动物胃肠道微生物元基因组学研究进展

动物胃肠道中寄居着庞大复杂的微生物,它们对宿主营养、健康和生产有着重要的作用.随着分子生物学的发展,未培养微生物的研究越来越被重视,宏基因组学方法研究胃肠道微生物不仅能了解未培养微生物多样性,还能获得微生物的遗传、代谢和生理等方面的信息.探讨了元基因组文库的构建和分析方法,并重点介绍了元基因组学在动物胃肠道尤其是反刍动物瘤胃微生物研究中的应用.     

白酒可培养微生物数据库构建

[目的] 白酒可培养微生物的分离筛选是白酒行业重要的研究内容,本文旨在构建中国白酒生产环境中可培养微生物信息数据库。[方法] 数据库信息主要来源于通过人工查阅和整理目前已发表的白酒微生物的相关的文献报道和菌种保藏中心相关的筛选自白酒生态系统的微生物信息。数据库主要设计3个功能：（1）白酒可培养微生物信息检索：通过菌株名称、分离位置、培养基、代谢产物等为条件检索相关的白酒可培养微生物信息,从而获取该白酒微生物详细的生理生化与分类学信息;（2）培养基信息检索：通过特定培养基成分,培养基编号和名称检索相关的培养基信息,包括培养的组成和配制方法。（3）数据更新：在线上传新的可培养微生物和培养基信息。[结果] 目前数据库共收1221种白酒可培养微生物和295种培养基信息,网址为：http：//cmbaijiu.i-sanger.com/。[结论] 本数据库是我国白酒领域首个可培养微生物信息的数据库,将有助于白酒微生物培养的相关研究工作开展。     

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。     

大连新港原油降解微生物的分离纯化及降解效率评价

目的 在大连新港原油污染海域分离纯化出可降解原油的“土著”微生物,评价其原油降解能力,并研究提高降解效率的方法.方法 取海水样品进行富集培养,分离纯化出“土著”原油降解微生物,以16S rDNA测序法鉴定微生物种类,并采用MEGA 5.0进行多序列比对分析,选用最大相似法构建系统发育树.在实验室纯培养的条件下以气相色谱法对微生物的原油降解能力进行分析,选出优势菌种,再将优势菌种混配分析最佳原油降解条件.结果 分离纯化得到的“土著”原油降解微生物分属枯草芽孢杆菌属、动性球菌属、嗜冷菌属等多个菌属,“土著”原油降解微生物资源丰富,优势菌种的混配有助于加快和提高原油降解效率,是有效且对生态环境友好的生物处理法.     

创新型实验教学项目建设的探索——土壤微生物分离实验

武汉大学生命科学学院微生物学实验教学中注重创新型实验教学项目的建设,创新型土壤微生物分离实验项目建设是培养学生创新能力的一次实践。从武汉大学校园和国家级自然保护区神农架采集土壤样品,分离土壤细菌、放线菌和霉菌。让学生了解土壤微生物数量测定的基本方法,学习了解平板计数的基本操作技术。在引申实验过程中又有机地结合与贯穿了微生物学的无菌操作技术、显微镜技术、纯培养技术、分离纯化技术等各项技术及最新的分子生物学研究方法和技术。本实验的创新点有三:首先,实验从室内延伸到室外;其次,从本科生的常规土壤微生物的分离实验衍生到科研实验(新菌种的分离、鉴定);最后,使共性教学衍生为个性化培养。     

未培养技术在瘤胃产甲烷菌群研究中的应用

瘤胃中的产甲烷菌为严格厌氧微生物,很难通过分离培养的方法进行研究。现已经培养并公布的瘤胃产甲烷菌只有4种,成千上万种类的瘤胃产甲烷菌不能培养出来。未培养技术的发展与应用突破了传统分离培养检测方法的限制,使瘤胃产甲烷菌的研究有了较大的进展。综述了实时定量PCR、16S rDNA文库、DGGE和高通量测序技术等6项具有代表性的未培养技术及其在瘤胃产甲烷菌研究中的应用,为以后瘤胃产甲烷菌的研究提供方法上的参考。     

微生物可培养性低的生态学释因与对策

纯培养技术一直是微生物学研究的基石,但其单一的营养结构和生境与自然环境中微生物多样性、协同代谢等明显矛盾,从而成为部分微生物难以复苏的主要障碍。细菌共同协作的自然生存方式的崩溃、生境的极度营养变化和生态位巨变等是微生物可培养性低的主要生态学原因。非培养技术、加富培养、混合培养、稀释培养、模拟自然培养和综合方法等是主要的研究手段和策略,可在不同程度上解决微生物可培养性低的缺陷和问题。     

追寻被“遗漏”的微生物

人类对生态环境中微生物的认识从依赖于纯培养微生物的研究阶段已进入到结合各种组学方法的微生物群落代谢机制的研究阶段.在微生物群落组成的研究中,基于“通用”引物的PCR扩增方法会“遗漏”很多种类微生物,因此需要探索一些方法,以找回这些被“遗漏”的微生物.目前生态环境中能培养的微生物种类较为有限,但是通过培养方法的改进,分离培养新的微生物或富集培养特殊功能的微生物依然是扩展微生物种类认知范围的重要途径.而且,通过元基因组数据库分析,可以了解常用的“通用”引物所不能覆盖的微生物范围,并能阐明不同生态环境中各种微生物类型的分布情况.由于元转录组中最多的是核糖体RNA,所以可通过改进的元转录组方法同时分析有活性的细菌、古生菌和真核微生物群落结构.追寻被“遗漏”的微生物不仅能扩展我们对微生物的认知范围,同时也是研究并正确理解全球物质循环过程的一个重要领域.     

水样保存方法对青藏高原湖泊可培养微生物的影响

青藏高原湖水中生存着大量适应极端环境的微生物,如耐冷及嗜冷菌。合适的湖水样品保存方法是野外取样的关键和开展深入研究的前提。本研究对比了冷冻和4℃冷藏两种保存方法对湖水中可培养微生物种类的影响。首先从不同保存方法样品中分离得到纯菌,然后对分离菌的16S rRNA测序进行菌种鉴定。结果表明,自冷冻保存湖水中分离得到7个不同的16S rRNA基因OUT(operational taxonomic unit),冷藏保存水样共分离得到14个不同的16S rRNA基因OTU。冷冻保存水样分离得到的可培养微生物包括芽孢杆菌、简单芽孢杆菌、微小杆菌、不动杆菌、苏云金芽孢杆菌;冷藏保存水样分离得到的微生物包括γ-变形菌、金黄杆菌、芽孢杆菌、短小芽孢杆菌、寡养单胞菌、假炭疽杆菌、缺陷假单胞菌、细杆菌。研究表明,冷藏保存方法比冷冻保存方法可分离得到更多种类可培养微生物,这两种保存方法均分离得到了芽孢杆菌。     

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。     

The role of microbes in rumen lipolysis and biohydrogenation and their manipulation

Despite the fact that the ruminant diet is rich in polyunsaturated fatty acids (PUFA), ruminant products – meat, milk and dairy – contain mainly saturated fatty acids (SFA) because of bacterial lipolysis and subsequent biohydrogenation of ingested PUFA in the rumen. The link between SFA consumption by man and coronary heart disease is well established. In contrast, ruminant products also contain fatty acids that are known to be beneficial to human health, namely conjugated linoleic acids (CLAs). The aims of research in this field have been to understand the microbial ecology of lipolysis and biohydrogenation and to find ways of manipulating ruminal microbes to increase the flow of PUFA and CLA from the rumen into meat and milk. This review describes our present understanding of the microbial ecology of ruminal lipid metabolism, including some apparently anomalous and paradoxical observations, and the status of how the metabolism may be manipulated and the possible consequential effects on other aspects of ruminal digestion. Intuitively, it may appear that inhibiting the ruminal lipase would cause more dietary PUFA to reach the mammary gland. However, lipolysis releases the non-esterified fatty acids that form the substrates for biohydrogenation, but which can, if they accumulate, inhibit the whole process. Thus, increasing lipase activity could be beneficial if the increased release of non-esterified PUFA inhibited the metabolism of CLA. Rumen ciliate protozoa do not carry out biohydrogenation, yet protozoal lipids are much more highly enriched in CLA than bacterial lipids. How could this happen if protozoa do not metabolise PUFA? The answer seems to lie in the ingestion of plant organelles, particularly chloroplasts, and the partial metabolism of the fatty acids by contaminating bacteria. Bacteria related to Butyrivibrio fibrisolvens are by far the most active and numerous biohydrogenating bacteria isolated from the rumen. But do we misunderstand the role of different bacterial species in biohydrogenation because there are uncultivated species that we need to understand and include in the analysis? Manipulation methods include dietary vegetable and fish oils and plant-derived chemicals. Their usefulness, efficacy and possible effects on fatty acid metabolism and on ruminal microorganisms and other areas of their metabolism are described, and areas of opportunity identified.     

Molecular genetics of obligate anaerobes from the rumen

Abstract The rumen is inhabited by a highly specialised microflora consisting of obligately anaerobic bacteria, fungi and protozoa. Rumen bacteria belong to many different phylogenetic groupings and many species exhibit a high degree of rRNA gene sequence diversity, whereas the rumen fungi are monophyletic. At least 21 genes concerned with the degradation and utilisation of plant cell wall polysaccharides, from five species of rumen bacteria and from rumen fungi, have been isolated and sequenced. In general, the catalytic domains of the encoded enzymes belong to enzyme families identified among non-rumen microorganisms, but some show unusual organisation, consisting of multiple catalytic domains. Several bacterial species have been used as recipients for gene transfer by electrotransformation or by conjugation, allowing development of methods for genetic analysis. The rumen is also considered as a potential site for natural gene transfer.     

The genome sequence of the capnophilic rumen bacterium Mannheimia succiniciproducens

The rumen represents the first section of a ruminant animal's stomach, where feed is collected and mixed with microorganisms for initial digestion. The major gas produced in the rumen is CO(2) (65.5 mol%), yet the metabolic characteristics of capnophilic (CO(2)-loving) microorganisms are not well understood. Here we report the 2,314,078 base pair genome sequence of Mannheimia succiniciproducens MBEL55E, a recently isolated capnophilic Gram-negative bacterium from bovine rumen, and analyze its genome contents and metabolic characteristics. The metabolism of M. succiniciproducens was found to be well adapted to the oxygen-free rumen by using fumarate as a major electron acceptor. Genome-scale metabolic flux analysis indicated that CO(2) is important for the carboxylation of phosphoenolpyruvate to oxaloacetate, which is converted to succinic acid by the reductive tricarboxylic acid cycle and menaquinone systems. This characteristic metabolism allows highly efficient production of succinic acid, an important four-carbon industrial chemical.     

瘤胃微生物与宿主互作及其日粮调控研究进展

瘤胃微生物与宿主间存在互作关系,宿主动物遗传信息影响瘤胃微生物,而瘤胃微生物变化也同样受到日粮原料、营养水平以及外源添加物质的调控。近年来,通过多组学技术分析瘤胃微生物与宿主关系及其内在机制已成为研究热点。综述了瘤胃微生物与宿主关系及受日粮调控作用研究进展,具体介绍了瘤胃微生物与宿主基因组关系,瘤胃微生物与动物生产性能关系,以及在日粮配置、益生菌益生元和植物次生代谢物添加等条件下对瘤胃微生物的影响;并对瘤胃微生物研究的发展趋势和应用前景进行了展望。     

厌氧真菌纤维降解酶及其应用潜力的研究进展

反刍动物的瘤胃是已知的纤维降解能力最强的天然发酵罐,其发酵粗纤维的能力很大程度上依赖于其中栖息的各类微生物的功能。厌氧真菌作为瘤胃内的一类低丰度菌群,最先定殖到宿主动物摄入的纤维质饲料上,并通过分泌大量高效的碳水化合物活性酶降解粗纤维。然而,由于缺乏足够的基因组信息和有效的厌氧真菌遗传操作系统,目前国内外对厌氧真菌分泌的纤维降解酶及其降解机制的研究进展有限。本文对厌氧真菌的分类及已发表的基因组信息进行概述,介绍了各类纤维降解酶及纤维小体的组成结构和催化机制特点,并对纤维降解酶在生物质能、饲料处理、纺织造纸及食品加工等方面的应用进行总结。研究厌氧真菌纤维降解酶的作用特性,将有助于完善其在瘤胃环境中有效竞争资源并降解粗纤维的知识体系,也将进一步了解其生物技术应用潜力,为工业生产中应用酶制剂提供新思路。     

牦牛瘤胃中产脂肪酶微生物的分离与鉴定

【背景】脂肪酶是一类特殊的酯键水解酶,广泛应用于工业化生产中,微生物是工业脂肪酶的主要来源。瘤胃中微生物种类繁多、数量庞大,已有关于瘤胃微生物产纤维素酶的报道,尚无产脂肪酶瘤胃微生物的分离筛选报道。【目的】从牦牛瘤胃中分离筛选出能够产脂肪酶的微生物,并进行菌株鉴定及其酶学性质的研究。【方法】以橄榄油为唯一碳源,通过中性红油脂平板进行初步筛选后,用改进铜皂-分光光度法测定酶活力进行复筛;再经形态学观察、生理生化实验和16S rRNA基因序列分析进行菌种鉴定;研究3种脂肪酶的最适作用温度、pH值及金属离子、有机溶剂和表面活性剂对酶活力的影响。【结果】筛选出6株酶活力较高的菌株,其中3株为液化沙雷氏菌,2株为白地霉,1株为卷枝毛霉。脂肪酶的酶学性质研究表明:液化沙雷氏菌、白地霉和卷枝毛霉所产脂肪酶的最适作用温度为45、35和40°C;最适pH为8.0、7.0和7.0;Ca2+和Mg2+对3种脂肪酶均有激活作用;Zn2+对3种脂肪酶有不同程度的抑制作用,EDTA、SDS可使3种脂肪酶失活;3种脂肪酶对丙三醇的耐受力较高,卷枝毛霉脂肪酶对甲醇、乙醇、丙酮的耐受力较高。【结论】从牦牛瘤胃中分离出3种产脂肪酶的微生物,且证实瘤胃微生物在脂肪酶研究方面具有较高的价值。     

瘤胃纤维素降解细菌的研究进展

反刍动物瘤胃是自然界中最有效的纤维素降解系统，其纤维素降解能力主要源于寄居于其中的纤维素降解细菌、真菌和原虫。其中，瘤胃纤维素降解细菌因数量庞大、种类繁多以及代谢途径丰富，在木质纤维素降解及利用方面发挥着重要作用。本文综述了国内外瘤胃纤维素降解细菌的种类，分析了瘤胃纤维素降解细菌的特性；阐述了瘤胃纤维素降解细菌通过纤维小体对纤维素的降解过程，以及瘤胃微生物之间的相互作用和相互制约关系；简述宏组学技术在开发新纤维素降解菌和新纤维素酶方面的应用，旨在为进一步研究纤维素降解细菌的降解机理，开发新的纤维素菌种和酶资源提供新的思路。     

盐碱环境放线菌多样性研究

放线菌因产生多种多样的生物活性物质而受到重视。但极端环境放线菌的研究甚少。采用DGGE、纯培养法,重点研究了新疆、青海及埃及的重盐碱环境的放线菌分布情况,种类组成,生物学特性。发现了1个新科,8个新属及30多个新种。从嗜(耐)盐碱放线菌筛选到许多带有PKS基因的菌株。认为极端环境放线菌是生物活性物质的重要来源;改进分离程序,分离未知放线菌,是放线菌多样性研究及开发利用的前提之一;并对极端环境放线菌研究作了论述。     

Comparison of Rumen Microbial Inhibition Resulting from Various Essential Oils Isolated from Relatively Unpalatable Plant Species

Essential oils were isolated from eight plant species which were relatively unpalatable to sheep and deer. The inhibitory potency of these essential oils upon sheep and deer rumen microorganisms was compared, in terms of total gas and volatile fatty acid (VFA) production, by use of an anaerobic manometric technique. Inhibitory effects of oils from the eight plant species may be placed in four groups: (i) essential oils from vinegar weed (Trichostema lanceoletum) and California bay (Umbellularia californica) inhibited rumen microbial activity most; (ii) lesser inhibition was exhibited by rosemary (Rosmarinus officinalis) and California mugwort (Artemisia douglasiana) oils, followed by (iii) blue-gum eucalyptus (Eucalyptus globulus) and sagebrush (Artemisia tridentata) oils; and (iv) oils from Douglas fir (Psuedotsuga menziesii) and Jerusalem oak (chenopodium botrys) resulted in the least inhibition, when 0.3 ml of each oil was used. A highly significant correlation coefficient (r = 0.98(**)) between total gas and VFA production indicated the validity of either method to measure the activity of rumen microorganisms. Our results are discussed in relation to the hypothesis that the selectivity and voluntary consumption of ruminants are related to the characteristic odor and antibacterial action of essential oils isolated from relatively unpalatable plant species.     

Isolation, Culture Characteristics, and Identification of Anaerobic Bacteria from the Chicken Cecum

Studies on the anaerobic cecal microflora of the 5-week-old chicken were made to determine a suitable roll-tube medium for enumeration and isolation of the bacterial population, to determine effects of medium components on recovery of total anaerobes, and to identify the predominant bacterial groups. The total number of microorganisms in cecal contents determined by direct microscope cell counts varied (among six samples) from 3.83 x 10(10) to 7.64 x 10(10) per g. Comparison of different nonselective media indicated that 60% of the direct microscope count could be recovered with a rumen fluid medium (M98-5) and 45% with medium 10. Deletion of rumen fluid from M98-5 reduced the total anaerobic count by half. Colony counts were lower if chicken cecal extract was substituted for rumen fluid in M98-5. Supplementing medium 10 with liver, chicken fecal, or cecal extracts improved recovery of anaerobes slightly. Prereduced blood agar media were inferior to M98-5. At least 11 groups of bacteria were isolated from high dilutions (10(-9)) of cecal material. Data on morphology and physiological and fermentation characteristics of 90% of the 298 isolated strains indicated that these bacteria represented species of anaerobic gram-negative cocci, facultatively anaerobic cocci and streptococci, Peptostreptococcus, Propionibacterium, Eubacterium, Bacteroides, and Clostridium. The growth of many of these strains was enhanced by rumen fluid, yeast extract, and cecal extract additions to basal media. These studies indicate that some of the more numerous anaerobic bacteria present in chicken cecal digesta can be isolated and cultured when media and methods that have been developed for ruminal bacteria are employed.