海洋环境中难培养微生物的寡营养培养

海洋中存在着丰富的微生物资源, 但迄今为止能够在实验室培养的微生物却不到1%, 而且能够通过培养得到的环境优势种更少, 这成为当代环境微生物学研究和海洋资源开发的最大障碍。过去十多年来, 通过不断改进培养方法和检测手段, 发明了许多新颖独特的技术, 提高了培养效率。特别是通过海洋微生物的寡营养培养技术, 分离并命名了一些难培养微生物, 给予人们极大的启发。海洋微生物资源的可持续性开发和利用, 是21世纪人类发展的重要方向, 是我们研究海洋微观世界的基础, 值得微生物学界同仁的共同关注。     

Isolation of novel bacteria, including a candidate division, from geothermal soils in New Zealand

We examined bacterial diversity of three geothermal soils in the Taupo Volcanic Zone of New Zealand. Phylogenetic analysis of 16S rRNA genes recovered directly from soils indicated that the bacterial communities differed in composition and richness, and were dominated by previously uncultured species of the phyla Actinobacteria , Acidobacteria , Chloroflexi , Proteobacteria and candidate division OP10. Aerobic, thermophilic, organotrophic bacteria were isolated using cultivation protocols that involved extended incubation times, low-pH media and gellan as a replacement gelling agent to agar. Isolates represented previously uncultured species, genera, classes, and even a new phylum of bacteria. They included members of the commonly cultivated phyla Proteobacteria , Firmicutes , Thermus/Deinococcus , Actinobacteria and Bacteroidetes , as well as more-difficult-to-cultivate groups. Isolates possessing < 85% 16S rRNA gene sequence identity to any cultivated species were obtained from the phyla Acidobacteria , Chloroflexi and the previously uncultured candidate division OP10. Several isolates were prevalent in 16S rRNA gene clone libraries constructed directly from the soils. A key factor facilitating isolation was the use of gellan-solidified plates, where the gellan itself served as an energy source for certain bacteria. The results indicate that geothermal soils are a rich potential source of novel bacteria, and that relatively simple cultivation techniques are practical for isolating bacteria from these habitats.     

未培养环境微生物培养方法的研究进展

自然界中微生物种类繁多、功能多样、分布广泛,对人类健康安全、生态稳定和物种进化等发挥不可替代的作用.尽管微生物培养技术至今已有一百多年,然而由于各种限制因素的制约,目前成功分离培养的微生物仅占0.1％-1.0％,自然界中仍有十分丰富的微生物资源有待挖掘和开发利用.如何理解难培养微生物的制约因素并探索作用机制,同时借此开...     

The filtration-acclimatization method for isolation of an important fraction of the not readily cultivable bacteria

We developed a novel method, the filtration-acclimatization method (FAM), which enables the isolation and cultivation of an important fraction of the bacterial diversity, which is not cultivable by standard methods. The method consists of a filtration step, which removes most of the readily cultivable bacteria able to overgrow slowly growing bacteria, and an acclimatization procedure that provides a slow transition from the low environmental substrate concentrations to the high concentration of standard microbial media. So far, we isolated in total 65 strains from surface freshwater habitats by utilizing FAM. The isolates are affiliated with Actinobacteria, Alpha-, Betaproteobacteria, Bacteroidetes, and Spirochaeta. All isolates are pure cultures and form visible colonies on agar plates with high substrate concentrations. For further analysis, strains sharing more than a 97% 16S rRNA gene sequence similarity were grouped into one taxon. Based on sequence similarities, 88% of the obtained taxa can be considered to be undescribed species (<97% similarity to closest species). The highest similarity value of the taxa to the respective closest related species ranged from 87.7% to 99.8%, and was on average 94.5%. For comparison we isolated, by direct plating of water samples on a rich agar medium, a similar number of taxa. Amongst these taxa the percentage of taxa, which can be considered to be undescribed species, was only half of the percentage found for the taxa isolated by FAM. More importantly, it was amongst the taxa obtained by the standard method no taxon that was closer related to an uncultured bacterium than to an isolate, while 56% of the taxa isolated by FAM were closely related to uncultured bacteria.     

Multiplex PCR screening of soil isolates for novel Bacillus-related lineages

A16S rDNA multiplex PCR-based high-throughput protocol is presented to screen bacterial isolates in large amounts for the appearance of novel lineages of bacteria, especially hitherto unknown Bacillus relatives. The 16S rDNAs of 4224 isolates from a comprehensive cultivation campaign were screened for similarity to predominant uncultured soil bacteria. Soil suspensions were plated in serial dilutions on various media. After 2, 4 and 6 weeks, colonies were collected with toothpicks and transferred to microtiterplates for cell lysis and storage plates for subculture. Cell lysis was a simple freeze-heating cycle in distilled water. The multiplex PCR was adapted to operate sufficiently for Gram positives under these conditions. Approximately 10.6% of all picked colonies reacted with a primer targeting a Bacillus fraction containing novel Bacillus benzoevorans-relatives previously detected as predominant soil bacteria by culture-independent studies. From these 446 colonies detected by multiplex PCR, 363 (81.4%) could be successfully used for continued subculture and 16S rDNA sequencing. All identification was done by 16S rDNA sequencing. This revealed that more than 60% of them represented a variety of candidates for potentially new species. Twelve colonies were identified as almost identical matches to 16S rDNA sequences of hitherto uncultured but apparently predominant soil bacteria cloned from directly extracted soil DNA. Also, novel lineages of unpredicted phylogenetic diversity like novel Paenibacillus, Sporosarcina and even Xanthomonads were represented.     

Detection of a pederin-like compound using a dilution-to-extinction-based platform for the isolation of marine bacteria in drug discovery strategies

The continued development of culturing technologies for the discovery of new molecules from marine microbes is of paramount importance for drug discovery. Coupled with this, the use of the high-throughput approach shows promise for increasing the number of Gram-negative and non-filamentous bacteria cultures that can be surveyed, since they show a lower potential of bioactivity. In this work, we propose a new strategy of high-throughput cultivation of bacteria inspired by a dilution-to-extinction (DTE) methodology for the isolation of, and screening for, new cytotoxic compound producing marine bacteria. A marine sponge tissue was directly used as inoculum and the results were compared with the data obtained through the direct plating isolation method. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) genomic fingerprinting indicated the isolation of four bioactive strains, three of them producers of a pederin-like compound, and the fourth one able to synthesize a different compound, still unidentified, rendered by the DTE approach, in comparison with one bioactive strain identified through the plating method. Analyses based on the 16S rRNA gene data showed the existence of two different species belonging to the genus Labrenzia. The efficiency and diversity ratio in the number of isolates and compounds are discussed. In view of the results, the proposed DTE approach proved to be efficient for the isolation of new cytotoxic compounds of marine origin and pave the way for future potential applications.     

海洋动物来源微生物分离培养的新方法

海洋动物体内有着丰富的微生物,它们可以帮助动物宿主合成一些营养物质或者抵御其他动物侵害所需的化合物。在海洋动物来源微生物的生物活性化合物中,目前已有功能较好且应用于临床治疗的化合物。由于实验室分离培养条件的限制,目前仅有小部分的微生物被分离利用。因此开展新颖而有效的海洋动物来源微生物分离培养方法的研究十分必要。本文概括了近些年从海洋动物中分离微生物的新方法的结果和不足,这些方法包括原位培养技术、电回收法和培养基的改良等,重点介绍了扩散盒技术、I-tip技术和微囊包埋技术等。这些新方法的应用有助于获得更多新的微生物菌种和微生物次生代谢产物,了解微生物与动物宿主之间的关系,以及扩大海洋微生物资源的开发和利用。     

To BAC or not to BAC: marine ecogenomics

Most microbes in the ocean are still resistant to our collective cultivation efforts. Environmental microbial genomics provides science with the means for accessing and assessing the genomes, diversity, evolution and population dynamics of uncultured microorganisms--the ocean's hidden majority.     

Assessment of Microbial Diversity in Saudi Springs by Culture-Dependent and Culture-Independent Methods

Culture-dependent and culture-independent methods were used to evaluate the microbial diversity in two hot springs of the Aljouf region in Saudi Arabia, including Qasr Kaff and Ain Hawas. Physicochemical characteristics of the springs were examined to establish their effect on the biodiversity of thermophilic bacteria and fungi. We employed culture-dependent techniques to study microbial diversity using four different complex media for bacteria and fungi. In addition, the direct count for algal populations from two springs was investigated. We surveyed the microbial diversity in water and sediment samples from both springs by denaturing gradient gel electrophoresis (DGGE) and clone library construction. Bacillariophycaea (18 species) was the most diverse group, followed by Cyanophyceaea. Bacterial isolates closer to the genera Bacillus spp., Geobacillus, Thermoactinomyces, and unidentified actinobacteria were recovered. Fungal isolates were related to Aspergillus, Pezizaceae, Penicillium, Acremonium, Fusarium, Chrysosporium, and Stachybotrys. Using molecular-based techniques, the results were slightly different from those obtained by culture-dependent methods, and more genera were obtained. However, most genera were uncultured microbes, particularly from bacterial communities.     

High-throughput methods for culturing microorganisms in very-low-nutrient media yield diverse new marine isolates

Microbial diversity studies based on the cloning and sequencing of DNA from nature support the conclusion that only a fraction of the microbial diversity is currently represented in culture collections. Out of over 40 known prokaryotic phyla, only half have cultured representatives. In an effort to culture the uncultured phylotypes from oligotrophic marine ecosystems, we developed high-throughput culturing procedures that utilize the concept of extinction culturing to isolate cultures in small volumes of low-nutrient media. In these experiments, marine bacteria were isolated and cultivated at in situ substrate concentrations-typically 3 orders of magnitude less than common laboratory media. Microtiter plates and a newly developed procedure for making cell arrays were employed to raise the throughput rate and lower detection sensitivity, permitting cell enumeration from 200-microl aliquots of cultures with densities as low as 10(3) cells/ml. Approximately 2,500 extinction cultures from 11 separate samplings of marine bacterioplankton were screened over the course of 3 years. Up to 14% of the cells collected from coastal seawater were cultured by this method, which was 14- to 1,400-fold higher than the numbers obtained by traditional microbiological culturing techniques. Among the microorganisms cultured were four unique cell lineages that belong to previously uncultured or undescribed marine Proteobacteria clades known from environmental gene cloning studies. These cultures are related to the clades SAR11 (alpha subclass), OM43 (beta subclass), SAR92 (gamma subclass), and OM60/OM241 (gamma subclass). This method proved successful for the cultivation of previously uncultured marine bacterioplankton that have consistently been found in marine clone libraries.     

Microbial communities in anaerobic digestion processes for waste and wastewater treatment: a microbiological update

Anaerobic digestion technology is the biological treatment of organic waste and wastewater without input of external electron acceptors (oxygen), offering the potential to reduce treatment cost and to produce energy as 'biogas' (methane) from organic waste. The technology has become enormously popular in the past two decades, and knowledge of microbiological aspects of the technology has also accumulated significantly. Major advances have been made in elucidating the diversity of yet-to-be cultured microbes in anaerobic digestion processes, and the cultivation of uncultured organisms is of great interest with regard to gaining insights into the function of these organisms. In addition, recent advances have been made in the development of microbial fuel cells as an alternative, direct energy-yielding treatment system.     

宏基因组技术在开拓天然产物新资源中的应用

微生物代谢产物具有巨大的化学多样性,是多种抗生素和其它药物的重要来源。由于现有培养手段的局限性,可培养的微生物不到微生物总数的1％,使绝大部分微生物资源的开发利用受到制约。近年来．直接提取环境样品中混合微生物总基因组DNA,利用可培养的宿主细菌构建宏基因组文库,通过筛选目的克隆,寻找活性代谢产物,取得瞩目进展。对这一新领域的研究进展结合我们的研究概况进行了简要综述。     

Enrichment of previously uncultured bacteria from natural complex communities by adhesion to solid surfaces

The adhesion to inert solid surfaces was explored as a novel approach for the enrichment of previously uncultured bacteria from natural microbial communities. Enrichments on solid steel, glass and synthetic polymeric surfaces were established using samples from five freshwater lakes, a marine microbial mat and an alpine soil, and were subsequently analysed by molecular fingerprinting and sequencing of their 16S rRNA gene fragments. The majority of the enriched phylotypes grouped with the Alphaproteobacteria, Betaproteobacteria or Bacteroidetes and in several cases were related to typical biofilm‐forming species and genera. Most enrichments were most closely related to previously uncultured phylotypes and none had previously been cultivated from the original environments even when applying improved high throughput liquid cultivation techniques. Of the 13 phylotypes enriched from freshwater samples, seven were previously unknown, three matched so‐far uncultured environmental clones, and three were identical to previously cultivated bacteria. Of the 17 phylotypes recovered from soil, 12 were previously unknown with five of these phylotypes representing novel genera, whereas five phylotypes were identical to previously cultured soil bacteria. The feasibility of the biofilm‐enrichment approach was exemplified by the successful isolation of a not‐yet cultured Betaproteobacterium that constituted a discernible component of the alpine soil microbial community in situ and exhibited only 93% similarity to its closest cultured relative. Based on these results, cultivation on solid surfaces represents a promising approach to recover isolates that have so far escaped cultivation as suspended cultures in liquid media.     

Activity-based cell sorting reveals responses of uncultured archaea and bacteria to substrate amendment

Metagenomic studies have revolutionized our understanding of the metabolic potential of uncultured microorganisms in various ecosystems. However, many of these genomic predictions have yet to be experimentally tested, and the functional expression of genomic potential often remains unaddressed. In order to obtain a more thorough understanding of cell physiology, novel techniques capable of testing microbial metabolism under close to in situ conditions must be developed. Here, we provide a benchmark study to demonstrate that bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing can be used to identify anabolically active members of a microbial community incubated in the presence of various growth substrates or under changing physicochemical conditions. We applied this approach to a hot spring sediment microbiome from Yellowstone National Park (Wyoming, USA) and identified several microbes that changed their activity levels in response to substrate addition, including uncultured members of the phyla Thaumarchaeota, Acidobacteria, and Fervidibacteria. Because shifts in activity in response to substrate amendment or headspace changes are indicative of microbial preferences for particular growth conditions, results from this and future BONCAT-FACS studies could inform the development of cultivation media to specifically enrich uncultured microbes. Most importantly, BONCAT-FACS is capable of providing information on the physiology of uncultured organisms at as close to in situ conditions as experimentally possible.Subject terms: Environmental microbiology, Microbial communities, Microbial ecology, Archaea     

宏基因组学在微生物活性物质筛选中的应用

采用传统分离培养筛选微生物新活性物质的方法受到很大制约,自然界99%以上的微生物不能培养,其资源开发受到很大限制。环境微生物宏基因组技术应用避开了微生物分离纯培养问题,极大拓展了微生物资源的利用空间,增加获得新活性物质的机会和途径。本文着重介绍宏基因组的概念、研究策略包括DNA提取、文库构建与筛选等及在微生物活性物质筛选中的应用,并对宏基因组研究中存在的问题进行探讨。     

未培养微生物的研究与微生物分子生态学的发展*

近年来现代分子技术和基因组学逐渐渗透到有关生命科学的整个领域,也为微生物生态学提供了新的研究方法和机遇。16S rRNA基因序列分析、DNA-DNA杂交、核酸指纹图谱以及宏基因组学等分子技术检查自然环境中的微生物,可以克服传统纯培养技术的不足,是一条探知未培养微生物、寻找新基因及其产物的新途径,开启了我们认识微生物多样性和获得新资源的大门。     

"Bring to lab" of 19 novel species among 60 isolates retrieved from a freshwater pond

We report here on the cultivation of numerous novel bacterial species from a eutrophic freshwater pond. A total of 60 strains, 15 strains per each culture medium, were obtained from the surface of a eutrophic freshwater pond by employing a conventional dilution-plating method with four different kinds of culture media, including R2A, 1/10R2A, PCA, and 1/10PCA. Among the 60 strains isolated, 27 strains showed less than 97% 16S rRNA gene sequence similarities to validly published species, and thus they are considered to comprise 19 novel species. Of the 27 strains assigned to the novel species, the majority of the strains (20 strains) were affiliated with the Alphaproteobacteria and Betaproteobacteria. The remaining 7 strains were affiliated with the Gammaproteobacteria, Firmicutes, Actinobacteria, and Deinococci. Because we have isolated 19 novel species from a usual freshwater pond using a conventional culturing technique, our results suggest that an unexplored ecosystem, even if it looks like a common ecosystem found elsewhere, harbors diverse unidentified microbes, which will be definitely further characterized.     

The logic of biologically active small molecules: amazing ability of microorganisms*

In this review article, I will outline my way of thinking about biologically active small molecules. The structure of liposidomycin B from Streptomyces species resulted in my initial sense that a structure tells its function. A biologically active small molecule may save directly or indirectly a number of people. Even if the molecule has not been used as a therapeutic agent, it can be used as a useful chemical probe for dissecting a living cell into different biochemical pieces. Such biologically active small molecules derived from microorganisms have been primarily found in cultivable microorganisms that make up only 1% of total microbes in nature. Discovery of novel growth factors, zincmethylphyrin, zinc coproporphyrin, and coproporphyrin enabled laboratory cultivation of previously uncultured Leucobacter sp. These findings might expand the possibility for further discovery of novel therapeutic agents or chemical probes.     

Metagenomics: Concept,methodology, ecological inference and recent advances

Microorganisms constitute two third of the Earth's biological diversity. As many as 99% of the microorganisms present in certain environments cannot be cultured by standard techniques. Culture-independent methods are required to understand the genetic diversity, population structure and ecological roles of the majority of organisms. Metagenomics is the genomic analysis of microorganisms by direct extraction and cloning of DNA from their natural environment. Protocols have been developed to capture unexplored microbial diversity to overcome the existing barriers in estimation of diversity. New screening methods have been designed to select specific functional genes within metagenomic libraries to detect novel biocatalysts as well as bioactive molecules applicable to mankind. To study the complete gene or operon clusters, various vectors including cosmid, fosmid or bacterial artificial chromosomes are being developed. Bioinformatics tools and databases have added much to the study of microbial diversity. This review describes the various methodologies and tools developed to understand the biology of uncultured microbes including bacteria, archaea and viruses through metagenomic analysis.     

一份南海深海沉积物样品中可培养细菌的多样性

海洋沉积环境蕴含丰富的微生物资源。对深海难培养微生物的分离培养,不仅有利于深海微生物资源的挖掘与利用,也有利于对深海微生物学的研究。本研究采用多种培养基分离获得细菌菌株纯培养,并通过16S r RNA基因序列鉴定,对我国南海海域1个4 000 m水深的深海表层沉积物样品的可培养细菌多样性进行初探。共设计23种分离培养基,经过选择性分离培养最终获得612株细菌菌株,分别隶属于厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)的9目10科27个属级类群,可培养优势类群为厚壁菌门,占所有分离物种数量的85.8%,包含13个16S rRNA基因序列相似性低于98%的潜在新物种。海洋琼脂类培养基适合培养不同种类的海洋细菌类群,放线菌选择性分离类合成培养基对放线菌类群的分离效果较好。最终获得一些与具有产抗生素、细胞毒素、高效酶活、耐受不良环境、降解污染物等特殊功能微生物相近的菌株。研究结果表明,该深海沉积物样品的可培养微生物资源、潜在新物种和微生物生理特性丰富多样,研究深海环境难培养微生物的分离策略及其微生物适应生理特性对研究极端环境微生物打下了基础。