新疆低阶煤本源生物甲烷转化中微生物群落组成及变化

【目的】探究新疆低阶煤生物甲烷转化过程微生物群落组成及多样性。【方法】采用厌氧培养方法和末端限制性片段长度多态性技术(Terminal restriction fragment length polymorphism,T-RFLP)分析新疆低阶煤本源微生物对甲烷转化及有机酸含量的影响,分析新疆哈密大南湖长焰煤生物甲烷转化过程中微生物群落动态变化。【结果】研究表明长焰煤和褐煤对本源微生物产甲烷影响较小,随着低阶煤生物甲烷转化时间的延长,甲烷产量呈上升趋势,转化60 d后长焰煤甲烷产量高达10.28 m L/g,挥发性有机酸(VFA)浓度则最低;微生物多样性指数变化不明显,不同转化时间微生物主要类群为放线菌门(Actinobacteria),拟杆菌门(Bacteroidetes),厚壁菌门(Firmicutes),变形菌门(Proteobacteria);甲烷菌的群落结构相对于细菌较简单,在整个低阶煤生物转化产甲烷过程中共有古菌类群为甲烷八叠球菌属(Methanosarcina)、甲烷盐菌属(Methanohalobium)、甲烷叶菌属(Methanolobus)、甲烷食甲基菌属(Methanomethylovorans),它们是构成群落结构的基本菌群。【结论】低阶煤生物甲烷转化过程微生物群落具有丰富的多样性,且不同时期多样性有较大差异。甲烷菌群落结构相对于细菌较简单,共有类群明显。     

青海湖与中国内陆盐湖古菌群落组成的比较

【目的】本研究旨在全面揭示中国最大的内陆咸水湖——青海湖的古菌群落结构,并对青海湖与盐湖的古菌多样性及群落结构进行比较。【方法】随机选取了青海省的茶卡盐湖、陕西省的花马池盐湖和苟池盐湖以及山西省的运城盐湖作为盐湖组。青海湖与盐湖组每个湖泊各采取5个样品,采用针对16S rRNA基因的高通量测序技术分析5个湖泊中的古菌群落组成。【结果】研究发现,青海湖的优势菌群为DHVEG-6_norank、Methanomicrobia_unclassified、Methanobacterium(甲烷细菌属)、Methanolobus(甲烷叶菌属)、Candidatus_Methanomethylophilus、Miscellaneous_Euryarchaeotic_Group(MEG)_norank、AMOS1A-4113-D04_norank、Methanosarcina(甲烷八叠球菌属)、Miscellaneous_Crenarchaeotic_Group_norank。其中,DHVEG-6_norank(70.46%)占绝对优势,但该类群在盐湖中含量极少。4个盐湖的共有优势属为Halonotius、Halorubrum(盐红菌属)、Natronomonas(嗜盐碱单孢菌属)、Halobellus和Haloarcula(盐盒菌属)。对于青海湖与盐湖之间的古菌群落多样性,影响最大的因素为湖水的矿化度,矿化度与5个湖泊的古菌多样性呈负相关,矿化度较低的青海湖群落组成与其他4个盐湖差异显著,无共同优势菌;其次为pH,pH与湖泊中古菌群落多样性呈微弱正相关,小幅度影响到某些菌属的丰度;而本文研究范围内的海拔与其群落结构及多样性没有明显相关性。【结论】青海湖与其他4个盐湖之间的群落结构及多样性有显著差异,矿化度对古菌群落多样性具有显著影响。另,本次测序发现5个湖泊中均有大量未分类的古菌,应为潜在的新种。     

浓香型白酒两个产区窖泥微生物群落结构分析

【目的】探索浓香型白酒两个典型产区窖泥微生物群落结构和多样性,分析窖泥微生物群落地域特征及对白酒风格形成的影响。【方法】分别提取四川和安徽两个产区窖泥样品总DNA,应用PCR-ARDRA和16S rRNA基因克隆测序技术对两个产区窖泥细菌和古菌进行研究。【结果】两个浓香型白酒产区窖泥细菌丰富,包括:厚壁菌门(Firmicute)、拟杆菌门(Bacteroidetes)、绿弯菌门(Chloroflexi)、互养菌门(Synergistetes)、Armatimonadetes类群和未分类细菌(Unclassified bacteria)。两个产区窖泥绝对优势种群均为厚壁菌门中梭菌纲(Clostridia)细菌,在四川产区窖泥中检出较多的互营单胞菌属(Synthrophomonas)和紫单胞菌属(Petrimonas)。古菌的群落组成较为简单,主要是甲烷囊菌属(Methanoculleus)、甲烷八叠球菌属(Methanosarcina)、甲烷鬃菌属(Methanosaeta)和甲烷杆菌属(Methanobacterium)4个产甲烷古菌类群,四川产区窖泥优势古菌为甲烷囊菌和甲烷八叠球菌,安徽产区则为甲烷八叠球菌属和甲烷鬃菌。【结论】四川和安徽两个产区窖泥微生物的16S rRNA基因克隆文库系统地反映了两者微生物群落的相似性和差异性,对揭示浓香型白酒两个产区的酒体风格差异形成有一定的参考价值。     

我国8个典型水稻土中产甲烷古菌群落组成的空间分异特征

【目的】产甲烷古菌主导稻田甲烷生成,是稻田生态系统的模式微生物类群之一,具有重要的生态学意义。然而,水稻土产甲烷古菌群落组成的空间分异却鲜有报告。【方法】本研究沿20.55°N至47.43°N梯度,采集了我国不同纬度上8个典型水稻土,利用PCR-DGGE指纹图谱和系统发育树分析揭示不同地点水稻土中产甲烷古菌群落的组成;结合多个环境因子,利用生物信息学,典范对应分析(Canonical Correspondence Analysis,CCA)和维恩图(Venn diagram)明确产甲烷古菌的空间分异规律。【结果】研究发现p H值是驱动水稻土中产甲烷古菌群落组成分异的主要因子;此外,沿纬度梯度,8个地区的产甲烷古菌群落组成也呈现出规律性变化。【结论】本研究首次阐明了稻田中产甲烷古菌群落分布情况,并揭示其主要驱动因子。该认知不仅有助于我们更好地了解产甲烷古菌的生物地理学分布,还有助于从微生物学机制上阐明我国温度梯度带上有机质转化空间的差异。     

不同风化年限碳酸岩风化壳真菌群落结构特征

【目的】岩生真菌是促进碳酸岩生物风化的重要推动者,研究黔中典型喀斯特区不同风化年限碳酸岩风化壳真菌群落结构特征有利于了解真菌对岩石的风化作用。【方法】选取位于黔中花溪区南部的废弃碳酸岩墓碑为调查对象,对不同风化年限碳酸岩风化壳进行采样,运用宏基因组测序方法对风化壳样品进行基因测序,并利用统计学方法对真菌群落结构特征及功能特征进行分析。【结果】18个风化壳样品中共获得1087种真菌,分属于9个门、44纲、538属。不同样本之间真菌群落数量和组成差异较大,在碳酸岩风化过程中,子囊菌门(Ascomycota)始终为优势类群,平均相对丰度达到95%以上,随风化年限的增加呈现显著下降的趋势。Shannon指数和Simpson指数显示,碳酸岩风化壳真菌群落多样性随风化年限的增加呈现先减小后增加再减小的趋势。从所有样本中共检测出3 379 478个KEGG pathway level 3通路相关基因,主要与物质能量的代谢、运输等功能相关。与碳循环、氮循环和硫循环相关的主要微生物属于子囊菌门,随着风化年限的增加呈现下降的趋势。冗余分析(redundancyanalysis,RDA)结果表明,三氧化二铁...     

基于高通量测序研究青藏高原茶卡盐湖微生物多样性

【目的】茶卡盐湖(Chaka Salt Lake,CSL)是青藏高原有名的天然结晶盐湖,具有独特的石盐盐湖矿床,盛产青盐。盐湖卤水环境中存在丰富的嗜盐菌资源和潜在的新种,细菌和古菌的群落结构特征和物种多样性尚不明确。【方法】采用Illumina高通量测序平台对茶卡盐湖水样和底泥混合物中的细菌和古菌群落进行16S r RNA基因(V3-V5区)高通量测序,检测4个样本的群落结构差异和微生物多样性。【结果】获得细菌和古菌总有效序列分别为117 192和110 571条。结果分析表明细菌和古菌的物种注释(Operational taxonomic unit,OTU)数目分别为421和317,获得分类地位明确的细菌种类为14门28纲170属,古菌为5门4纲34属。细菌的优势类群是厚壁菌门(Firmicutes),所占比例为68.37%,其次为变形菌门Proteobacteria(20.49%);优势种属依次为芽孢杆菌属Bacillus(41.94%)、海洋芽孢杆菌属Oceanobacillus(8.03%)、假单胞菌属Pseudomonas(7.67%)、盐厌氧菌属Halanaerobium(7.42%)和乳球菌属Lactococcus(7.38%);古菌的优势类群以广古菌门(Euryarchaeota)盐杆菌纲(Halobacteria)为主,优势菌是Halonotius(17.21%)和盐红菌属Halorubrum(16.23%)。【结论】揭示了茶卡盐湖中细菌和古菌的群落结构及物种多样性,为嗜盐菌的开发及后续微生物资源的挖掘提供了理论依据。     

褐煤强化产甲烷菌群的群落分析及条件优化

【目的】以白音华褐煤为底物,利用从我国多地煤矿及污水处理厌氧罐中富集-混合-驯化得到的高效混合菌群进行产气,分析其群落组成并优化产气条件。【方法】采用Miseq高通量测序分析混合菌群结构,通过Plackett-Burman(P-B)和Box-Behnken(B-B)试验对褐煤产气影响因素和条件进行筛选和优化。【结果】本源和外源微生物样本混合样品(HN+MD+WT)经驯化后菌群产气效率最高。该样品菌群中细菌群落多样性丰富,以变形菌门的脱硫弧菌属Desulfovibrio(15.07%)、拟杆菌门的屠场杆状菌属Macellibacteroides(14.6%)、厚壁菌门的梭菌属Clostridiaceae(9.77%)、互营菌门的脱硫代硫酸盐弧菌属Dethiosulfovibrio(8.76%)以及热袍菌门Oceanotoga属(8.66%)为主。古菌全部为广古菌门,其群落多样性则较为单一,其中甲烷卵圆形菌属Methanocalculus(80.28%)占据绝对优势。Plackett-Burman(P-B)试验结果表明温度、CoCl_2添加量和NiCl_2添加量是影响褐煤产气的关键因素;Box-Behnken(B-B)试验结果表明最优产气条件为:温度36°C,CoCl_2添加量0.17 g/L,NiCl_2添加量0.02 g/L,最优条件下褐煤累计产甲烷量(周期20 d)达到159.33μmol/g。【结论】经过驯化可以得到高效的产气菌群,优化培养条件可使产气效率明显提高。     

青海湖河源湿地甲烷氧化菌群落对温度升高的响应

甲烷氧化菌对甲烷的氧化能力显著影响全球甲烷排放总量。研究河源湿地甲烷氧化菌群落对温度升高的响应有助于深入探究河源湿地生态系统的甲烷循环机制。采用高通量测序方法研究了温度升高后青海湖河源湿地甲烷氧化菌群落特征的变化。青海湖河源湿地甲烷氧化菌的优势菌门为变形菌门, 增温处理没有显著影响河源湿地甲烷氧化菌的群落多样性。河源湿地甲烷氧化菌的群落结构对温度升高响应明显, 增温显著增加了属水平优势菌群(相对丰度 > 0.1%)的相对丰度。LEfSe分析表明, 增温处理组与自然对照组共存在23个差异菌群, 增温显著增加了甲基球菌属的相对丰度, 显著降低了甲基杆菌属的相对丰度。河源湿地甲烷氧化菌群落功能与碳、氮代谢过程密切相关。整体而言, 河源湿地甲烷氧化菌的群落结构对温度升高较为敏感, 部分菌群的相对丰度变化显著。     

寺河矿煤地质产甲烷微生物菌群的保藏和产甲烷性能

【背景】煤地质产甲烷微生物菌群可以代谢煤基质产生甲烷,对于实现煤层气资源的再利用具有重要意义。【目的】检测产甲烷菌群在保藏过程中群落结构的动态变化以及在产气实验中甲烷气的生成情况,以验证保藏方法的可行性,同时为煤层气的微生物增产奠定基础。【方法】分别于不同温度条件下比较3种菌种保藏方法,即甘油/L-半胱氨酸法、富营养法和煤基-基础盐法。通过产气实验检测不同保藏条件下产甲烷菌群的活力。同时,采用454高通量测序技术测定16S r RNA基因序列,分析25°C条件下煤基-基础盐菌种保藏过程中微生物群落结构的变化。【结果】比较了9组菌种保藏方法,发现菌种最佳保藏条件为25°C的煤基-基础盐保藏。在该条件下保藏的产甲烷菌群活性最高,甲烷生成量最大。以无烟煤为碳源进行产气实验时甲烷生成量为12%-25%,而以褐煤为碳源时甲烷生成量可达24%-73%。在25°C的煤基-基础盐菌种保藏条件下,保藏初期细菌的主要优势菌为假单胞菌属(Pseudomonas),而古菌的主要优势菌为甲烷八叠球菌属(Methanosarcina)。随着保藏时间的增加,细菌的群落结构变化显著,发酵细菌及产氢产乙酸细菌成为优势细菌,古菌的群落结构则相对稳定。【结论】菌种保藏的最佳条件为25°C的煤基-基础盐,保藏的产甲烷菌群能长期维持在较高的活性状态,具有较好的产甲烷能力。     

基于mcrA基因的沁水盆地煤层气田产甲烷菌群与途径分析

【目的】分析沁水盆地煤层气田不同煤层气井产出水样中产甲烷菌群和生物成因气的生成途径。【方法】以甲基辅酶M还原酶基因(mcr A)作为目标基因,采用454焦磷酸高通量测序方法,同时比对NCBI功能基因文库中的mcr A序列,分析不同煤层气井产出水中的产甲烷菌群。【结果】高通量测序表明,5个出水样产甲烷菌群OTUs(Operational taxonomic units)数为64–157个,共有的为22个,各占样品总数14%-34%;样品共检测到4种已知菌属,即甲烷杆菌属(Methanobacterium)、甲烷微菌属(Methanomicrobium)、甲烷叶菌属(Methanolobus)和甲烷螺菌属(Methanospirillum),优势菌属均为Methanobacterium。系统发育分析表明,未明确地位的菌属主要与Methanobacterium、Methanomicrobium、产甲烷球菌属(Methanococcus)和甲烷囊菌属(Methanoculleus)有较近的亲缘关系。5个样品中菌属所占比例不同,检测到的菌属类别大致相同。所有检测样品生物成因煤层气(Coalbed methane,CBM)的生成途径主要为氢营养型产甲烷途径。【结论】沁水盆地不同煤层气田产甲烷菌群菌种差异比较大,但生物成因气生成途径基本相似,与地理位置和煤藏条件没有相关性。     

河南卢氏和江西宜春两处锂矿山地表微生物群落分布特征差异及成因分析

[目的] 揭示地表锂矿石表面和风化产物中细菌群落多样性特征。[方法] 针对细菌16S rRNA片段扩增进行高通量测序,分析不同锂矿石表面及其风化产物中细菌群落组成、多样性及功能属性等信息。[结果] 河南卢氏南阳山伟晶岩型锂矿石和江西宜春花岗岩型锂矿石表面及其风化产物的细菌群落多样性有差异。南阳山伟晶岩矿石与其风化产物、宜春花岗岩矿石表面和风化产物（NK-1、NK-1F、YK-1、YK-1F、YK-2、YK-2F、YK-3）的OTUs分别是1010、540、835、828、1117、974和604,其差异与不同的矿物组成显著关联。两矿山均有其优势微生物,在门水平上,两矿山均以放线菌门（Actinobacteria）、变形菌门（Proteobacteria）为优势菌门。同时两矿区微生物群落组成具有显著差异性（P<0.05）,不同地理位置风化产物样本之间差异尤为显著（P<0.001）;在属水平上,NK-1中相对丰度大于5%的属为鞘氨醇单胞菌属（Sphingomonas）、马赛菌属（Massilia）;NK-1F为类芽孢杆菌属（Paenibacillus）、杆状细菌属（Bacillus）、马赛菌属（Massilia）;YK-1F为芽球菌属（Blastococcus）、念珠菌固体杆菌属（Candidatus-Solibacter）、Noviherbaspirillum属、伯克霍尔德氏菌属（Burkholderia-Caballeronia-Paraburkholderia）,YK-2为unidentified-Chloroplast属,YK-2F为北里孢菌属（Kitasatospora）,YK-3为1174-901-12属、甲基杆菌属（Methylobacterium）。不同地理位置的矿石及其风化物样本的功能注释均涉及代谢、遗传信息处理、环境信息处理等6个代谢通路。[结论] 16S rRNA高通量测序揭示不同地区锂矿石及其风化产物的细菌多样性存在差异,各具优势类群,样本间菌落组成、多样性及功能属性的差异与锂矿石化学组成、风化程度和地理分布密切相关。这项研究揭示了优势微生物类群的元素地球化学功能与含锂矿物地表风化的潜在联系,可为微生物生态分布研究及相关微生物资源开发提供新数据。     

Phylum-Level Archaeal Distributions in the Sediments of Chinese Lakes With a Large Range of Salinity

Uncovering microbial diversity and their influencing factors is a primary goal for microbial ecology. In comparison with studies on bacterial diversity, limited is known about archaeal diversity and its response to influencing factors in lakes. Here, we investigated the archaeal community compositions (ACCs) and their correlation with spatial/environmental factors in the sediments from 38 Chinese lakes with a large range of salinity (0.2–363.1 g/l) and pairwise geographic distance (3–3656 km). Illumina-Miseq sequencing was employed to characterize the ACCs in the lakes samples. The results showed that Euryarchaeota, Bathyarchaeota, Thaumarchaeota, and Woesearchaeota were the dominant archaeal phyla in the studied samples, and they each can occur in the samples with a wide range of salinity (0.2–363.1 g/l) although their abundance was relatively low (<1%) in certain samples. The Thaumarchaeota and Woesearchaeota phyla dominated (up to 90% of total sequences) some lake sediments. Mantel test indicated that compositions of total archaeal community and the Euryarchaeota and Woesearchaeota populations were significantly (p < 0.05) correlated with geographic distance in the studied lake sediments. Salinity was the most important environmental factor influencing the compositions of the total archaeal community and the Euryarchaeota population, while it did not show significant influence on the distribution of the Woesearchaeota and Thaumarchaeota populations. Taken together, this survey expands our current knowledge on the ecology of lacustrine archaea and give clues for studying the archael role in biogeochemical cycles in lakes.     

艾比湖湿地芦苇根际土壤氨氧化古菌的多样性和群落结构

【目的】旨在揭示耐盐植物芦苇根际与非根际土壤AOA群落结构间的差异,为深入研究盐生植物根际土壤微生物与耐盐性之间的关系提供理论基础。【方法】应用高通量测序技术以氨单加氧酶基因(amoA)为分子标记,对新疆艾比湖湿地荒漠生态系统不同季节(春、夏、秋)芦苇根际与非根际土壤氨氧化古菌(AOA)的多样性和群落结构进行研究。【结果】结果表明,不同季节芦苇根际土壤AOA多样性和丰富度存在差异,相比非根际土壤,夏季和秋季芦苇根际土壤AOA多样性较低丰富度较高,春季多样性较高丰富度较低。芦苇根际土壤中AOA的多样性为春季夏季秋季。AOA群落组成分析表明,土壤样品中AOA群落主要集中在泉古菌门(Crenarchaeota)和奇古菌门(Thaumarchaeota),其中泉古菌门为主要优势菌门。RDA分析表明,含水量(SM)、有机质(SOM)、总氮(TN)和pH是影响芦苇根际土壤AOA群落多样性和丰富度的主要环境因子。【结论】不同季节芦苇根际土壤AOA多样性及丰富度存在差异,相比非根际土壤,芦苇根际土壤AOA更丰富。     

贵州典型煤矸石堆场微生物群落结构及功能特征

【背景】煤矸石堆场用于堆放煤矿开采过程中产生的一种热值低、含重金属的固体废弃物,会产生大量的酸性废水,对堆场周边的生态环境造成严重影响。【目的】探究煤矸石堆场的微生物群落结构和功能特征。【方法】选择贵州省六枝特区典型煤矸石堆场作为研究对象,采集堆场表层土壤、矸石层土壤、废水浸出口沉积物和堆场下游河道沉积物,通过宏基因组学技术进行分析。【结果】细菌的优势菌门为变形菌门(Proteobacteria)和放线菌门(Actinobacteria),优势菌属为钩端螺菌属(Leptospirillum)和硫化杆菌属(Sulfobacillus);古菌的优势菌门为Candidatus＿Thermoplasmatota和泉古菌门(Crenarchaeota),优势菌属为热原体属(Thermoplasma)和金属球菌属(Metallosphaera)。不同采样点细菌和古菌的优势菌属存在显著差异,矸石层土壤和废水浸出口沉积物中的铁氧化细菌和硫氧化细菌比其他2个样点丰富。煤矸石堆场中微生物的碳、氮、硫代谢基因丰度较高,共检测到6条固碳途径、6条氮代谢途径和3条硫代谢途径。主要的固碳基因为ACAT和E2.2....     

New Undescribed Lineages of Non-extremophilic Archaea Form a Homogeneous and Dominant Element Within Alpine Moonmilk Microbiomes

The moonmilk deposits within the alpine Hundsalm cave in Austria offered the opportunity to investigate anthropogenically uninfluenced microbiomes. Via cultivation experiments we were able to show that the communities were cold-adapted and oligotrophic. Combined qPCR, DGGE, cloning and sequencing data further highlighted that the archaeal community basically comprises a low number of species, though highly abundant. These organisms are assumed to form new lineages within the Euryarchaeota, while the detected Thaumarchaeota, closely related to ammonium oxidizers, form a second, but minor, abundant group within the moonmilk deposits. Moreover, in terms of abundance the archaeal community clearly outnumbers bacteria (e.g., genera Pseudomonas, Flavobacterium and Rhodococcus) and fungi within the investigated microbiomes. In contrast to the highly complex bacterial and fungal communities, only a low number of archaeal species form a constant and essential element within the moonmilk speleothems and other cave-internal habitats.     

Vertical profiles of microbial communities in perfluoroalkyl substance-contaminated soils

Poly- and perfluoroalkyl compounds (PFASs) are ubiquitous in the environment, but their influences on microbial community remain poorly known. The present study investigated the depth-related changes of archaeal and bacterial communities in PFAS-contaminated soils. The abundance and structure of microbial community were characterized using quantitative PCR and high-throughput sequencing, respectively. Microbial abundance changed considerably with soil depth. The richness and diversity of both bacterial and archaeal communities increased with soil depth. At each depth, bacterial community was more abundant and had higher richness and diversity than archaeal community. The structure of either bacterial or archaeal community displayed distinct vertical variations. Moreover, a higher content of perfluorooctane sulfonate (PFOS) could have a negative impact on bacterial richness and diversity. The rise of soil organic carbon content could increase bacterial abundance but lower the richness and diversity of both bacterial and archaeal communities. In addition, Proteobacteria, Actinobacteria, Chloroflexi, Cyanobacteria, and Acidobacteria were the major bacterial groups, while Thaumarchaeota, Euryarchaeota, and unclassified Archaea dominated in soil archaeal communities. PFASs could influence soil microbial community.     

柴达木盆地尕斯库勒盐湖区邻近水体对湖泊微生物群落的贡献

【目的】探究尕斯库勒盐湖生态系统中邻近水体对湖泊微生物的贡献。【方法】采集尕斯库勒盐湖区湖水、沉积物以及邻近的泉水、河水和盐田的水样,对其进行地球化学分析;通过16S rRNA基因的Illumina MiSeq高通量测序分析样品的微生物群落组成。【结果】尕斯库勒盐湖区水体和沉积物中的优势门是变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)和广古菌门(Euryarchaeota)。盐度和pH是影响尕斯库勒盐湖区群落组成的最主要环境因素。邻近水体对湖泊水体和沉积物的贡献分别为12.94%和7.53%。【结论】邻近水体对尕斯库勒盐湖微生物群落的贡献有限。     

Diversity of Archaea in Bottom Sediments of the Discharge Areas With Oil- and Gas-Bearing Fluids in Lake Baikal

Using massively parallel sequencing (the Roche 454 platform) we have studied the diversity of archaeal 16S rRNA gene sequences in oxic and anoxic sediments at six sites in Lake Baikal with oil- and gas-bearing fluids discharge. Archaeal communities appeared to be represented mainly by five phyla: Euryarchaeota, Crenarchaeota, Thaumarchaeota, Bathyarchaeota (miscellaneous Crenarchaeotic group), and Woesearchaeota (deep sea hydrothermal vent group 6). Among them we detected sequences of methanogens of the orders Methanomicrobiales, Methanosarsinales, Methanococcales, as well as representatives of the following uncultured archaeal lineages: Group C3, Marine Benthic Group D, and Terrestrial Miscellaneous Group. We have also identified sequences of ammonia-oxidizing archaea of the phyla Crenarchaeota and Thaumarchaeota. Phylogenetic analysis showed the presence ANME-2d-related sequences. However, the analysis of mcrA genes libraries has not revealed typical representatives of ANME groups. Comparison of amplicon libraries 16S rRNA gene fragments from different samples proved the widespread presence of previously detected Baikal archaeal lineages, which are members of the phylum Crenarchaeota and Thaumarchaeota (formerly Group C3 of Crenarchaeota).     

Novel primers for 16S rRNA-based archaeal community analyses in environmental samples

Next generation sequencing technologies for in depth analyses of complex microbial communities rely on rational primer design based on up-to-date reference databases. Most of the 16S rRNA-gene based analyses of environmental Archaea community composition use PCR primers developed from small data sets several years ago, making an update long overdue. Here we present a new set of archaeal primers targeting the 16S rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340F-1000R showed a high archaeal specificity (< 1% bacteria amplification) covering 93 and 97% of available sequences for Crenarchaeota and Euryarchaeota respectively. In silico tests of the primers revealed at least 38% higher coverage for Archaea compared to other commonly used primers. Empirical tests with clone libraries confirmed the high specificity of the primer pair to Archaea in three biomes: surface waters in the Arctic Ocean, the pelagic zone of a temperate lake and a methanogenic bioreactor. The clone libraries featured both Euryarchaeota and Crenarchaeota in variable proportions and revealed dramatic differences in the archaeal community composition and minimal phylogenetic overlap between samples.