖水草区、湖心区和河口区共9个采样点表层沉积物细菌的16S rRNA基因进行序列测定.结果发现：不同湖区的物种丰富度和均匀度为水草区 > 湖心区 > 河口区;不同湖区优势细菌群落组成存在差异.其中,硝化螺旋菌门（Nitrospirae）和酸杆菌门（Acidobacteria）2类门水平优势细菌以及厌氧绳菌纲（Anaerolineae）和硝化螺菌纲（Nitrospira）2类纲水平优势细菌在不同湖区之间差异显著.冬季太湖沉积物细菌优势类群为绿弯菌门（Chloroflexi）、变形菌门（Proteobacteria）、蓝藻门（Cyanobacteria）和硝化螺旋菌门（Nitrospirae）;在属分类水平上,主要优势类群为unidentified_Chloroplast和微囊藻属（Microcystis）.Cyanobacteria和Microcystis在所有采样点均有检出,平均丰度均以湖心区最高.对沉积物细菌群落与环境因子的关系进行冗余分析,结果表明：营养盐水平、水温和pH值均能影响沉积物细菌群落结构,NO₃^--N和水温是Microcystis的主要影响因子.     

蓝藻水华形成过程对氮磷转化功能细菌群的影响

为探寻蓝藻水华形成过程中细菌群落结构和氮、磷转化功能菌群的动态变化,利用细菌16S rRNA基因的高通量测序和功能基因的荧光定量PCR(qPCR)方法,分析了在蓝藻水华过程中水体细菌群落组成及功能菌群的变化情况.高通量测序结果证明了蓝藻水华形成过程中细菌群落的多样性降低,细菌群落在水华期和非水华期具有不同的结构.随着水华过程中蓝藻密度的增加,水体中Actinobacteria和Bacteroidetes相对丰度减少,而Firmicutes相对丰度增加;蓝藻水华对聚磷菌(PAOs)具有富集作用,对硝化细菌具有抑制作用,而反硝化细菌的数量在中度水华条件下显著增加. qPCR结果显示,随着蓝藻水华的持续发展,硝化和反硝化功能基因丰度下降甚至消失,表明水体中氮转化过程可能受到抑制,此过程也有利于水华过程中微囊藻快速增殖对氮的需求,对水华蓝藻的生长具有正反馈作用.     

南京玄武湖微囊藻水华种类组成的研究

2005-08～2006-10采集蓝藻水华样品, 通过形态学特征和ITS序列分子标记的方法对南京玄武湖水华的优势种类进行了鉴定. 结果表明,玄武湖水华主要由微囊藻构成, 通过形态特征鉴定主要种类为铜绿微囊藻(M. aeruginosa)、M. novacekii 和惠氏微囊藻(M. wesenbergii). 3种藻的生物量在水华中的相对比例分别为30%～45%、35%～40%和10%～15%. 采用分子标记的方法对２种优势种类进行了分子鉴定. PCR扩增ITS序列并进行测序, 通过基因序列比对和基于ITS序列的系统发生树分析, 进一步确定了优势种类为M. aeruginosa和M. novacekii; 通过检测产毒基因mcyB来确认2株优势微囊藻的产毒特性, PCR扩增结果均出现了大约为780 bp的特异性片段, 表明这2种藻均为产毒种. 对水华提取物的高效液相色谱(HPLC)测定表明, 玄武湖水华中微囊藻毒素的种类主要是MC-LR和MC-RR.     

原油降解中微生物群落的16S rDNA-PCR-DGGE分析

为对原油降解过程中微生物群落结构的变化,本文采用直接向原油中加入营养物刺激的方法,获得原油降解菌体系。提取不同培养时间条件下降解菌体系的总DNA,并以之为模板采用PCR-DGGE技术研究了在原油降解过程中微生物群落的组成,结果表明,随着培养时间的不同,微生物的群落结构表现出差异,随着时间的变化其微生物群落也会发生改变。回收了DGGE图谱中的18个条带,测序结果经过Blast比对表明其中10个条带代表的细菌是不可培养的,显示了DGGE技术的优越性。同时采用气相色谱技术对降解过程中的原油烃的变化进行了分析,结果显示原油烃类组分会随着降解菌多样性的变化而发生变化,降解菌体系表现出了其对烷烃类物质的降解能力。     

铜绿微囊藻与藻际细菌Ma-B1菌株的相互作用

藻与细菌通常共生于淡水生境,形成藻-菌共生体系,藻际细菌是水体生态系统中的重要组成部分,对藻的消长起重要的调控作用,但有关藻际微环境中藻与细菌的互作机制还不清楚. 采用传统的细菌平板培养方法,从太湖优势水华藻——铜绿微囊藻(Microcystis aeruginosa)细胞表面分离出一株藻际细菌Ma-B1,基于生理、生化试验和16S rRNA基因序列分析,初步鉴定为甲基营养芽孢杆菌(Bacillus methylotrophicus). 通过测定细胞生长,分析藻-菌相互作用机理. 结果表明:一定浓度(＞60 μg/mL)的Ma-B1的胞外代谢物可显著抑制铜绿微囊藻的生长(培养基为BG11,28 ℃/日,22 ℃/夜,3 000 lx,光暗比为14 h∶10 h);铜绿微囊藻的胞外滤液(500 μL/mL)对Ma-B1的生长有一定的促进作用,但其总滤液(500 μL/mL)显著促进Ma-B1的生长;Ma-B1细胞对铜绿微囊藻的生长没有显著影响,而高浓度(藻菌比10∶1)的铜绿微囊藻细胞则可显著抑制Ma-B1的生长. 铜绿微囊藻与Ma-B1之间存在复杂的相互抑制或促进关系,共同影响着藻、菌在自然水体生态系统中的消长.      

压力下伪空胞破裂对3种水华蓝藻生长及光合作用的影响

采用批量培养方式,研究了伪空胞的破裂对3种典型水华蓝藻——铜绿微囊藻(Microcystis aeruginosa)、螺旋鱼腥藻(Anabaena spiroides)和孟氏浮游蓝丝藻(Plankothrix mougeotii)生长的影响,同时分析了伪空胞破裂对3种藻光合作用及细胞形态的影响.结果表明,伪空胞破裂对3种藻的生长影响不同,铜绿微囊藻和孟氏浮游蓝丝藻的细胞生长不受伪空胞破裂的影响,而螺旋鱼腥藻生长初期出现了5 d的延滞期;伪空胞破裂对3种藻光合作用影响较小,其中铜绿微囊藻和螺旋鱼腥藻的光合作用没有受到影响,而孟氏浮游蓝丝藻在饱和光强时最大光合作用速率降低了12.6%.铜绿微囊藻、孟氏浮游蓝丝藻及螺旋鱼腥藻细胞在伪空胞破裂后恢复较快,在3～5 d内又重新生成了大部分的伪空胞.      

Bacterial diversity in soils around a lead and zinc mine

Five samples of soil collected from a lead and zinc mine were used to assess the effect of combined contamination of heavy metals on soil bacterial communities using a polyphasic approach including characterization of isolates by culture method, community level catabolic profiling in BIOLOG GN microplates, and genetic community fingerprinting by denaturing gradient gel electrophoresis of 16S rDNA fragments amplified by PCR from community DNA （PCR-DGGE）. The structure of the bacterial community was affected to a certain extent by heavy metals. The PCR-DGGE analysis of 16S rRNA genes showed that there were significant differences in the structure of the microbial community among the soil samples, which were related to the contamination levels. The number of bacteria and the number of denaturing gradient gel electrophoresis （DGGE） bands in the soils increased with increasing distance from the lead and zinc mine tailing, whereas the concentration of lead （Pb） and cadmium （Cd） was decreased. Heavily polluted soils could be characterized by a community that differs from those of lightly polluted soils in richness and structure of dominating bacterial populations. The clustering analysis of the DGGE profiles showed that the bacteria in all the five samples of soil belonged to three clusters. The data from the BIOLOG analysis also showed the same result. This study showed that heavy metal contamination decreased both the biomass and diversity of the bacterial community in soil.     

Modeling mitigation strategies for toxic cyanobacteria blooms in shallow and eutrophic Lake Kasumigaura, Japan

This paper explores mitigation scenarios for toxic cyanobacteria blooms in Lake Kasumigaura is located about 60–90 km northeast of Tokyo, in the southeast of Ibaraki Prefecture, Japan. Dominant species of cyanobacteria (Microcystis aeruginosa, Microcystis viridis and Microcystis ichthyoblabe) produce highly potent toxins as (Microcystin-(Leucine + Arginine, MC-LR), Microcystin-(Arginine + Arginine, MC-RR) and Microcystin-(Tyrosine + Arginine, MC-YR) in the Lake. Toxin production is generally the result of two major factors-natural processes and human interferences. Both factors have an extreme influence on the generation of cyanobacteria toxins within lake ecosystems. To address these factors, we propose two concepts for mitigation. The first concept is intended for examining the natural process of toxin production behavior within the lake and the second concept is used for evaluating inflow of wastes and nutrients from human activities that form toxins. Our research aims to combine both strategies to mitigate impacts of toxins, by examining trends of cyanobacteria nutrient sources, buoyancy regulations that influence bloom formation, and the environmental conditions that spur blooms. This study proposes a simple IMPACT (Integrating Mitigation Policies for Aquatic Cyanobacteria Toxin) model for diminution strategies of harmful algal blooms and their toxins. A mixed-methods approach is employed, nested within the Environmental Systems Analysis (ESA) tools e.g. scenario analysis and stakeholder analysis. The quality of the lake is assessed through a combination of observation and field study analysis. The findings suggest that successful mitigation of cyanobacteria toxins is highly dependent on multi-functional, multi-stakeholder involvement, and relevant intergovernmental policy. Without integrating approaches among different stakeholders, diverse socioeconomic activists, local-national policymakers and effective policy measures, prevention of cyanobacteria toxin production within lakes becomes extremely complex and difficult. The proposed IMPACT model could be a decision framework for identifying suitable policies that mitigate cyanobacteria impacts.     

Cyanobacterial flora and the physico-chemical environment of six tropical fresh water lakes of Udaipur, India

The cyanobacteria and physico-chemical environments of sic tropical fresh water lakes of Udaipur,India were investigated.These lakes receive varying nutrient inputs from different sourcesi.Altogether 51 species of cyanobacteria were recorded.Species composition varied between lakes and between seasons.Lake VI(Beghdara),which receives nutrients from natural sources only,differed considerably from the others in water chemistry and composition of dominated species.Lake Ⅱ(Swaroop Sagar),eutrophied due to sewage inputs,was species poor.Non-diazotrophs,represented by 27 species,dominated during summer.With few exceptions,N2-fixing species,bothe heterocystous and unicellular diazotrophs (represented by 24 species),were dominant during winter.Microcystis aeruginosa,Phormidium sp.and Anabaena flos-aque were ther dominant taxa of lakes characterized by sewage eutrophication.The study shows that both species diversity and community composition were affected by water chemistry.     

Analysis of bacterial community structures in two sewage treatment plants with different sludge properties and treatment performance by nested PCR-DGGE method

The bacterial community structures in two sewage treatment plants with different processes and performance were investigated by denaturing gradient gel electrophoresis （DGGE） of nested polymerase chain reaction （nested PCR） amplified 16S rRNA gene fragments with group-specific primers. Samples of raw sewage and treated effluents were amplified using the whole-cell PCR method, and the activated sludge samples were amplified using the extracted genomic DNA before the PCR products were loaded on the same DGGE gel for bacterial community analysis. Ammonia-oxidizing bacterial and actinomycetic community analysis were also carried out to investigate the relationship between specific population structures and system or sludge performance. The two plants demonstrated a similarity in bacterial community structures of raw sewage and activated sludge, but they had different effluent populations. Many dominant bacterial populations of raw sewage did not appear in the activated sludge samples, suggesting that the dominant bacterial populations in raw sewage might not play an important role during wastewater treatment. Although the two plants had different sludge properties in terms of settleability and foam forming ability, they demonstrated similar actinomycetic community structures. For activated sludge with bad settling performance, the treated water presented a similar DGGE pattern with that of activated sludge, indicating the nonselective washout of bacteria from the system. The plant with better ammonium removal efficiency showed higher ammonia-oxidizing bacteria species richness. Analysis of sequencing results showed that the major populations in raw sewage were uncultured bacterium, while in activated sludge the predominant populations were beta proteobacteria.     

不同植被群落表层土壤中细菌群落多样性

对3种不同植被群落(海桐、雪松、桂花)表层土壤中的pH、w(TOC)、w(TN)和w(TP)进行了测定,采用DGGE(变性梯度凝胶电泳)方法对各植被群落表层土壤细菌的16S rDNA V3~V6可变区扩增片段进行了分析,利用分析得到的图谱数据与表层土壤中的pH、w(TOC)、w(TN)和w(TP)进行了群落-理化因子相关性分析以及CCA(典范对应分析). 结果表明,不同植被群落表层土壤的pH和养分含量不同,其中w(TOC)和w(TN)的差异显著(P<0.05),而pH和w(TP)的差异不显著(P>0.05). DGGE图谱分析结果揭示,不同植被群落表层土壤的细菌群落结构明显不同. 聚类分析显示,海桐和雪松的细菌群落组成和结构最为相似(82%),而它们与桂花样地的相似性(55%)相对较低,桂花样地的Shannon-Wiener指数最高且与其他2个样地差异显著,雪松样地的均匀度指数最高. 相关性分析和CCA结果进一步表明,w(TOC)和w(TN)是表层土壤细菌群落结构和多样性的显著影响因素,并且植被群落可能通过其根系分泌物产生间接影响.      

Characterization of depth-related microbial communities in lake sediment by denaturing gradient gel electrophoresis of amplified 16S rRNA fragments

The characterization of microbial communities of different depth sediment samples was examined by a culture-independent method and compared with physicochemical parameters, those are organic matter （OM）, total nitrogen （TN）, total phosphorus （TP）, pH and redox potential （Eh）. Total genomic DNA was extracted from samples derived from different depths. After they were amplified with the GC-341 f/907r primer sets of partial bacterial 16S rRNA genes, the products were separated by denaturing gradient gel electrophoresis （DGGE）. The profile of DGGE fingerprints of different depth sediment samples revealed that the community structure remained relatively stable along the entire 45 cm sediment core, however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. The principle coordinates analysis suggested that the bacterial communities along the sediment core could be separated into two groups, which were located 0-20 cm and 21-45 cm, respectively. The sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belonged to Bacillus, Bacterium, Brevibacillus, Exiguobacterium, γ-Proteobacterium, Acinetobacter sp. and some uncultured or unidentified bacteria. The results indicated the existence of highly diverse bacterial community in the lake sediment core.     

Environmental factors influencing cyanobacteria community structure in Dongping Lake, China

The present study was conducted to provide a detailed understanding of the variation in cyanobacterial communities of Dongping Lake, which is the final water volume adjusting and storing lake in the east route of the South-to-North Water Diversion Project in China. The spatial and temporal distribution of cyanobacteria was assessed from May 2010 to October 2012 based on monthly samples collected from three stations. Over the 30-month survey, 15 genera and 25 species of cyanobacteria were identified, with cyanobacterial abundance at each monitoring station ranging from undetected to 3.04×107 cells/L, average of 4.27×106 cells/L. The dominant cyanobacterial species were Pseudanabaena limnetica and Aphanizomenon issatschenkoi and not the usual bloom-forming genera such as Microcystis and Anabaena. Cyanobacterial community structure and water quality variables exhibited substantial changes over the period of survey. Redundancy analysis, Pearson correlations, and regression analysis were applied to analyze the relationships among the variables. The results suggested that temperature and chemical oxygen demand were key drivers of the cyanobacterial community composition in Dongping Lake. In addition, the concentration of inorganic nitrogen in the lake had a profound efect on the cyanobacterial abundance as a non-limiting factor in warm periods.     

藻类的光谱吸收特征及其混合藻吸收系数的分离

利用定量滤膜技术测定了实验室培养的聚球藻(Synechococcus 7942)、铜绿微囊藻(Microcystis aeruginosa)、斜生栅藻(Scenedesmusobliquus)、蛋白核小球藻(Chlorella pyrenoidosa)、直链硅藻的吸收光谱(Melosira granulate Var.angustissima),通过其标准化吸收谱,分析了同种藻不同生长时期的吸收谱特征,并比较分析了不同藻类的标准化吸收谱.结果表明,同一藻的标准化吸收光谱在不同生长时期基本恒定不变.色素组成及其比例的不同是引起藻类间吸收光谱变化的主要原因,同时也是大型浅水湖泊中浮游植物种群演替的原因之一.蓝藻光谱主要存在620nm左右的吸收峰,使得其在水体中较易获取生长所需的光能.直链硅藻在短波范围内稍强的吸收能力被非藻类颗粒物、黄质所弱化,而其在630nm的吸收峰较低,因而难以成为浅水湖泊中的优势种;绿藻在655nm处的吸收肩峰有利于其光能竞争.通过纯种藻的标准化吸收谱,较为成功地对混合藻的吸收谱进行了分离.     

鄱阳湖蓝藻分布及其影响因素分析

利用鄱阳湖的原位监测数据,分析鄱阳湖水华蓝藻的分布现状及其影响因素,探索鄱阳湖水华蓝藻的源头.研究结果表明,鄱阳湖浮游植物的优势种为硅藻,蓝藻为鄱阳湖的次级优势种,蓝藻在浮游植物总生物量的比例有逐年增加的趋势.水华蓝藻的主要优势种为鱼腥藻,其次为微囊藻和浮游蓝丝藻. 鄱阳湖蓝藻水华形成初期的基本规律为水华蓝藻在营养盐浓度相对较高且水流较缓的内湾及尾闾区生长分布,在夏秋季水位较高时在水流和风的作用下向主航道输移聚集. 结合鄱阳湖水文特点,主航道的水华蓝藻聚集有可能是上游四个湖区的蓝藻向下游漂移综合作用的结果.研究成果可为控制鄱阳蓝藻水华区域风险灾害提供基础数据.     

环境DNA宏条形码技术在蓝藻群落监测中的应用

全球气候变化下的蓝藻水华大规模暴发成为重要的环境问题,对蓝藻准确、高效和实时的监测是蓝藻水华防控的关键.近年来,环境DNA（environmental DNA,eDNA）宏条形码技术开始应用于蓝藻群落监测,弥补了显微镜镜检法物种鉴定难和受主观经验影响大的缺点.eDNA宏条形码为快速和大规模蓝藻群落监测提供了可能,其应用尚处于初步探索阶段,数据处理方法尚不成熟,因此有必要从引物选择、序列聚类、注释方法和绝对定量4个层面系统综述eDNA宏条形码技术在蓝藻群落监测中的研究进展,以推进eDNA宏条形码在蓝藻群落监测中的应用.引物选择应针对目标蓝藻类群,16S rRNA基因数据库涵盖蓝藻物种范围广,是最常用的eDNA宏条形码,但16S-23S rRNA基因间隔区域（internal transcribed spacer,ITS）和功能基因在属内物种间具有较好的区分效果,为eDNA宏条形码在蓝藻物种水平注释提供可能.序列聚类一般通过设定物种间分类阈值进行OTUs聚类,但可能丢失序列相似度高于分类阈值的不同物种;序列差异低至单核苷酸变异方法的应用进一步区分了隐蔽的物种和生态型,产生的序列具有生物学意义,可以实现跨数据集间的比较分析.在注释方法上,基于数据库参考序列距离相似度的注释方法,注释结果的分辨率和准确度不高;基于系统进化位置的注释方法,提升了注释结果的准确度,反映了物种间的进化关系.加入外源DNA的内标法,以及基于细胞体积和基因拷贝数目关系的细胞体积校正系数法为eDNA宏条形码物种丰度的绝对定量提供了可能.