自然界中不可培养微生物的研究进展

尽管微生物培养技术已经发展了几十年,但是环境中可培养的微生物比例仍然较低。目前研究者对微生物不可培养的原因有了进一步的了解,其关键在于：高浓度的营养基质、无法实现原位培养、不明确环境中微生物之间的相互作用、缺乏尖端的微生物检测方法等。研究者为了克服这些培养障碍,不断研究出许多提高微生物培养效率的方法,简要介绍改进培养基、发展新的培养条件等提高微生物可培养性的方法。     

口腔未培养微生物分离培养策略的研究进展

口腔微生物是人体微生物组的重要组成部分,其群落组成丰富且独特。现有研究显示,口腔微生物与龋病、牙周炎等口腔健康问题有直接的联系,因而具有重要的研究价值。随着高通量测序技术的发展,人们对口腔中未培养微生物多样性的认识不断加深,这进一步催生对微生物分离培养技术需求的增加。为此,本文将围绕口腔未培养微生物及其分离培养策略的研究进展,首先介绍口腔中未培养微生物的研究现状;其次分析口腔微生物分离培养中可能的限制因素;最后综述微生物分离培养技术发展及其在口腔未培养微生物研究中的应用。全文旨在为口腔未培养微生物的分离培养提供思路和技术参考。     

微生物油脂的研究进展及展望

综述了培养微生物生产油脂的发展历史及研究现状,讨论了产油影响因素,对于其瓶颈因素碳源和氮源的影响。可采用培养后期限制氮源并补加碳源的措施,从而解决增加微生物油脂的含量。展望了采取微生物混合培养方法生产油脂技术研究的发展前景。     

未/难培养微生物可培养策略研究：机遇与挑战

微生物分布广泛、种类众多、功能多样,虽体积微小但功能强大,关乎人类的安全健康和生态的稳定发展,在整个地球生命系统中起着举足轻重的作用。17世纪以来,研究者们一直努力获得、了解和利用这些微生物,然而目前分离方法的局限性使得环境中绝大部分微生物仍不能被纯培养,严重阻碍了我们对微生物生命活动规律的认知。因此,如何分离获得这些仍未被培养出来的“暗物质”是微生物研究面临的严峻挑战和重大机遇。本文分析了环境中制约微生物分离培养的因素,综述未/难培养微生物可培养研究的最新进展,着重论述优化的传统培养方法及网络导向培养、膜扩散培养、微流控分选培养和细胞分选培养等新型技术的应用,并对未来研究进行展望,探索多技术联合使用策略,为未/难微生物资源的挖掘及开发利用提供借鉴。     

肠道微生物培养的研究进展及应用

肠道中栖居着组成复杂、功能多样的微生物群,这些微生物群在宿主免疫、营养吸收、代谢调节等方面发挥着重要作用。随着测序技术的快速发展,肠道微生物研究通过16S rRNA基因测序和宏基因组测序产生了大量的数据,其中许多未组装的序列成为微生物“暗物质”。近年来,不少研究利用多种不同微生物分离培养方法,结合高通量鉴定技术,从人、小鼠、猪肠道中分离了大量的微生物,丰富了菌株资源,为解析微生物“暗物质”以及后续肠道微生物功能和应用研究提供了基础和保障。尽管微生物的可培养性受到多种因素的影响,大部分微生物尚处于“未培养”的状态,但无论是病因研究还是生理和遗传特征的解析都离不开微生物实体资源的获取。肠道微生物的分离培养对微生物研究从关联分析向菌群功能验证、因果机制解析和功能菌株开发的深入研究具有重要意义。本文旨在探讨和综述影响微生物可培养性的因素,总结回顾肠道微生物的培养方法并阐述肠道微生物培养研究的进展,以期为肠道微生物培养研究提供新的视角。     

单细胞分选技术在微生物分离与培养中的应用与展望

自然界相当多的微生物是未培养的,对这些"微生物界暗物质"进行研究,对于研究微生物的进化过程,充分利用微生物资源具有重要意义。对未培养微生物的分离与培养已成为当前国际研究热点,本文对显微操作、荧光活化分选、微流控分选、光镊技术、激光诱导向前转移等可用于微生物分离与培养的技术原理、发展历程进行简单介绍,对比分析了每种分选技术的优点与不足,重点阐述了激光诱导向前转移技术应用于微生物分离的技术优势,并进一步对未来应用于未培养微生物的研究进行了初步展望。     

未培养微生物研究:方法、机遇与挑战

自然界中绝大部分的微生物仍是未培养的,称之为未培养微生物或微生物"暗物质"。对其进行研究不仅有助于认识微生物多样性及其代谢特征,加深对环境中微生物参与的生态学过程的理解,还有利于重构生命之树,揭示微生物的进化历程,具有重要的科学意义。同时未培养微生物是发现新基因资源和新活性物质的巨大宝库。随着现代分子生物学研究方法和培养技术的成熟和完善,从环境中直接破译未培养微生物的遗传信息,并实现培养逐渐成为可能。本文主要介绍了基于宏基因组技术和单细胞基因组技术或两者结合运用,研究环境中未培养微生物的主要方法和挑战,总结分析了目前已经解析的未培养微生物的主要类群,并对未来研究的机遇进行了展望。     

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。     

连续流生物反应器技术在微生物培养中的应用

自然环境中99%微生物在实验室条件下仍是不能被培养的,称之为"未培养"微生物或微生物"暗物质"。对其进行研究不仅有助于认识环境中微生物代谢多样性,丰富生命之树,同时未培养微生物还蕴含着巨大的新基因和新天然产物资源。但传统培养技术的局限性阻碍了"未培养"微生物资源的开发和利用。虽然随着分子生物学技术的发展,可以直接从环境中获得未培养微生物的遗传信息,分析微生物的广泛代谢多样性,但微生物的生理特征和代谢产物等分析仍然需要建立在研究纯菌株的基础上。目前,已经有很多新颖的培养技术被研发,如原位培养技术、共培养技术和连续流生物反应器培养技术等用于挖掘未培养微生物资源。本文主要介绍了连续流生物反应器培养新技术的发展与改进,探讨了"未培养"微生物培养技术及设备的发展方向,以进一步促进"未培养"微生物资源的开发与利用。     

微生物单细胞分离方法研究进展

自然界中大多数微生物处于未培养状态,被称为“微生物暗物质”。随着微生物单细胞分离方法的不断更新,利用新技术、新方法应对微生物纯培养的挑战获得了重要进展,这些新的分离及培养策略对推动微生物资源学的发展具有重要意义。尽管宏基因组学和基因组学数据相关成果日益增多,但微生物单细胞的分离与培养对于系统研究微生物的生态功能、遗传进化等仍至关重要。本文主要概述了目前使用的或正在研发的膜扩散培养法、微流控分选、荧光激活细胞分选、单细胞拉曼分选、光镊技术、显微操作技术等单细胞分离技术的原理与应用,及其在微生物单细胞分离和培养方面的优点与不足,同时展望了这些单细胞分离技术未来的发展和应用前景。     

Spatial organization of intestinal microbiota in the mouse ascending colon

Complex microbial populations are organized in relation to their environment. In the intestine, the inner lining (mucosa) is a potential focal point for such organization. The proximal murine colon contains mucosal folds that are known to be associated with morphologically distinct microbes. To identify these microbes, we used the technique of laser capture microdissection (LCM) to sample microbes associated with these folds (interfold region) and within the central lumen (digesta region). Using 16S rRNA gene tag pyrosequencing, we found that microbes in the interfold region were highly enriched for the phylum Firmicutes and, more specifically, for the families Lachnospiraceae and Ruminococcaceae. Other families such as Bacteroidaceae, Enterococcaceae and Lactobacillaceae were all enriched in the digesta region. This high-resolution system to capture and examine spatial organization of intestinal microbes should facilitate microbial analysis in other mouse models, furthering our understanding of host–microbial interactions.     

近自然纯培养法对细菌培养的初步研究

依据微生物在自然生境中协作生长的基本特性,提出一种对传统纯培养技术的改进思路及方法：设计一种有孔培养皿,皿内覆盖有不允许细菌通过、但营养物质可以自由流动的微孔滤膜。培养时将该培养皿放入被分离微生物所需生境中,可以克服传统纯培养难以提供外源活性物质的缺陷,一定程度上弥补了混合培养法和传统纯培养法的弱点,从而达到增强部分微生物可培养性、甚至培养出未培养微生物的目的。     

Metagenomics--the key to the uncultured microbes

It is widely accepted that up to 99.8% of the microbes present in many environments are not readily culturable. 'Metagenome technology' tries to overcome this bottleneck by developing and using culture-independent approaches. From the outset, metagenome-based approaches have led to the accumulation of an increasing number of DNA sequences, but until this time the sequences retrieved have been those of uncultured microbes. These genomic sequences are currently exploited for novel biotechnological and pharmaceutical applications and to increase our knowledge on microbial ecology and physiology of these microbes. Using the metagenome sequences to fully understand how complex microbial communities function and how microbes interact within these niches represents a major challenge for microbiologists today.     

南极海洋生物肠道微生物研究进展

生物的胃肠道微生物数量众多,各种菌群之间互相制约,与宿主共同进化。南大洋作为一个巨大的生物资源库,繁衍生存着大量的生物,其生活环境的多样性及特殊性,使得其胃肠道微生物较为特殊,肠道微生物群落也进化到适应宿主的各种营养生活方式。从不同营养级具有代表性的南极海洋生物出发,以南极磷虾,鱼类,企鹅,海豹为主线,综述这些生物胃肠道微生物的研究概况以及相关研究方法的优势与局限性,以期揭示肠道微生物与宿主的关系,为更加有效开发利用微生物资源提供借鉴。     

Relationship between aquifer biofilms and unattached microbial indicators of urban groundwater contamination

Aquifers, springs and other groundwater‐dependent ecosystems are threatened by urban land use, which causes water quality deterioration through nutrient loading, sewage infiltration, groundwater extraction and, along coasts, seawater intrusion. The presence of certain microbes in groundwater can indicate that an aquifer is anthropogenically contaminated. Interpretations made from observations of indicator microbes in groundwater are limited because the relationship between the presumably allochthonous indicator microbes and relevant autochthonous microbial communities has not been characterized. This study addressed whether autochthonous aquifer biofilms can influence the presence of presumed microbial indicators in groundwater, and simultaneously used microbial indicators to trace sources of urban contamination at a karst spring of conservation concern. These questions were approached using a 17‐month time series analysis of attached biofilm and adjacent unattached bacteria in the submerged karst aquifer conduit associated with this spring. Environmental 16S rRNA gene sequencing was performed to characterize these communities, and community structure data were contextualized with groundwater geochemical and hydrogeological measurements. Linear regression models were developed to explain the relative abundance patterns of indicator microbes and other unattached microbes at this site. The results of this study suggest that dominant aquifer biofilms do not influence the presence of unattached microbial taxa that are presumed to be indicators of groundwater contamination, and generated new information about the origin of coliform bacteria at the study site. These results build confidence in the use of microbial indicators in groundwater‐dependent ecosystem conservation strategies and inform future management plans for urban aquifers and springs worldwide.     

The physiology and ecological implications of efficient growth

The natural habitats of microbes are typically spatially structured with limited resources, so opportunities for unconstrained, balanced growth are rare. In these habitats, selection should favor microbes that are able to use resources most efficiently, that is, microbes that produce the most progeny per unit of resource consumed. On the basis of this assertion, we propose that selection for efficiency is a primary driver of the composition of microbial communities. In this article, we review how the quality and quantity of resources influence the efficiency of heterotrophic growth. A conceptual model proposing innate differences in growth efficiency between oligotrophic and copiotrophic microbes is also provided. We conclude that elucidation of the mechanisms underlying efficient growth will enhance our understanding of the selective pressures shaping microbes and will improve our capacity to manage microbial communities effectively.     

Applications of the rep-PCR DNA fingerprinting technique to study microbial diversity, ecology and evolution

A large number of repetitive DNA sequences are found in multiple sites in the genomes of numerous bacteria, archaea and eukarya. While the functions of many of these repetitive sequence elements are unknown, they have proven to be useful as the basis of several powerful tools for use in molecular diagnostics, medical microbiology, epidemiological analyses and environmental microbiology. The repetitive sequence-based PCR or rep-PCR DNA fingerprint technique uses primers targeting several of these repetitive elements and PCR to generate unique DNA profiles or 'fingerprints' of individual microbial strains. Although this technique has been extensively used to examine diversity among variety of prokaryotic microorganisms, rep-PCR DNA fingerprinting can also be applied to microbial ecology and microbial evolution studies since it has the power to distinguish microbes at the strain or isolate level. Recent advancement in rep-PCR methodology has resulted in increased accuracy, reproducibility and throughput. In this minireview, we summarize recent improvements in rep-PCR DNA fingerprinting methodology, and discuss its applications to address fundamentally important questions in microbial ecology and evolution.     

采油微生物在微驱过程中的生长、运移及分布规律

[目的]深入了解现场微生物驱油机理、效果评价标准及影响因素.[方法]结合现场微生物驱油过程产出液的跟踪监测及室内物模实验对微生物在地层中的生长繁殖、运移及分布规律进行研究.[结果]结果表明,通过从水井注入的外源微生物在油藏中能够有效生长繁殖,而且注入的营养液也能够激活内源微生物,但由于地层渗透率及营养液浓度的影响,产出液菌浓要比注入菌浓低1-2个数量级;葡萄糖的快速降解以及地层对微生物的过滤及吸附作用使大量的微生物停留在近井地带,仅有部分微生物能够从生产井采出,而且其运移速度要比营养液慢.[结论]地层渗透率和产出液中营养物浓度是影响微生物数量及分布的两个关键因素,现场微生物驱油产出液中的菌浓一般很难达到106个/mL以上,该研究结果对微生物驱油技术的发展和应用具有重要意义.     

Effect of Various Essential Oils Isolated from Douglas Fir Needles upon Sheep and Deer Rumen Microbial Activity

The effects of essential oils isolated from Douglas fir needles on sheep and deer rumen microbial activity were tested by use of an anaerobic manometric technique. Rumen microorganisms were obtained from a sheep which had been fed mainly on alfalfa hay and dried range grass. One deer used in this study had access to Douglas fir trees the year around, whereas the other deer had no access to Douglas fir. All of the monoterpene hydrocarbons isolated from Douglas fir needles—α-pinene, β-pinene, limonene, myrcene, camphene, Δ³-carene, and terpinolene—promoted only slightly or had no effect on deer rumen microbial activity, whereas all of them promoted activity in sheep rumen microbes, except Δ³-carene and terpinolene, which inhibited activity. Of the oxygenated monoterpenes, all monoterpene alcohols—α-terpineol, terpinen-4-ol, linalool, citronellol, and fenchyl alcohol—strongly inhibited the rumen microbial activity of both sheep and deer. Monoterpene esters (bornyl acetate) produced mild inhibition for both sheep and deer microbes, and citronellyl acetate inhibited rumen microbial activity in sheep, whereas it promoted activity in both deer. Monoterpene aldehyde (citronellal) inhibited the activity of rumen microbes from both sheep and deer having no access to Douglas fir from the Hopland Field Station, whereas they produced no effect upon the deer having access to Douglas fir from the Masonite forest. Rumen microbial activity for sheep and deer was promoted slightly with aliphatic ester (ethyl-n-caproate). There was a marked difference between sheep and deer rumen microbes as affected by addition of the various essential oils. The monoterpene hydrocarbons promoted activity more on sheep rumen microbes than on deer, and the monoterpene alcohols inhibited sheep rumen microbial activity more than that of deer. Furthermore, the deer rumen microbes from Hopland Field Station were affected more than the deer from Masonite forest.