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。     

中国南海沉积环境可培养细菌多样性研究

【目的】探索海洋沉积环境中可培养细菌的多样性。【方法】采用纯培养分离及16S rRNA基因序列鉴定的方法,对我国南海海域20个沉积物样品进行细菌多样性分析。【结果】共获得200株细菌,分属于47个属,99个种。经系统进化分析,可培养菌株主要分布于4个类群:厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes),优势类群为Firmicutes,其中芽孢杆菌属(Bacillus)所占比例为55.6%;而Actinobacteria和Bacteroidetes两个类群获得菌株较少;在Firmicutes和Actinobacteria两个类群中发现8个潜在新种和3个潜在新属级类群。【结论】初步研究结果表明,南海海洋沉积环境可培养微生物资源丰富,新物种资源多样;其中,芽孢杆菌为海洋沉积环境中的优势类群,随着样品深度的增加,细菌多样性呈现递减的趋势,深度可能是影响细菌多样性的一个重要因素;其次,分离培养基和分离方法直接关系到样品中可培养微生物多样性的发现,有待深入研究。     

中国南海部分深海沉积物真菌多样性及其抗菌活性

【目的】从南海4个站位的深海沉积物中分离真菌,揭示其多样性并测定抗菌活性。【方法】使用4种培养方法和8种培养基,从12个深海沉积物样本中分离培养真菌,通过菌落形态观察和ITS序列系统发育分析进行鉴定。采用滤纸片扩散法和生长速率法分别测试真菌小量发酵液粗浸膏的抗细菌和抗真菌活性。【结果】共分离到125株纯培养真菌,基于形态和ITS序列分析,排重后得到18个种类型,这些真菌可以划分到12个属,大多数属于子囊菌门(Ascomycota),只有2株属于担子菌门(Basidiomycota)。4个站位可培养真菌多样性具有差异性。抑菌活性筛选显示,大多数真菌具有较好的抑菌活性;链格孢属(Alternaria)、青霉属(Penicillium)、匐柄霉属(Stemphylium)这几个属的真菌表现出对多种指示细菌有抑制作用,尤其是Alternaria tenuissima DN09、Alternaria alternata DN14和Penicillium chrysogenum DN16对G~+和G~–细菌均表现出抑制作用。【结论】本研究揭示了南海深海沉积物可培养真菌多样性和抑菌活性,为进一步利用深海沉积物来源真菌奠定了基础。     

中国典型冻土区土壤可培养细菌多样性

[目的]对比分析中国典型高纬度冻土区和高海拔冻土区土壤可培养细菌的多样性.[方法]采用NM、TSA 、R2A 3种培养基分离培养不同冻土区土壤可培养细菌,用通用引物扩增分离的细菌16S rRNA基因,根据系统发育分析进行鉴定.[结果]从6个样品中得到冻土土壤可培养细菌的菌落数量为4.70×103 -2.57×105 cfu/g(土壤干重),根据不同的菌落形态分离出144株可培养细菌.纯培养物的16S rRNA基因部分序列分析表明:我国高纬度冻土区土壤样品中的细菌分别属于Firmicutes分支(59.52％)、Gammaproteobacteria 分支(38.10％)、Betaproteobacteria分支(2.38％),其中假单胞菌属(Pseudomonas)、芽胞杆菌属(Bacillus)、类芽胞杆菌属(Paenibacillus)的菌株为该区域的三大优势菌群.我国高海拔冻土区土壤样品中分离细菌属于Gammaproteobacteria分支(89.22％)、Firmicutes分支(8.82％)和Bacteroidetes分支(1.96％)o优势菌群为假单胞菌属( Pseudomonas).[结论]我国高纬度冻土区和高海拔冻土区土壤具有较高的可培养细菌多样性;不同类型冻土区土壤可培养细菌群落组成不同.本文研究结果将为我国冻土区土壤细菌资源研究与利用提供理论依据.     

南海深海沉积物烷烃降解菌的富集分离与多样性初步分析

通过培养和非培养2种手段研究了南海沉积物中石油降解菌的多样性。通过烷烃富集培养,从2个站点不同深度的南海沉积物样品中富集筛选出48株深海细菌,其中27株对十六烷有降解能力。表面张力测定结果表明,4株降解菌同时具有较强的表面活性剂产生能力,2株Dietzia maris菌能使水的表面张力降至33mN/m左右,这是该种微生物产表面活性剂的首次报道。通过变性梯度凝胶电泳(DGGE)分析显示,南海沉积物富集物中的烷烃降解菌优势菌是芽孢杆菌,而且有多种。其中,Bacillus aquimaris在两个站点的7个样品的富集物中都是优势菌。此外,Sporosarcina,Halomona以及Brevibacterium属的细菌在不同样品中也表现为除Bacillus之外的优势菌。     

普通卷甲虫肠道可培养细菌的分离、鉴定及产消化酶活性

目的分离培养普通卷甲虫肠道中的可培养细菌,筛选有产消化酶活性的细菌,推测其在协助普通卷甲虫消化食物中的作用。方法通过传统分离培养法分离普通卷甲虫肠道中的可培养细菌,利用平板透明圈法筛选产淀粉酶、蛋白酶、纤维素酶和脂肪酶活性的细菌,利用水解圈与菌落直径的比值,比较不同细菌的产消化酶活性。利用SPSS 20.0软件进行统计学分析,组间比较采用单因素方差分析。结果在普通卷甲虫肠道中分离出4个属9种细菌,其中气单胞菌属3种,假芽胞杆菌属和柠檬酸杆菌属各2种,芽胞杆菌属和假单胞菌属各1种。9种细菌中弗氏柠檬酸杆菌、豚鼠气单胞菌、南海假芽胞杆菌等3种细菌可产蛋白酶,嗜水气单胞菌、波特卡伦柠檬酸杆菌、水生气单胞菌、豚鼠气单胞菌和南海假芽胞杆菌等5种细菌可产纤维素酶,嗜水气单胞菌、波特卡伦柠檬酸杆菌、印度芽胞杆菌、水生气单胞菌、嗜盐假芽胞杆菌、豚鼠气单胞菌和南海假芽胞杆菌等7种细菌可产淀粉酶,未筛选到产脂肪酶细菌。统计学分析表明,3种产蛋白酶细菌和5种产纤维素酶细菌的产酶活性差异无统计学意义。而7种产淀粉酶细菌产酶的活力间差异有统计学意义,水生气单胞菌的产淀粉酶活性能力最强。结论普通卷甲虫肠道可培养细菌结构简单,但有消化酶活性的细菌种类多,5种细菌有产2种以上消化酶功能,说明肠道细菌可能在普通卷甲虫食物消化中起着重要作用。     

基于可培养方法分析云南腾冲小空山火山谷芽胞杆菌分布特征

【目的】为了解云南腾冲小空山火山谷土壤中可培养芽胞杆菌种类分布特征。【方法】采用可培养手段对小空山火山谷阳坡、谷底和阴坡土壤中的芽胞杆菌进行分离培养,根据16S rRNA基因序列同源性对分离菌株进行鉴定,并分析系统发育地位。利用Canoco5软件分析采样点芽胞杆菌种类分布特征与土壤样品理化性质的相关性。【结果】从火山谷土壤样品中共分离获得180株芽胞杆菌,16S rRNA基因测序鉴定结果表明分离菌株隶属于芽胞杆菌纲2个科(芽胞杆菌科和类芽胞杆菌科)、6个属、34个种,其中芽胞杆菌属(Bacillus) 11个种,类芽胞杆菌属(Paenibacillus) 14个种,短芽胞杆菌属(Brevibacillus)3个种,赖氨酸芽胞杆菌属(Lysinibacillus)4个种,嗜冷芽胞杆菌属(Psychrobacillus)1个种和绿芽胞杆菌属(Viridibacillus)2个种,其中7个菌株与其最近模式菌株16SrRNA相似性低于种的界定阈值(98.65%),为芽胞杆菌潜在新物种。优势属为芽胞杆菌属和类芽胞杆菌属,优势种为蕈状芽胞杆菌(Bacillusmycoides),图瓦永芽胞杆菌(Bacillustoyonensis),蜡状芽胞杆菌(Bacilluscereus),解木糖赖氨酸芽胞杆菌(Lysinibacillusxylanilyticus),蜂房类芽胞杆菌(Paenibacillusalvei)和沙地绿芽胞杆菌(Viridibacillus arenosi)。其中16个种分离自阳坡,29个种分离自阴坡,9个种分离自谷底,三者共同种类为6种。阳坡、谷底和阴坡的芽胞杆菌种群分布Bray-Curtis相似性为62.4%,多样性分析结果表明,Shannon指数(H′)大小次序为阴坡阳坡谷底。环境因子分析发现,芽胞杆菌种群分布多样性特征与其土壤的海拔高度、碳氮比和硫含量呈负相关,而和碳源和氮源含量呈正相关。【结论】从以上结果得出,云南腾冲火山谷有着较为丰富的芽胞杆菌资源,且还存在可分离培养的芽胞杆菌的潜在新物种,为利用火山微生物资源提供了保障。     

明永冰川垂直气候带中可培养低温细菌多样性分析

本研究对位于云南滇西北的明永冰川地区暖温带、中温带和寒温带三个不同垂直气候带中可培养低温细菌的多样性进行了研究。利用四种不同培养基对该地区可培养低温细菌进行了分离纯化,共得到细菌37 513株,根据菌落形态特征分为了391种,其中LB培养基分离到99种,Organic培养基分离到78种,PSG培养基分离到96种,PYGV培养基分离得到118种,可以看出寡营养培养基PYGV分离得到的细菌种类多于LB和Organnic等富营养培养基,表明PYGV针对冰川地区细菌的分离与鉴定更为合适;通过革兰氏染色和扫描电镜观察表明大部分菌株为革兰阴性杆菌;对已分离得到的优势菌进行了16S rRNA基因测序并构建系统发育树,分析得出:假单胞菌属(Pseudomonas)、耶尔森氏菌属(Yersinia)和黄杆菌属(Flavobacterium)在明永冰川不同垂直气候带上均有分布,其中假单胞菌属最多占据35%;而寡养单胞菌属(Stenotrophomonas)是寒温带上特有的菌属。本研究证明明永冰川地区垂直气候带中可培养低温细菌多样性非常丰富,也为下一步了解这一特殊地理生态环境下微生物的群落演替规律、研究冰川环境中微生物群落如何响应气候变化提供了参考。     

广东南岭森林土壤中可培养细菌多样性

广东南岭森林土壤中蕴藏着丰富的生物资源,但对其中的可培养细菌种类仍缺乏系统了解。本研究采用贫营养型的R2A培养基和富营养型的TSA培养基对南岭森林土壤中细菌进行了分离,获得细菌408株,分别从属于厚壁菌门、变形菌门、放线菌门和拟杆菌门的35属。其中的优势类群为厚壁菌门,占分离总数量的71%。在属水平,芽胞杆菌及其近缘属为优势类群。除芽胞杆菌外,假单胞菌、伯克霍尔德氏菌、草酸杆菌科Collimonas属和罗丹诺杆菌科Dyella属是分离获得的主要类群。R2A培养基在分离革兰氏阴性的变形菌门菌株方面表现出一定的偏好性,而TSA培养基分离得到的更多为快速生长的芽胞杆菌及其近缘的革兰氏阳性细菌。发现了15属的菌株具有一定的水解酶活性,大多表现出对淀粉和牛奶的水解活性,对有机磷的水解性能优于对无机磷的水解。降解纤维素的菌株则主要集中于芽胞杆菌及其近缘属中。发现了潜在新物种26株,分布于芽胞杆菌、Dyella、类芽孢杆菌等9属中。本研究仅使用了两种营养类型的培养基,进一步借助培养组学技术有望能更加全面反映南岭森林土壤中的可培养微生物多样性。     

伊犁河流域春季可培养细菌多样性及其胞外酶检测

利用可培养法对新疆伊犁河流域水体和沉积物中细菌多样性进行分析，以期初步阐明流域河流可培养细菌群落结构。采用5种琼脂培养基分离纯化可培养细菌，依据其16S rRNA基因序列进行系统发育分析，并运用平板法对纯化菌株的胞外酶产生情况进行检测。序列分析结果表明，225株细菌分别属于变形菌门γ亚群(Gamma-pseudomonadota, 56.44%)、放线菌门(Actinomycetota, 18.22%)、厚壁菌门(Firmicutes, 14.22%)、变形菌门α亚群(Alpha-pseudomonadota, 4.89%)、变形菌门β亚群(Beta-pseudomonadota, 4%)、拟杆菌门(Bacteroidota, 0.44%)和异常球菌-栖热菌门(Deinococcota, 0.44%)等7个大的系统发育类群，41个属84个种。其中假单胞菌属(Pseudomonas,42.22%)、不动杆菌属(Acinetobacter,9.33%)和芽胞杆菌属(Bacillus,9.33%)为优势菌属。菌种分布结果显示，伊犁河流域主要支流和干流中可培养细菌地域分布性强。分离菌株产胞外酶...     

More refined diversity of anammox bacteria recovered and distribution in different ecosystems

A newly reported 16S rRNA gene-based PCR primer set was successfully applied to detect anammox bacteria from four ecosystem samples, including sediments from marine, reservoir, mangrove wetland, and wastewater treatment plant sludge. This primer set showed ability to amplify a much wider coverage of all reported anammox bacterial genera. Based on the phylogenetic analyses of 16S rRNA gene of anammox bacteria, two new clusters were obtained, one closely related to Candidatus Scalindua, and the other in a previously reported novel genus related to Candidatus Brocadia. In the Scalindua cluster, four new subclusters were also found in this study, mainly by sequences of the South China Sea sediments, presenting a higher diversity of Candidatus Scalindua in marine environment. Community structure analyses indicated that samples were grouped together based on ecosystems, showing a niche-specific distribution. Phylogenetic analyses of anammox bacteria in samples from the South China Sea also indicated distinguished community structure along the depth. Pearson correlation analysis showed that the amount of anammox bacteria in the detected samples was positively correlated with the nitrate concentration. According to Canonical Correspondence Analysis, pH, temperature, nitrite, and nitrate concentration strongly affected the diversity and distribution of anammox bacteria in South China Sea sediments. Results collectively indicated a promising application of this new primer set and higher anammox bacteria diversity in the marine environment.     

Eukaryotic diversity in late Pleistocene marine sediments around a shallow methane hydrate deposit in the Japan Sea

Marine sediments contain eukaryotic DNA deposited from overlying water columns. However, a large proportion of deposited eukaryotic DNA is aerobically biodegraded in shallow marine sediments. Cold seep sediments are often anaerobic near the sediment–water interface, so eukaryotic DNA in such sediments is expected to be preserved. We investigated deeply buried marine sediments in the Japan Sea, where a methane hydrate deposit is associated with cold seeps. Quantitative PCR analysis revealed the reproducible recovery of eukaryotic DNA in marine sediments at depths up to 31.0 m in the vicinity of the methane hydrate deposit. In contrast, the reproducible recovery of eukaryotic DNA was limited to a shallow depth (8.3 m) in marine sediments not adjacent to the methane hydrate deposit in the same area. Pyrosequencing of an 18S rRNA gene variable region generated 1,276–3,307 reads per sample, which was sufficient to cover the biodiversity based on rarefaction curves. Phylogenetic analysis revealed that most of the eukaryotic DNA originated from radiolarian genera of the class Chaunacanthida, which have SrSO₄ skeletons, the sea grass genus Zostera, and the seaweed genus Sargassum. Eukaryotic DNA originating from other planktonic fauna and land plants was also detected. Diatom sequences closely related to Thalassiosira spp., indicative of cold climates, were obtained from sediments deposited during the last glacial period (MIS‐2). Plant sequences of the genera Alnus, Micromonas, and Ulmus were found in sediments deposited during the warm interstadial period (MIS‐3). These results suggest the long‐term persistence of eukaryotic DNA from terrestrial and aquatic sources in marine sediments associated with cold seeps, and that the genetic information from eukaryotic DNA from deeply buried marine sediments associated with cold seeps can be used to reconstruct environments and ecosystems from the past.     

Seasonal and spatial diversity of microbial communities in marine sediments of the South China Sea

This study was conducted to characterize the diversity of microbial communities in marine sediments of the South China Sea by means of 16S rRNA gene clone libraries. The results revealed that the sediment samples collected in summer harboured a more diverse microbial community than that collected in winter, Deltaproteobacteria dominated 16S rRNA gene clone libraries from both seasons, followed by Gammaproteobacteria, Acidobacteria, Nitrospirae, Planctomycetes, Firmicutes. Archaea phylotypes were also found. The majority of clone sequences shared greatest similarity to uncultured organisms, mainly from hydrothermal sediments and cold seep sediments. In addition, the sedimentary microbial communities in the coastal sea appears to be much more diverse than that of the open sea. A spatial pattern in the sediment samples was observed that the sediment samples collected from the coastal sea and the open sea clustered separately, a novel microbial community dominated the open sea. The data indicate that changes in environmental conditions are accompanied by significant variations in diversity of microbial communities at the South China Sea.     

Symbiotic diversity in marine animals: the art of harnessing chemosynthesis

Chemosynthetic symbioses between bacteria and marine invertebrates were discovered 30 years ago at hydrothermal vents on the Galapagos Rift. Remarkably, it took the discovery of these symbioses in the deep sea for scientists to realize that chemosynthetic symbioses occur worldwide in a wide range of habitats, including cold seeps, whale and wood falls, shallow-water coastal sediments and continental margins. The evolutionary success of these symbioses is evident from the wide range of animal groups that have established associations with chemosynthetic bacteria; at least seven animal phyla are known to host these symbionts. The diversity of the bacterial symbionts is equally high, and phylogenetic analyses have shown that these associations have evolved on multiple occasions by convergent evolution. This Review focuses on the diversity of chemosynthetic symbionts and their hosts, and examines the traits that have resulted in their evolutionary success.     

Archaeal Diversity and Spatial Distribution in the Surface Sediment of the South China Sea

Archaea represent a significant portion of biomass in the marine sediments and may play an important role in global carbon cycle. However, the identity and composition of deep sea sediment Archaea are unclear. Here, we used the archaeal 16S rRNA gene primers to determine the diversity and community structure of Archaea from shallow water (<100 m) and deep water (>1500 m) sediments in the South China Sea. Phylogenetically the archaeal community is separated between the shallow- and deep sea sediments, with the former being dominated by the Thaumarchaeota and the latter by the Marine Benthic Group B, E and the South African GoldMine Euryarchaeotal Group as well as Thaumarchaeota. Sand content showed significant correlation with Thaumarchaeota, suggesting that the porous media may create an oxic environment that allowed these aerobic organisms to thrive in the surface sediments. The carbon isotope composition of total organic carbon was significantly correlated to the distribution of archaeal groups, suggesting that Archaea overall may be constrained by the availability or sources of organic carbon in the sediments of the South China Sea.     

黄海海域海洋沉积物细菌多样性分析

【背景】海洋独特的环境造就了海洋生物的多样性,海洋沉积物中细菌对海洋环境具有至关重要的作用。【目的】研究陆地土壤和海洋沉积物间细菌群落相似性和差异性,以便更好地认识海洋细菌多样性,深入了解沉积物细菌在海洋环境中的潜在作用。【方法】从中国黄海海域及大连市大黑山脚下分别采集样品,以陆地土壤为对照,采用16SrRNA基因高通量测序技术分析海洋沉积物的细菌群落结构。【结果】海洋沉积物样品中芽孢杆菌纲(Bacilli)、鞘氨醇单胞菌属(Sphingomonas)和芽孢杆菌属(Bacillus)丰度高于陆地土壤样品;海洋沉积物中亚硝化单胞菌(unculturedbacterium f. Nitrosomonadaceae)和厌氧绳菌(uncultured bacterium f. Anaerolineaceae)丰度虽低于陆地土壤,但丰度值也均高于1%;样品分类学统计显示酸杆菌门(Acidobacteria)在海洋沉积物和陆地土壤样品中的序列丰度比例都较大,鞘氨醇单胞菌属(Sphingomonas)在海洋沉积物样品中的序列丰度大于陆地土壤样品。【结论】海洋沉积物细菌多样性可作为海洋环境恢复情况的重...     

中国东海和南海海域可培养烃类降解细菌的筛选及功能

【背景】海洋环境中蕴藏着丰富的微生物资源,其种类繁多而且功能多样,在驱动物质循环及能量流动等方面起着重要的作用。目前,海洋中烷烃化合物降解菌的分离筛选和降解功能研究已有文献报道;但是对海洋中尤其是我国东海和南海海域,具有降解芳香烃类化合物功能的菌株分离筛选及其多样性研究鲜有报道。【目的】分离筛选我国东海和南海海域具有烃类降解能力的可培养菌株,并对其降解功能和多样性进行初步研究。【方法】分别从东海和南海海底沉积物样品中筛选菌株,选择不同的烃类化合物为菌株筛选的唯一碳源,采用梯度稀释和平板划线法分离纯化得到单菌落,并利用相应烃类为唯一碳源进行生长验证获得该化合物降解菌。【结果】以肉桂酸、碱木素、十六烷等12种烃类化合物为唯一碳源,从样品中共分离到63株具有烃类化合物降解能力的菌株,分别属于3个门4个纲8个目10个属,主要为红球菌属(Rhodococcus)、不动杆菌属(Acinetobacter)、弧菌属(Vibrio)、盐单胞菌属(Halomonas)、假单胞菌属(Pseudomonas)。两大海域优势降解菌差别较大,其中东海沉积物降解菌株主要为不动杆菌属(Acinetobacter),而南海沉积物降解菌株主要为红球菌属(Rhodococcus)。【结论】我国东海和南海海域蕴藏着丰富的烃类化合物降解菌株资源,两大海域优势降解菌种类存在明显差异,这将为我国未来可能的海洋环境石油污染的微生物治理储备菌种资源。     

Microbial diversity in sediments associated with a shallow methane seep in the tropical Timor Sea of Australia reveals a novel aerobic methanotroph diversity

This study examined the diversity of Bacteria, Archaea and in particular aerobic methanotrophs associated with a shallow (84 m) methane seep in the tropical Timor Sea, Australia. Seepage of thermogenic methane was associated with a large carbonate hardground covered in coarse carbonate-rich sediments and various benthic organisms such as solitary corals. The diversity of Bacteria and Archaea was studied by analysis of cloned 16S rRNA genes, while aerobic methanotrophic bacteria were quantified using real-time PCR targeting the α-subunit of particulate methane monooxygenase ( pmoA ) genes and diversity was studied by analysis of cloned pmoA genes. Phylogenetic analysis of bacterial and archaeal 16S rRNA genes revealed diverse and mostly novel phylotypes related to sequences previously recovered from marine sediments. A small number of bacterial 16S rRNA gene sequences were related to aerobic methanotrophs distantly related to the genera Methylococcus and Methylocaldum . Real-time PCR targeting pmoA genes showed that the highest numbers of methanotrophs were present in surface sediments associated with the seep area. Phylogenetic analysis of pmoA sequences revealed that all phylotypes were novel and fell into two large clusters comprised of only marine sequences distantly related to the genera Methylococcus and Methylocaldum that were clearly divergent from terrestrial phylotypes. This study provides evidence for the existence of a novel microbial diversity and diverse aerobic methanotrophs that appear to constitute marine specialized lineages.