稳定性同位素探测技术在微生物生态学研究中的应用

稳定性同位素标记技术同分子生物学技术相结合而发展起来的稳定性同位素探测技术（stableisotope probing,SIP）,在对各种环境中微生物群落组成进行遗传分类学鉴定的同时,可确定其在环境过程中的功能,提供复杂群落中微生物相互作用及其代谢功能的大量信息,具有广阔的应用前景.其基本原理是：将原位或微宇宙（microcosm）的环境样品暴露于稳定性同位素富集的基质中,这些样品中存在的某些微生物能够以基质中的稳定（性同位素为碳源或氮源进行物质代谢并满足其自身生长需要,基质中的稳定性同位素被吸收同化进入微生物体内,参与各类物质如核酸（DNA和RNA）及磷脂脂肪酸（PLFA）等的生物合成,通过提取、分离、纯化、分析这些微生物体内稳定性同位素标记的生物标志物,从而将微生物的组成与其功能联系起来.在介绍稳定性同位素培养基质的选择及标记方法、合适的生物标志物的选择及提取分离方法的基础上,举例阐述了此项技术在甲基营养菌、有机污染物降解菌、根际微生物生态、互营微生物、宏基因组学等方面的应用.     

DNA稳定同位素探针技术在宏基因组学中的应用及前景

DNA稳定同位素探针 (DNA-SIP) 是一种新兴的技术,通过将同位素稳定结合到特定的底物来确定环境中微生物的作用。DNA-SIP与宏基因组学结合可以让某些微生物的特性与其特殊新陈代谢联系在一起,不仅可以从宏基因组库里检测到低含量的微生物,而且加速了对新的酶类和其他生物活性物质的发现。以下总结了SIP-宏基因组学技术的原理、应用及研究进展,并讨论了其在环境微生物学和生物技术的应用前景。     

CARD-FISH技术及其在微生物生态学研究中的应用

催化报告沉积荧光原位杂交技术(Catalyzed reporter deposition-fluorescence in situ hybridization,CARDFISH)是基于传统的FISH技术发展而来,由于其较高的灵敏度及稳定性,可以检测微生物的rRNA、mRNA和DNA上的目标基因等,获得环境微生物的群落及功能信息,现已成为微生物生态学研究领域中的重要技术手段。近些年,CARD-FISH与同位素示踪技术、纳米二次离子质谱技术(Nano SIMS)、扫描电子显微镜(SEM)、流式细胞仪等技术方法的联合使用,不仅可以研究复杂环境中微生物的物种组成、数量及其高分辨形态学信息,而且可以获得微生物在单细胞水平的生理代谢信息及其活性,对在单细胞水平认识原位环境微生物的生理生态功能具有重要意义。本文重点介绍了CARD-FISH的技术路线和要点,并探讨CARD-FISH与相关技术联用在环境微生物生态学研究中的应用及进展。     

环境中微生物原位检测方法研究进展

微生物是生态系统物质循环和能量流动的主要参与者,在生态系统中起着重要的作用。但在现有技术条件下可培养微生物所占比例极小,限制了微生物资源的开发利用。目前有许多方法,可以避开微生物不可培养的问题,直接对微生物进行原位检测。对此,将前人关于微生物生态的原位检测研究方法进行了综述,方便以后对这些方法的合理应用。分别从DNA水平、RNA水平和蛋白质水平,介绍了对应的原位微生物检测方法(如Brd U标记、DNA-SIP、FISH和环境转录物组等),比较了它们的优缺点,并介绍了如何将这些方法与目前流行的高通量测序、单细胞测序等技术相结合来捕获原位活性微生物组等。同时,还对这些方法的特点进行了比较,使得人们可以更清楚地了解在不同场景下对不同方法的选择。这些经过改进的新兴方法及其与其它方法的结合使用将有助于解决微生物生态学研究中出现过或即将出现的很多问题。地球上的各种生态系统复杂而庞大,包含的微生物种群也各有差异。各种原位检测方法对微生物生理生态做出了更加真实有效的描述,必将成为研究微生物生态的有力手段。     

纳米二次离子质谱技术(NanoSIMS)在微生物生态学研究中的应用

超高分辨率显微镜成像技术与同位素示踪技术相结合的纳米二次离子质谱技术(NanoSIMS)具有较高的灵敏度和离子传输效率、极高的质量分辨率和空间分辨率(＜ 50 nm),代表着当今离子探针成像技术的最高水平.利用稳定性或者放射性同位素在原位或者微宇宙条件下示踪目标微生物,然后将样品进行固定、脱水、树脂包埋或者导电镀膜处理,制备成可供二次离子质谱分析的薄片,进一步通过NanoSIMS成像分析,不仅能够在单细胞水平上提供微生物的生理生态特征信息,而且能够准确识别复杂环境样品中的代谢活跃的微生物细胞及其系统分类信息,对于认识微生物介导的元素生物地球化学循环机制具有重要意义.介绍了纳米二次离子质谱技术的工作原理和技术路线,及其与同位素示踪技术、透射电子显微镜(TEM)、扫描电子显微镜(SEM)、荧光原位杂交技术(FISH)、催化报告沉积荧光原位杂交技术(CARD-FISH)、卤素原位杂交技术(Halogen In Situ Hybridization,HISH)等联合使用在微生物生态学研究方面的应用.     

新一代高通量测序与稳定性同位素示踪DNA/RNA技术研究稻田红壤甲烷氧化的微生物过程

[目的]利用新一代高通量测序技术分析复杂土壤环境中整体微生物群落结构的变化规律,研究特定功能微生物生理过程的分子机制;利用稳定性同位素示踪微生物核酸DNA/RNA,研究复杂土壤中关键元素转化的微生物调控机制.[方法]针对我国第四纪红色粘土母质发育的3种稻田红壤,围绕13C-甲烷好氧氧化的微生物过程,在DNA和RNA水平高通量测序土壤微生物群落16S rRNA基因和16S rRNA,通过超高速密度梯度离心土壤微生物总核酸获得13C-标记的DNA/RNA,进一步采用克隆文库技术研究稻田红壤甲烷好氧氧化的微生物作用者.[结果]新一代高通量测序结果表明,3种稻田红壤甲烷的好氧氧化过程中,甲烷好氧氧化菌占土壤整体微生物群落的丰度显著增加,RNA水平的增幅显著高于DNA水平,能够更为灵敏地反映土壤甲烷好氧氧化的微生物过程.3种稻田红壤甲烷的好氧氧化过程中,类型Ⅰ和类型Ⅱ甲烷好氧氧化菌在湖南古市土壤中显著增加,湖南桃源土壤中类型Ⅱ甲烷好氧氧化菌增加明显,而类型Ⅰ甲烷好氧氧化菌在广东雷州土壤中增幅最大.进一步利用13C-DNA和13C-RNA分别构建pmoA基因和16S rRNA克隆文库,发现类型Ⅰ甲烷好氧氧化菌主导了湖南古市和广东雷州稻田红壤甲烷的好氧氧化过程,类型Ⅱ甲烷好氧氧化菌主导了湖南桃源稻田红壤甲烷的好氧氧化过程.[结论]新一代高通量测序技术能够在整体微生物群落水平,清楚反映复杂土壤中特定功能微生物的生理生态过程,而RNA较DNA水平的分析更为灵敏;稳定性同位素示踪微生物核酸DNA/RNA技术能够准确地揭示复杂土壤重要过程的微生物作用者.     

蚯蚓环境生态作用研究进展

蚯蚓的环境生态作用有促进微生物和其它土壤动物活动、破碎和消化分解枯落物、提高土壤肥力及环境指示等方面.目前,国内蚯蚓环境生态作用研究侧重于污染生态系统,湿地和森林两大天然生态系统的相关研究较少.国外,20世纪70年代的研究主题与我国现状相似,80年代从蚯蚓生理生态、种群生态、群落生态、生态系统生态等多尺度展开研究,90年代以来向蚯蚓生态学微观尺度的分子生态、宏观尺度的景观生态拓展.今后,继续研究蚯蚓促进物质循环、治理污染、提高土壤肥力及蚯蚓与农业、与微生物的关系等热点问题的同时,还应利用新技术提高研究精度,注意室内外研究相结合及特殊蚓种的环境适应性比较与接种研究以提高研究成果的实用性.     

稳定性同位素技术与Keeling曲线法在陆地生态系统碳/水交换研究中的应用

稳定性同位素技术和Keeling曲线法是现代生态学研究的重要手段和方法之一。稳定性同位素能够整合生态系统复杂的生物学、生态学和生物地球化学过程在时间和空间尺度上对环境变化的响应。Keeling曲线法是以生物过程前后物质平衡理论为基础,将CO₂或H₂O的同位素组成(δD、δ¹³C或δ¹⁸O)与其对应浓度测量结合起来,将生态系统净碳通量区分为光合固定和呼吸释放通量,或将整个生态系统水分蒸散区分为植物蒸腾和土壤蒸发。在全球尺度上,稳定性同位素技术、Keeling曲线法与全球尺度陆地生态系统模型相结合,还可区分陆地生态系统和海洋生态系统对全球碳通量的贡献以及不同植被类型(C₃或C₄)在全球CO₂同化量中所占的比例。然而,生态系统的异质性使得稳定性同位素技术和Keeling曲线法从冠层尺度外推到生态系统、区域或全球尺度时存在有一定程度的不确定性。此外,取样时间、地点的选取也会影响最终的研究结果。尽管如此,随着分析手段的不断精确和研究方法的日趋完善,稳定性同位素技术和Keeling曲线法与其它测量方法(如微气象法)的有机结合将成为未来陆地生态系统碳/水交换研究的重要手段和方法之一。     

稳定性同位素技术在生物科学中的应用

稳定同位素技术是一项相对新兴的技术,其最早主要应用于地质学和地球化学.近些年来,稳定性同位素技术在农业、医学、环境学、海洋学、石油、化工、冶金等方面的应用也日益广泛.利用稳定性同位素技术还可以解决生物科学中一些传统方法难以解决的问题.对稳定性同位素的相关概念进行了介绍,并对其在植物、动物和微生物生理、生态学领域研究中的应用进行了综述,同时对其在环境保护、兴奋剂检测、毒品和炸药的产地辨别等领域的应用作了概述.     

树木年轮年内高分辨率稳定同位素记录:方法、进展和展望

树轮稳定同位素比率能有效地记录树木生长过程中气候环境变化信息及树木的生理响应机制。年内高分辨率树轮稳定同位素比率则能够提供更为详细的气候环境信息,揭示树木对季节尺度气候环境变化的生理生态响应机制,在古气候和全球变化生态学研究方面显示出巨大的潜力。本文收集了1990年以来发表的树轮年内高分辨率稳定同位素比率研究论文,从样品剥离方法、α-纤维素化学提取方法以及应用等方面综述了其研究进展,展望了年内高分辨率树轮稳定同位素记录研究的潜力和未来发展方向。     

提高微生物可培养性的方法和措施

目前自然界中只有极少部分微生物能够得到培养,严重阻碍了对微生物生命活动规律的研究和微生物资源的开发。改进传统培养方法,采用新型培养技术,提高微生物可培养性,大量培养自然界中存在的微生物,从而更全面、准确地了解微生物细胞的生命规律、获悉微生物群落中各种微生物之间的动态相互作用和相互协调的规律,对环境微生物工艺进行准确地设计、精细地调控和高效地利用。简要介绍了微生物不可培养的原因,系统总结了有关提高微生物可培养性方法的最新研究进展,提出研究中存在的问题,并阐述了模拟自然环境条件、强调微生物相互关系是提高微生物可培养性的关键。     

The host selects mucosal and luminal associations of coevolved gut microorganisms: a novel concept

Along the human gastrointestinal tract, microorganisms are confronted with multiple barriers. Besides selective physical conditions, the epithelium is regularly replaced and covered with a protective mucus layer trapping immune molecules. Recent insights into host defense strategies show that the host selects the intestinal microbiota, particularly the mucosa-associated microbial community. In this context, humans coevolved with thousands of intestinal microbial species that have adapted to provide host benefits, while avoiding pathogenic behavior that might destabilize their host interaction. While mucosal microorganisms would be crucial for immunological priming, luminal microorganisms would be important for nutrient digestion. Further, we propose that the intestinal microorganisms also coevolved with each other, leading to coherently organized, resilient microbial associations. During disturbances, functionally redundant members become more abundant and are crucial for preserving community functionality. The outside of the mucus layer, where host defense molecules are more diluted, could serve as an environment where microorganisms are protected from disturbances in the lumen and from where they can recolonize the lumen after perturbations. This might explain the remarkable temporal stability of microbial communities. Finally, commensals that become renegade or a decreased exposure to essential coevolved microorganisms may cause particular health problems such as inflammatory bowel diseases, obesity or allergies.     

The Earth's bounty: assessing and accessing soil microbial diversity.

The study of microbial diversity represents a major opportunity for advances in biology and biotechnology. Recent progress in molecular microbial ecology shows that the extent of microbial diversity in nature is far greater than previously thought. Here, we discuss methods to analyse microorganisms from natural environments without culturing them and new approaches for gaining access to the genetic and chemical resources of these microorganisms.     

油藏微生物及其压力适应机制

目前我国油田开发主要处于高含水后期,微生物驱提高石油采收率技术(MEOR)以低成本、环境友好等独特的优势引起了石油工业界的重视。实际上,经过半个多世纪的发展,MEOR已经成为提高采收率的重要前沿技术。高压是油藏的主要环境特征,在影响油藏微生物生存与活性等方面具有重要作用。本文从油藏及其微生物的主要特征、微生物对高压环境的适应机制以及高压下微生物降解烃的代谢特征等方面进行了综述。介绍了对油藏微生物资源、群落结构、微生物在油水相中分布的认识,微生物乳化原油机制,以及微生物在油藏厌氧环境中协同代谢、受温度和压力影响的特点,并列举了MEOR的矿场应用。在高压适应机制上,微生物主要通过改变和调整细胞膜结构、增加胞内脂质组分和表达胞内特殊酶等作用来实现对压力的适应;在高压下烃降解微生物代谢速率低于常压,而且耐压菌和嗜压菌具有不同的烃降解效率。     

油藏微生物群落研究的方法学

油藏微生物群落的解析和认知是开发和应用微生物采油技术的基础。利用各种提高油藏微生物可培养性的方法和非培养技术解析不同油藏微生物的群落结构、功能和多样性,对定向调控油藏微生物群落、开发和应用有效微生物驱油技术具有重要的指导意义。通过调查新近发展的提高微生物可培养性的方法和措施以及不依赖于培养的分子微生物生态学技术,总结了油藏微生物群落研究方法学的最新进展。提高微生物可培养性的方法和措施主要通过模拟微生物的生存环境,减少富营养的毒害作用、添加信号分子维持微生物细胞间的作用和提供新型电子供体和受体等手段采用稀释法、高通量培养法等方法得以实现;不依赖于培养的分子微生物生态学技术主要包括荧光原位杂交、末端限制性片断长度多态性分析、变性梯度凝胶电泳和构建克隆文库等技术。这些方法学的进展为更有效的获得各种油藏微生物资源、调控油藏微生物群落以提高石油采收率提供理论指导。     

Microorganisms in the atmosphere over Antarctica

Antarctic microbial biodiversity is the result of a balance between evolution, extinction and colonization, and so it is not possible to gain a full understanding of the microbial biodiversity of a location, its biogeography, stability or evolutionary relationships without some understanding of the input of new biodiversity from the aerial environment. In addition, it is important to know whether the microorganisms already present are transient or resident – this is particularly true for the Antarctic environment, as selective pressures for survival in the air are similar to those that make microorganisms suitable for Antarctic colonization. The source of potential airborne colonists is widespread, as they may originate from plant surfaces, animals, water surfaces or soils and even from bacteria replicating within the clouds. On a global scale, transport of air masses from the well-mixed boundary layer to high-altitude sites has frequently been observed, particularly in the warm season, and these air masses contain microorganisms. Indeed, it has become evident that much of the microbial life within remote environments is transported by air currents. In this review, we examine the behaviour of microorganisms in the Antarctic aerial environment and the extent to which these microorganisms might influence Antarctic microbial biodiversity.     

Dealing with water stress and microbial preservation

The relevance of preserving microorganisms has been well accepted for several decades. Interest is now shifting towards investigating adequate preservation methods to improve microbial survival rates and to preserve new taxa of previously considered unculturable microorganisms. In addition, a growing interest in preserving fragile microbial consortia or communities with biotechnological interest motivates the improvement of preservation methods. In the present study, we reviewed the effect of water availability in microbial diversity shift. We describe the effect of drought on microorganisms at the molecular level and their molecular responses to this life-threatening challenge focusing on the production of xeroprotectants. We also review the interspecies interactions of those drought-tolerant microorganisms with other sensitive organisms including neighbouring prokaryotes and eukaryotes such as plants, and the potential role of these microorganisms at determining the ecological composition of stressed environments. We emphasize the importance of applying the knowledge derived from the molecular mechanisms used by desiccation-tolerant microorganisms for the improvement of the preservation techniques. An overview of the current and newer techniques for preserving microorganisms and microbial communities is provided. The biotechnological interest in preserving pure cultures, microbial consortia and communities is also discussed.     

Effects of polymicrobial communities on host immunity and response

Microorganisms grow as members of microbial communities in unique niches, such as the mucosal surfaces of the human body. These microbial communities, containing both commensals and opportunistic pathogens, serve to keep individual pathogens 'in check' through a variety of mechanisms and complex interactions, both between the microorganisms themselves and the microorganisms and the host. Recent studies shed new light on the diversity of microorganisms that form the human microbial communities and the interactions these microbial communities have with the host to stimulate immune responses. This occurs through their recognition by dendritic cells or their ability to induce differential cytokine and defensin profiles. The differential induction of defensins by commensals and pathogens and the ability of the induced defensins to interact with the antigens from these microorganisms may attenuate proinflammatory signaling and trigger adaptive immune responses to microbial antigens in a multistep process. Such an activity may be a mechanism that the host uses to sense what is on its mucosal surfaces, as well as to differentiate among commensals and pathogens.     

Response of microbial communities of Lake Baikal to extreme temperatures

The survival rate, metabolic activity, and ability for growth of microbial communities of Lake Baikal have been first studied after exposure to extremely low temperatures (freeze-thawing) for different lengths of time. It has been shown that short-term freezing (1–3 days) inhibits the growth and activity of microbial communities. The quantity of microorganisms increased after 7-and 15-day freezing. In the periods of maximums, the total number of microorganisms in the test samples was twice as high as in the control. It was established that after more prolonged freezing the microorganisms required more time after thawing to adapt to new conditions. In the variants with 7-and 15-day freezing, the activities of defrosted microbial communities were three or more times higher than in the control. The survival rate and activity of Baikal microorganisms after freeze-thawing confirms the fact that the Baikal microbial communities are highly resistant to this type of stress impact.     

Survival of a bacterial/archael consortium on building materials as revealed by molecular methods

The analysis of microbial communities using molecular techniques has become a common method to describe their components. In this study some building materials were inoculated with a microbial consortium of 14 microorganisms, subjected to alternate wetting/drying cycles for six months, and left to rest for eight years. At the end of the resting period, most members of the consortium were detected by molecular methods, which indicate a remarkable preservation of the DNA, although only one bacterium was able to grow in a culture medium. The experimental approach we carried out indicates that the list of microorganisms obtained from DNA analyses reflects not only the actual composition of the microbial communities, but is also a reflection of the microorganisms that were once active on the building materials.