Activity and metabolic regulation of methane production in deep peat profiles of boreal bogs

The potential activity of methane production was determined in the vertical profiles of the peat deposits of three bogs in Tver oblast, which were representative of the boreal zone. In the minerotrophic fen, the rates of methane production measured throughout the profile did not change significantly with depth and comprised 3–6 ng CH₄-C g^?1 h^?1. In ombrotrophic peat bogs, the rate did not exceed 5 ng CH₄-C g^?1 h^?1 in the upper layer of the profile (up to 1.5 m) and increased to 15–30 ng CH₄-C g^?1 h^?1 in the deep layers of the peat deposits. The distribution of fermentative microorganisms and methanogens in the profiles of peat deposits was uniform in all the studied bogs. In bog water samples, the presence of butyrate (up to 14.1 mg 1^?1) and acetate (up to 2.4 mg 1^?1) was revealed throughout the whole profile; in the upper 0.5-m layer of the ombrotrophic bogs, formate (up to 8.9 mg 1^?1) and propionate (up to 0.3 mg 1^?1) were detected as well. The arrangement of local maxima of the fatty acid content and methanogenic activity in the peat deposits, as well as the decrease in the acetate concentrations during summer, support the hypothesis that the initial substrates for methanogenesis come from the upper peat layers. It was established that the addition of sulfate and nitrate inhibits methane production in peat samples; the changes in the concentrations, recorded in situ, may also influence the methane content in peat layers.     

新疆顿巴斯他乌盐湖沉积物免培养古菌多样性

【目的】了解新疆顿巴斯他乌盐湖沉积物免培养古菌组成及多样性。【方法】利用免培养法直接从顿巴斯他乌盐湖沉积物样品中提取环境总DNA,采用古菌通用引物对16S rRNA基因进行扩增,构建基因克隆文库。对随机挑选的59个阳性克隆进行HaeⅢ限制性酶切分型并测序、BLAST比对及构建16S rRNA基因系统发育树。【结果】文库覆盖率为89%,Shannon-Wiener指数为2.69,共得到21个不同的可操作分类单元,分属于广古菌门(Euryarchaeota,92%)和泉古菌门(Crenarchaeota,8%),其中多数为盐杆菌科(Halobacteriaceae,88%)的盐杆菌属(Halobacterium,24%)、盐盒菌属(Haloarcula,18%)、盐碱红菌属(Natronorubrum,14%)、盐红菌属(Halorubrum,8%)等,与海盐环境(thalassohaline)获得的16S rRNA基因序列相似性最高(﹥95%);整个文库中约11%的克隆与可培养古菌多个属的相似性小于97%。【结论】顿巴斯他乌盐湖古菌多样性略低于同类高盐环境,组成较为一致,只是各类群所占百分比稍有不同,且可能存在一些潜在新物种或新类群。     

象山港网箱养殖区沉积物的古菌空间分布

对象山港网箱养殖区及其周边沉积物中古菌群落的空间分布进行研究,应用基于16S rRNA基因的T-RFLP(末端限制性片段多态性分析)技术分析象山港网箱养殖区及其周边不同深度沉积物中古菌的群落结构和多样性,并构建克隆文库进行系统发育学分析。测定沉积物各项理化因子,通过PCA和RDA分析了古菌群落分布及其与环境因子之间的关系。结果表明,泉古菌是港口沉积物中的优势古菌群,占古菌群落的50%以上。网箱养殖区沉积物的古菌群落结构较非养殖区简单,多样性降低。非养殖区古菌群落随深度呈现有规律的变化。营养盐类和pH是造成养殖区域古菌群落结构区别于非养殖区域的主要环境因素。     

Archaeal Lipids and 16S rRNA Genes Characterizing Non-hydrate and Hydrate-Impacted Sediments in the Gulf of Mexico

This study reports the intact lipids and the phylogenetic compositions of archaea from marine sediments adjacent to or within a region of methane seeps and hydrate mounds in the Mississippi Canyon (MC) Block 118 in the Gulf of Mexico. An aliquot of lyophilized sediment (～5 g) was extracted for total lipids. Fractions of the glycerol dialkyl glycerol tetraethers (GDGTs) were obtained through column fractionation and determined using liquid-chromatography-mass spectrometry. DNA was extracted from a different aliquot of the sample (～7 g) that was kept at ?80°C. GDGTs showed distinct patterns between non-hydrate and hydrate-impacted samples, suggesting dramatically different archaeal communities caused by the presence of gas hydrates or cold seeps. Clone libraries of 16S rRNA genes were constructed to provide a phylogenetic explanation of the archaeal populations possibly causing the variation in lipid profiles. In contrast to the non-thermophilic crenarchaeota-dominant species in the normal marine sediment, the hydrate-impacted samples showed the predominance of ANME-1 subgroups with Thermoplasmatales being secondarily abundant; both of them are known to produce tetraether lipids and may be responsible for the enhanced archaeal lipids in the hydrate samples. MC 118 is designed to be a seafloor observatory in the Gulf of Mexico and our study represents the initial efforts in characterizing archaeal populations and their role in carbon cycle at this location.     

Bacterial,archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis

We studied the microbial diversity in the sediment from the Kongsfjorden, Svalbard, Arctic, in the summer of 2005 based on the analysis of 16S rRNA and 18S rRNA gene clone libraries. The sequences of the cloned 16S rRNA and 18S rRNA gene inserts were used to determine the species identity or closest relatives by comparison with sequences of known species. Compared to the other samples acquired in Arctic and Antarctic, which are different from that of ours, the microbial diversity in our sediment is much higher. The bacterial sequences were grouped into 11 major lineages of the domain Bacteria: Proteobacteria (include α-, β-, γ-, δ-, and ε-Proteobacteria); Bacteroidetes; Fusobacteria; Firmicutes; Chloroflexi; Chlamydiae; Acidobacteria; Actinobacteria; Planctomycetes; Verrucomicrobiae and Lentisphaerae. Crenarchaeota were dominant in the archaeal clones containing inserts. In addition, six groups from eukaryotes including Cercozoa, Fungi, Telonema, Stramenopiles, Alveolata, and Metazoa were identified. Remarkably, the novel group Lentisphaerae was reported in Arctic sediment at the first time. Our study suggested that Arctic sediment as a unique habitat may contain substantial microbial diversity and novel species will be discovered.     

Archaeal Community Revealed by 16s rRNA and Fluorescence in situ Hybridization in a Sulphuric Hydrothermal Hot Spring, Northern Taiwan

Summary The archaeal community composition of Yangmingshan National Park in northern Taiwan was investigated by 16S rRNA and fluorescence in situ hybridization (FISH). Optimization of tetrameric restriction enzyme (TRE) was performed to achieve efficient digestion and differentiation in the restriction fragment length polymorphism (RFLP) fragments, and AciI, BstUI and RsaI were shown to be the optimal TREs for TRE-RFLP. Nine clones were obtained in the studies, with clones M70 and M6 being found to be phylogenetically affiliated to Sulfolobus and Caldisphaera in domain Crenarchaeota, respectively, whereas seven other clones were found to be affiliated to an uncultured and unidentified archaeon isolated from thermoacidic environments. In FISH, soil and water region cells were hybridized with DAPI (4′, 6-diamidino-2-phenylindole) and specific fluorescently labelled probes. 15.69 and 7.16% of the DAPI-stained cells hybridized with universal archaeal probe ARC915 and sulphate-reducing bacterial probe SRB385, respectively.     

DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities

A protocol was devised which permitted the extraction of DNA from deep marine sediments up to 503 m below the sea floor. These sediments have been laid down over the last 3 million years. 16S rRNA gene sequences were amplified from the DNA by the polymerase chain reaction. The details of the successful extraction and polymerase chain reaction methodology varied between samples from different depths. This emphasizes the attention to detail required to allow the diversity of bacteria in these deep sediments to be studied.     

新疆两典型微咸水湖水体免培养古菌多样性

微咸水湖是湖泊演化过程中的一个重要中间状态,以新疆两典型微咸水湖-赛里木湖和柴窝堡湖水为研究对象,采用微孔滤膜收集菌体,SDS-酚-氯仿抽提法直接提取湖水总DNA,利用古菌16S rRNA基因通用引物进行PCR扩增,分别构建两湖古菌16S rRNA基因克隆文库。用限制性内切酶Hae Ⅲ对随机挑选的阳性克隆子进行酶切分型,分别得到7个和8个可操作分类单元(Operational Taxonomic Units, OTUs),两文库覆盖率均大于98%。BLAST比对和系统发育分析表明赛里木湖全部克隆子归属于泉古菌门(Crenarchaeota),97%的克隆子与不同环境免培养氨氧化泉古菌有较高的序列相似性(>97%)。柴窝堡湖水古菌归为3个门:Thaumarchaeota (81.2%)、广古菌门(Euryarchaeota)(13%)和泉古菌门(Crenarchaeota) (5.8%),81.2%的克隆子与具有氮代谢功能的氨氧化古菌纯培养物具有较高的序列相似性(97%-98%),13%的克隆子与已分离到的产甲烷古菌序列同源性大于97%。研究发现新疆微咸水湖可能存有大量新划分的古菌Thaumarchaeota门类群、可培养氨氧化及产甲烷古菌类群,两典型微咸水湖泊中古菌类群多样性较低且群落组成差异大。     

Comparative analysis of archaeal 16S rRNA and <Emphasis Type="Italic">amoA</Emphasis> genes to estimate the abundance and diversity of ammonia-oxidizing archaea in marine sediments

Considering their abundance and broad distribution, non-extremophilic Crenarchaeota are likely to play important roles in global organic and inorganic matter cycles. The diversity and abundance of archaeal 16S rRNA and putative ammonia monooxygenase alpha-subunit (amoA) genes were comparatively analyzed to study genetic potential for nitrification of ammonia-oxidizing archaea (AOA) in the surface layers (0-1 cm) of four marine sediments of the East Sea, Korea. After analysis of a 16S rRNA gene clone library, we found various archaeal groups that include the crenarchaeotal group (CG) I.1a (54.8%) and CG I.1b (5.8%), both of which are known to harbor ammonia oxidizers. Notably, the 16S rRNA gene of CG I.1b has only previously been observed in terrestrial environments. The 16S rRNA gene sequence data revealed a distinct difference in archaeal community among sites of marine sediments. Most of the obtained amoA sequences were not closely related to those of the clones retrieved from estuarine sediments and marine water columns. Furthermore, clades of unique amoA sequences were likely to cluster according to sampling sites. Using real-time PCR, quantitative analysis of amoA copy numbers showed that the copy numbers of archaeal amoA ranged from 1.1 x 10(7) to 4.9 x 10(7) per gram of sediment and were more numerous than those of bacterial amoA, with ratios ranging from 11 to 28. In conclusion, diverse CG I.1a and CG I.1b AOA inhabit surface layers of marine sediments and AOA, and especially, CG I.1a are more numerous than other ammonia-oxidizing bacteria.     

Phylogenetic Diversity of the Archaeal Component in Microbial Mats on Coral-like Structures Associated with Methane Seeps in the Black Sea

With the use of molecular ecology methods, the archaeal component of microbial mats on coral-like structures associated with methane seeps occurring at a depth of about 200 m in the Black Sea was investigated without the isolation of pure cultures. Using archaea-specific 16S rDNA–targeted oligonucleotide primes, long fragments of genes were amplified, cloned, and sequenced and their phylogenetic analysis was carried out. It was shown that archaea in microbial mats on coral-like structures are represented by two dominant phylotypes that belong to the kingdoms Crenarchaeota and Euryarchaeota and are not specifically related to any described archaeal species. The possible role of the revealed archaea in the process of anaerobic methane oxidation is discussed.     

Methanogenic communities in permafrost-affected soils of the Laptev Sea coast, Siberian Arctic, characterized by 16S rRNA gene fingerprints

Permafrost environments in the Arctic are characterized by extreme environmental conditions that demand a specific resistance from microorganisms to enable them to survive. In order to understand the carbon dynamics in the climate-sensitive Arctic permafrost environments, the activity and diversity of methanogenic communities were studied in three different permafrost soils of the Siberian Laptev Sea coast. The effect of temperature and the availability of methanogenic substrates on CH4 production was analysed. In addition, the diversity of methanogens was analysed by PCR with specific methanogenic primers and by denaturing gradient gel electrophoresis (DGGE) followed by sequencing of DGGE bands reamplified from the gel. Our results demonstrated methanogenesis with a distinct vertical profile in each investigated permafrost soil. The soils on Samoylov Island showed at least two optima of CH4 production activity, which indicated a shift in the methanogenic community from mesophilic to psychrotolerant methanogens with increasing soil depth. Furthermore, it was shown that CH4 production in permafrost soils is substrate-limited, although these soils are characterized by the accumulation of organic matter. Sequence analyses revealed a distinct diversity of methanogenic archaea affiliated to Methanomicrobiaceae, Methanosarcinaceae and Methanosaetaceae. However, a relationship between the activity and diversity of methanogens in permafrost soils could not be shown.     

Hysteresis in the temperature response of carbon dioxide and methane production in peat soils

The ability to predict the effects of climate change on trace gas fluxes requires a knowledge of microbial temperature responses. However, the response of a microbial community to temperature in a given substrate may be complicated by its thermal history. To examine the effect of sequentially changing temperature on methane and carbon dioxide production in different peat types, we incubated anaerobic peat samples from 3 types of northern peatlands, a bog, a sedge fen and a cedar swamp, in both rising and falling temperature regimes. Graphic and statistical comparisons of the different temperature regimes suggest hysteresis in microbial response to temperature, although the absolute rates at any given temperature often did not differ. Where regressions for temperature response (Arrhenius plots) were significant, they generally differed between temperature regimes. The greatest differences among treatments occurred during the first half of the 40-d incubation. Increases in carbon dioxide production were similar across all peat types, but methanogenesis varied widely: methane production was uniformly low in the bog peat but increased sharply with temperature in the other two peat types. The complicating effect of history or chronology on substrate responses to environmental stimuli may restrain our ability to model the responses of complex systems to changing conditions.     

垃圾填埋场渗滤液中古细菌群落16S rRNA基因的ARDRA分析

利用特异性的引物对,选择性扩增垃圾填埋场渗滤液中古细菌群落的18S rRNA基因片断,在此基础上建立16S rDNA克隆文库,经古细菌通用寡核苷酸探针的原位杂交筛选后,克隆文库内古细菌16S rDNA扩增片断的多样性通过ARDRA分析（amplified rDNA restriction analysis)而获得,利用PCR将各组重克隆子内的16S rDNA外源片断再扩增出来后,两种限制性内切酶－Hha I和HaeⅢ－被分别用于16S rDNA克隆片断的限制酶切分析,结果表明,随机选出的70个古细菌16S rDNA克隆片断被妥为21个不同的ARDRA型（组）,其中的两个优势型总共占了所有被分析克隆子的60％,而其余19个型的相对丰度均处于较低的水平,当中的14个型更仅含有1个克隆子,通过对16S rRNA基因的PCR扩增,克隆及其ARDRA分析,能快速地获得有关填埋场渗滤液中古细菌群落的结构及其多样性的初步信息。     

枯草菌素产生菌芽孢杆菌FB123的发酵条件优化及其16S rRNA序列分析

对芽孢杆茵FB123产枯草菌素发酵条件进行了优化并对菌株进行了16S rRNA分子鉴定.采用不同培养基配方、单因素及正交设计等试验对FBl23培养基、发酵条件进行了优化,FBl23的枯草菌素产量(透明圈直径:cm)从1.1 cm增加到1.67 cm;生物量提高了2.5倍.最佳培养基为(%):葡萄糖0.5,蛋白胨1.0,牛肉膏0.5,酵母膏0.1,pH 7.5;培养条件:培养温度28℃,培养时间32 h.进一步克隆测定了该茵16S rRNA基因序列,系统进化树分析表明该茵与枯草芽孢杆菌(Bacillus subtilis)具有最紧密亲缘关系.     

新疆艾比湖和伊吾湖可培养嗜盐古菌多样性

新疆地区盐湖密布,蕴藏着丰富的微生物资源。为保护和利用微生物物种与基因资源,作者从新疆准噶尔盆地的艾比湖和天山山间盆地的伊吾湖分离纯化嗜盐微生物。采用PCR方法扩增其中65株嗜盐古菌16SrRNA基因序列。序列分析表明,分离的嗜盐古菌分属6个属,艾比湖以Haloterrigena和Natrinema属的菌株为主,伊吾湖由Haloarcula和Halorubrum两个属的菌株构成。通过多样性指数、丰富度指数、均匀度指数和物种相对多度模型对分离的菌株进行多样性分析和比较,结果表明,盐湖嗜盐古菌的多样性指数、丰富度指数和均匀度指数具有一定相关性,艾比湖可培养嗜盐古菌的多样性高于伊吾湖。研究发现了一些新的物种资源,表明新疆盐湖中孕育的特色微生物资源亟待保护与利用。     

Effect of sample handling on estimation of bacterial diversity in marine sediments by 16S rRNA gene sequence analysis

Abstract The diversity of bacterial communities in deep marine sediments, up to 503 metres below the sea floor of the Japan Sea, was investigated by sequence analysis of amplified 16S rRNA genes. The use of different sample handling procedures greatly affected the types and diversity of sequences obtained. DNA from sediment samples stored aerobically for up to 24 h before freezing was dominated by sequences belonging to the β- and γ-proteobacteria, many of which appeared to originate from aerobic bacteria. Sub-samples equilibrated anaerobically at 16°C, were then injected with a radiotracer and immediately frozen, to simulate the conditions of a typical control sample from a radiotracer based activity assay, contained mostly α-proteobacterial sequences. Pristine sediment samples taken anaerobically and frozen within 2 h contained the widest diversity of sequences from α-, γ-, δ-proteobacteria and Gram-positive bacteria, which appeared to have originated from predominantly anaerobic or facultative bacteria. It was clear that both samples that were not frozen immediately (within 2 h) showed signs of enrichment of specific bacterial groups. Our results strongly suggest that immediate freezing should always be employed when sediment samples are to be used to assess bacterial diversity by molecular methods.     

Activity and metabolic regulation of methane production in deep peat profiles of boreal bogs

The potential activity of methane production was determined in the vertical profiles of the peat deposits of three bogs in Tver oblast, which were representative of the boreal zone. In the minerotrophic fen, the rates of methane production measured throughout the profile did not change significantly with depth and comprised 3-6 ng CH4-C g(-1) h(-1). In ombrotrophic peat bogs, the rate did not exceed 5 ng CH4-C g(-1) h(-1) in the upper layer of the profile (up to 1.5 m) and increased to 15-30 ng CH4-C g(-1) h(-1) in the deep layers of the peat deposits. The distribution of fermentative microorganisms and methanogens in the profiles of peat deposits was uniform in all the studied bogs. In bog water samples, the presence of butyrate (up to 14.1 mg l(-1)) and acetate (up to 2.4 mg l(-1)) was revealed throughout the whole profile; in the upper 0.5-m layer of the ombrotrophic bogs, formate (up to 8.9 mg l(-1)) and propionate (up to 0.3 mg l(-1)) were detected as well. The arrangement of local maxima of the fatty acid content and methanogenic activity in the peat deposits, as well as the decrease in the acetate concentrations during summer, support the hypothesis that the initial substrates for methanogenesis come from the upper peat layers. It was established that the addition of sulfate and nitrate inhibits methane production in peat samples: the changes in the concentrations, recorded in situ, may also influence the methane content in peat layers.     

双歧杆菌地高辛标记寡核苷酸基因探针制备和应用研究

目的探讨地高辛标记寡核苷酸基因探针应用于微生态研究的可行性和实用性。方法制备双歧杆菌属和部分种的地高辛标记16S rRNA寡核苷酸探针,初步应用于微生态制剂鉴定和临床肠道微生态检测,评价寡核苷酸探针杂交在肠道微生态研究和检测中的应用价值。结果地高辛标记寡核苷酸探针具有较好的特异性与灵敏度：地高辛标记的双歧杆菌属和种的共6种寡核苷酸基因探针与标准菌株杂交后灵敏度和特异度分别为属探针95％、75％,青春双歧87．5％、90％,两歧双歧87．5％、87．5％,短双歧87．5％、92．5％,婴儿双歧75％、95％,长双歧75％、100％。结论寡核苷酸基因探针用于肠道细菌的鉴定显示出一定前景,加大探针的种类与扩大调查范围有可能使该技术替代现有细菌培养技术。     

Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP analysis of 16S rRNA genes and functional genes over the last 10 years and evaluate the performance of this technique when used in conjunction with different statistical methods. Web-based tools designed to perform virtual polymerase chain reaction and restriction enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysis. Significant improvements have also been made in the statistical analysis of T-RFLP profiles such as the introduction of objective procedures to distinguish between signal and noise, the alignment of T-RFLP peaks between profiles, and the use of multivariate statistical methods to detect changes in the structure and composition of microbial communities due to spatial and temporal variation or treatment effects. The progress made in T-RFLP analysis of 16S rRNA and genes allows researchers to make methodological and statistical choices appropriate for the hypotheses of their studies.     

人体肠道微生物多样性和功能研究进展

人体肠道中庞大而复杂的微生物群落对人体自身代谢表型有深远的影响.肠道微生物群落在亚种或菌株水平上表现出极大的多样性.利用微生物分子生态学、元基因组学和代谢组学研究方法,发现肠道微生物与宿主表现出共进化的特点,肠道微生物群落及其基因组为宿主提供了互补的遗传和代谢功能,表现出互惠共生关系.但是,肠道微生物群落中影响宿主代谢表型的关键功能菌鉴定及其作用模式问题仍然悬而未决,综合运用多种高通量研究方法和多维数据分析方法可能成为解决这个问题的突破口.