Detection of fatty acids from intact microorganisms by molecular beam static secondary ion mass spectrometry

We report the use of a surface analysis approach, static secondary ion mass spectrometry (SIMS) equipped with a molecular (ReO(4)(-)) ion primary beam, to analyze the surface of intact microbial cells. SIMS spectra of 28 microorganisms were compared to fatty acid profiles determined by gas chromatographic analysis of transesterfied fatty acids extracted from the same organisms. The results indicate that surface bombardment using the molecular primary beam cleaved the ester linkage characteristic of bacteria at the glycerophosphate backbone of the phospholipid components of the cell membrane. This cleavage enables direct detection of the fatty acid conjugate base of intact microorganisms by static SIMS. The limit of detection for this approach is approximately 10(7) bacterial cells/cm(2). Multivariate statistical methods were applied in a graded approach to the SIMS microbial data. The results showed that the full data set could initially be statistically grouped based upon major differences in biochemical composition of the cell wall. The gram-positive bacteria were further statistically analyzed, followed by final analysis of a specific bacterial genus that was successfully grouped by species. Additionally, the use of SIMS to detect microbes on mineral surfaces is demonstrated by an analysis of Shewanella oneidensis on crushed hematite. The results of this study provide evidence for the potential of static SIMS to rapidly detect bacterial species based on ion fragments originating from cell membrane lipids directly from sample surfaces.     

A meta-analysis of changes in bacterial and archaeal communities with time

Ecologists have long studied the temporal dynamics of plant and animal communities with much less attention paid to the temporal dynamics exhibited by microbial communities. As a result, we do not know if overarching temporal trends exist for microbial communities or if changes in microbial communities are generally predictable with time. Using microbial time series assessed via high-throughput sequencing, we conducted a meta-analysis of temporal dynamics in microbial communities, including 76 sites representing air, aquatic, soil, brewery wastewater treatment, human- and plant-associated microbial biomes. We found that temporal variability in both within- and between-community diversity was consistent among microbial communities from similar environments. Community structure changed systematically with time in less than half of the cases, and the highest rates of change were observed within ranges of 1 day to 1 month for all communities examined. Microbial communities exhibited species–time relationships (STRs), which describe the accumulation of new taxa to a community, similar to those observed previously for plant and animal communities, suggesting that STRs are remarkably consistent across a broad range of taxa. These results highlight that a continued integration of microbial ecology into the broader field of ecology will provide new insight into the temporal patterns of microbial and ‘macro''-bial communities alike.     

Analysis of archaeal core ether lipids using Time of Flight–Secondary Ion Mass Spectrometry (ToF-SIMS): Exploring a new prospect for the study of biomarkers in geobiology

The capability of Time of Flight–Secondary Ion Mass Spectrometry (ToF‐SIMS) of analysing molecular archaeal biomarkers in geobiological samples was tested and demonstrated. Using a bismuth cluster primary ion source, isopranyl glycerol di‐ and tetraether core lipids were detected in small amounts of total organic extracts from methanotrophic microbial mats, simultaneously and without further chemical treatment and chromatographic separation. ToF‐SIMS was also employed to track the distribution of fossilized ether lipids in a massive carbonate (aragonite) microbialite that precipitated as a result of the microbial anaerobic oxidation of methane. An unambiguous signal was obtained when analysing a freshly broken rock surface (base of a microdrill core). Though some limitation occurred due to µm‐topographical effects (sample roughness), it was possible to display the abundance of high molecular weight (C₈₆) of tetraethers exposed in particular regions of the rock surface. ‘Molecular mapping’ revealed that a part of these molecules was encased within the rock fabric in a cluster‐like distribution that might trace the arrangement of the calcifying microbial colonies in the once active mat system. The results reveal promising perspectives of ToF‐SIMS for (i) the quasi‐nondestructive analysis of lipids in extremely small geobiological samples at low concentrations; (ii) resolving the spatial distribution of these compounds on a µm²‐ to cm²‐scale; and (iii) the more exact assignment of lipid biomarkers to their biological source.     

Light exposure mediates circadian rhythms of rhizosphere microbial communities

Microbial community circadian rhythms have a broad influence on host health and even though light-induced environmental fluctuations could regulate microbial communities, the contribution of light to the circadian rhythms of rhizosphere microbial communities has received little attention. To address this gap, we monitored diel changes in the microbial communities in rice (Oryza sativa L.) rhizosphere soil under light–dark and constant dark regimes, identifying microbes with circadian rhythms caused by light exposure and microbial circadian clocks, respectively. While rhizosphere microbial communities displayed circadian rhythms under light–dark and constant dark regimes, taxa possessing circadian rhythms under the two conditions were dissimilar. Light exposure concealed microbial circadian clocks as a regulatory driver, leading to fewer ecological niches in light versus dark communities. These findings disentangle regulation mechanisms for circadian rhythms in the rice rhizosphere microbial communities and highlight the role of light-induced regulation of rhizosphere microbial communities.Subject terms: Microbial ecology, Community ecology     

Phenotypic biomonitoring using multivariate flow cytometric analysis of multi-stained microorganisms

A new method for monitoring phenotypic profiles of pure cultures and complex microbial communities was evaluated. The approach was to stain microorganisms with a battery of fluorescent dyes prior to flow cytometry analysis (FCM) and to analyse the data using multivariate methods, including principal component analysis and partial least squares. The FCM method was quantitatively evaluated using different mixtures of pure cultures as well as microbial communities. The results showed that the method could quantitatively and reproducibly resolve both populations and communities of microorganisms with 5% abundance in a diverse microbial background. The feasibility of monitoring complex microbial communities over time during the biodegradation of naphthalene using the FCM method was demonstrated. The biodegradation of naphthalene occurred to differing extents in microcosms representing three different types of aromatic-contaminated groundwater and a sample of bio-basin water. The FCM method distinguished each of these four microbial communities. The phenotypic profiles were compared with genotypic profiles generated by random-amplified polymorphic DNA analysis. The genotypic profiles of the microbial communities described only the microbial composition, and not their functional change, whereas the phenotypic profiles seemed to contain information on both the composition and the functional change of the microorganisms. Furthermore, event analysis of the FCM data showed that microbial communities with initially differing compositions could converge towards a similar composition if they had a capacity for high levels of degradation, whereas microbial communities with similar initial compositions could diverge if they differed in biodegrading ability.     

垃圾堆肥复合菌剂对干旱胁迫下草坪植物生理生态特性的影响

从生活垃圾堆肥中提取有益微生物菌种,配成不同浓度的复合微生物菌剂（CM）,施入草坪基质,研究了复合微生物菌剂对干旱胁迫下草坪植物生理生态特性的影响。结果表明：在干旱胁迫下,接种过复合微生物菌剂的草坪植物叶片的丙二醛（MDA）含量显著低于未接种菌剂的对照,超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）活性显著高于对照。不同草坪植物品种生理生态特性在接种CM后对干旱胁迫的响应程度不同。即高羊茅（Festuca arundinacea L.）,当接种200倍稀释液的菌剂,叶片SOD、POD和CAT活性达到最大,分别是对照的8.13、1.53和2.46倍;而黑麦草（Lolium perenne L.）则当接种100倍稀释液的菌剂,POD和CAT活性分别高出对照64.4%和56.1%,而SOD活性是对照的6.50倍。和对照相比,各接种菌剂处理的脯氨酸（Pro）含量、可溶性蛋白含量明显降低,离体叶片持水力却保持了较高的水平。上述结果表明,接种复合微生物菌剂后,植物能够通过自身的保护酶活性和渗透调节物质含量来减轻干旱伤害,维持植物体的正常生理代谢功能,从而有效缓解干旱胁迫对草坪植物的伤害,提高草坪植物的抗旱性,为干旱环境草坪植物的建植提供依据。     

Response of Microbial Community Composition and Function to Soil Climate Change

Soil microbial communities mediate critical ecosystem carbon and nutrient cycles. How microbial communities will respond to changes in vegetation and climate, however, are not well understood. We reciprocally transplanted soil cores from under oak canopies and adjacent open grasslands in a California oak–grassland ecosystem to determine how microbial communities respond to changes in the soil environment and the potential consequences for the cycling of carbon. Every 3 months for up to 2 years, we monitored microbial community composition using phospholipid fatty acid analysis (PLFA), microbial biomass, respiration rates, microbial enzyme activities, and the activity of microbial groups by quantifying ¹³C uptake from a universal substrate (pyruvate) into PLFA biomarkers. Soil in the open grassland experienced higher maximum temperatures and lower soil water content than soil under the oak canopies. Soil microbial communities in soil under oak canopies were more sensitive to environmental change than those in adjacent soil from the open grassland. Oak canopy soil communities changed rapidly when cores were transplanted into the open grassland soil environment, but grassland soil communities did not change when transplanted into the oak canopy environment. Similarly, microbial biomass, enzyme activities, and microbial respiration decreased when microbial communities were transplanted from the oak canopy soils to the grassland environment, but not when the grassland communities were transplanted to the oak canopy environment. These data support the hypothesis that microbial community composition and function is altered when microbes are exposed to new extremes in environmental conditions; that is, environmental conditions outside of their “life history” envelopes.     

Spatial heterogeneity of eukaryotic microbial communities in an unstudied geothermal diatomaceous biological soil crust: Yellowstone National Park, WY, USA

Knowledge of microbial communities and their inherent heterogeneity has dramatically increased with the widespread use of high-throughput sequencing technologies, and we are learning more about the ecological processes that structure microbial communities across a wide range of environments, as well as the relative scales of importance for describing bacterial communities in natural systems. Little work has been carried out to assess fine-scale eukaryotic microbial heterogeneity in soils. Here, we present findings from a bar-coded 18S rRNA survey of the eukaryotic microbial communities in a previously unstudied geothermal diatomaceous biological soil crust in Yellowstone National Park, WY, USA, in which we explicitly compare microbial community heterogeneity at the particle scale within soil cores. Multivariate analysis of community composition showed that while subsamples from within the same soil core clustered together, community dissimilarity between particles in the same core was high. This study describes a novel soil microbial environment and also adds to our growing understanding of microbial heterogeneity and the scales relevant to the study of soil microbial communities.     

Response of microbial community structure to microbial plugging in a mesothermic petroleum reservoir in China

Microbial plugging, a microbial enhancement of oil recovery (MEOR) technique, has been applied in a candidate oil reservoir of Daqing Oil Field (China). The goal of this study is to monitor the survival of injected bacteria and reveal the response of microbial communities in field trial of microbial plugging through injection of selected microbial culture broth and nutrients. Culture-dependent enrichment and culture-independent 16S rDNA clone library methods were used. The results show that it was easy to activate targeted biopolymer-producing bacteria in a laboratory environment, and it was difficult for injected exogenous bacteria to survive. In addition, microbial communities in the oil reservoir also changed before and after the field trial. However, microbial communities, activated by fermentative medium for biopolymer-producing bacteria, appeared to show greater differences in the laboratory than in the natural reservoir. It was concluded that microbial populations monitoring was important to MEOR; results of response of microbial communities could provide a guide for the future field trials.     

Hypolithic and soil microbial community assembly along an aridity gradient in the Namib Desert

The Namib Desert is considered the oldest desert in the world and hyperarid for the last 5 million years. However, the environmental buffering provided by quartz and other translucent rocks supports extensive hypolithic microbial communities. In this study, open soil and hypolithic microbial communities have been investigated along an East–West transect characterized by an inverse fog-rainfall gradient. Multivariate analysis showed that structurally different microbial communities occur in soil and in hypolithic zones. Using variation partitioning, we found that hypolithic communities exhibited a fog-related distribution as indicated by the significant East–West clustering. Sodium content was also an important environmental factor affecting the composition of both soil and hypolithic microbial communities. Finally, although null models for patterns in microbial communities were not supported by experimental data, the amount of unexplained variation (68–97 %) suggests that stochastic processes also play a role in the assembly of such communities in the Namib Desert.     

Molecular and Functional Assessment of Bacterial Community Convergence in Metal-Amended Soils

Species diversity and the structure of microbial communities in soils are thought to be a function of the cumulative selective pressures within the local environment. Shifts in microbial community structure, as a result of metal stress, may have lasting negative effects on soil ecosystem dynamics if critical microbial community functions are compromised. Three soils in the vicinity of a copper smelter, previously contaminated with background, low and high levels of aerially deposited metals, were amended with metal-salts to determine the potential for metal contamination to shape the structural and functional diversity of microbial communities in soils. We hypothesized that the microbial communities native to the three soils would initially be unique to each site, but would converge on a microbial community with similar structure and function, as a result of metal stress. Initially, the three different sites supported microbial communities with unique structural and functional diversity, and the nonimpacted site supported inherently higher levels of microbial activity and biomass, relative to the metal-contaminated sites. Amendment of the soils with metal-salts resulted in a decrease in microbial activity and biomass, as well as shifts in microbial community structure and function at each site. Soil microbial communities from each site were also observed to be sensitive to changes in soil pH as a result of metal-salt amendment; however, the magnitude of these pH-associated effects varied between soils. Microbial communities from each site did not converge on a structurally or functionally similar community following metal-salt amendment, indicating that other factors may be equally important in shaping microbial communities in soils. Among these factors, soil physiochemical parameters like organic matter and soil pH, which can both influence the bioavailability and toxicity of metals in soils, may be critical.     

Visualization, modelling and prediction in soil microbiology

The introduction of new approaches for characterizing microbial communities and imaging soil environments has benefited soil microbiology by providing new ways of detecting and locating microorganisms. Consequently, soil microbiology is poised to progress from simply cataloguing microbial complexity to becoming a systems science. A systems approach will enable the structures of microbial communities to be characterized and will inform how microbial communities affect soil function. Systems approaches require accurate analyses of the spatio-temporal properties of the different microenvironments present in soil. In this Review we advocate the need for the convergence of the experimental and theoretical approaches that are used to characterize and model the development of microbial communities in soils.     

凋落物分解过程中土壤微生物群落的变化

凋落物分解是生态系统碳循环和营养物质循环的关键过程, 受多种因素共同影响。土壤微生物是影响凋落物分解的重要因素, 其群落组成在一定程度上依赖于所处植物群落的特征。因此, 研究分解过程中微生物群落组成的变化及其对植物多样性的响应, 有利于对凋落物分解机制的理解。本文采用分解袋野外原位分解的方法, 对凋落物分解过程中微生物群落的变化及其对所处森林环境中树木的种类和遗传多样性的响应进行了研究。结果表明: (1)凋落物分解183天后, 土壤中微生物群落的多样性降低, 并且森林群落的物种多样性与微生物群落多样性呈负相关关系; (2)凋落物分解前后, 土壤中真菌和细菌群落的磷脂脂肪酸(PLFA)量均有所增加, 说明凋落物分解为微生物生存和繁殖提供了养分; (3)地形因素是影响微生物群落变化最显著的因素, 可解释微生物群落变化的29.55%; 其次是凋落物的基质质量, 可以解释15.39%; 最后是森林群落的多样性, 可以解释8.45%; 这3种因素共同解释率为2.97%。综上所述, 与森林群落的植物多样性相比, 样地的地形因素与凋落物的基质质量对微生物群落的影响更显著。     

Two Invasive Plants Alter Soil Microbial Community Composition in Serpentine Grasslands

Plant invasions pose a serious threat to native ecosystem structure and function. However, little is known about the potential role that rhizosphere soil microbial communities play in facilitating or resisting the spread of invasive species into native plant communities. The objective of this study was to compare the microbial communities of invasive and native plant rhizospheres in serpentine soils. We compared rhizosphere microbial communities, of two invasive species, Centaurea solstitialis (yellow starthistle) and Aegilops triuncialis (barb goatgrass), with those of five native species that may be competitively affected by these invasive species in the field (Lotus wrangelianus, Hemizonia congesta, Holocarpha virgata, Plantago erecta, and Lasthenia californica). Phospholipid fatty acid analysis (PLFA) was used to compare the rhizosphere microbial communities of invasive and native plants. Correspondence analyses (CA) of PLFA data indicated that despite yearly variation, both starthistle and goatgrass appear to change microbial communities in areas they invade, and that invaded and native microbial communities significantly differ. Additionally, rhizosphere microbial communities in newly invaded areas are more similar to the original native soil communities than are microbial communities in areas that have been invaded for several years. Compared to native plant rhizospheres, starthistle and goatgrass rhizospheres have higher levels of PLFA biomarkers for sulfate reducing bacteria, and goatgrass rhizospheres have higher fatty acid diversity and higher levels of biomarkers for sulfur-oxidizing bacteria, and arbuscular mycorrhizal fungi. Changes in soil microbial community composition induced by plant invasion may affect native plant fitness and/or ecosystem function.     

长期不同施肥制度下几种土壤微生物学特征变化

 为阐明土壤微生物对土壤健康的生物指示功能, 以国家褐潮土肥力与肥料效益监测基地的长期肥料试验为平台, 应用BIOLOG ECO微平板培养法与常规分析法研究了长期施肥15年后不同施肥制度对土壤微生物生物量、活性、群落代谢功能多样性及土壤肥力的影响。研究结果表明, 与对照(CK)相比, 长期化肥与有机肥配施土壤中土壤有机质(SOM)、全氮(STN)、全磷(STP)含量升高, 土壤C/N与pH值降低, 土壤微生物量碳(Soil microbial biomass carbon, SMBC)、生物微生物量氮(Soil microbial biomass nitrogen, SMBN)、微生物商(qMB)及脲酶(Urease)活性升高, BIOLOG ECO微平板平均颜色变化率(Average well color development, AWCD)、土壤微生物代谢功能多样性指数变化不明显。和长期单施化肥处理(NPK)相比, 长期化肥与有机肥配施处理中上述几种微生物学特征(SMBC、SMBN、qMB、Urease及AWCD、代谢功能多样性指数)均呈极显著增加。NPK处理与CK相比虽然SOM、STN、STP含量稍有升高, 土壤C/N与pH值降低, SMBC、SMBN、qMB及Urease活性增高, 但是AWCD、土壤微生物代谢功能多样性指数却显著下降。过氧化氢酶活性(Catalase)在各处理土壤中的差异不显著。土壤微生物碳源利用的主成分分析表明, 长期不同施肥各处理在土壤微生物利用碳源的种类和能力上有差异。此试验说明, 土壤微生物受农业管理措施和多种环境因素的影响, 土壤微生物学特征可作为土壤质量的敏感指标, 为提高作物产量、增强肥力提供理论参考。     

Abiotic drivers and plant traits explain landscape‐scale patterns in soil microbial communities

The controls on aboveground community composition and diversity have been extensively studied, but our understanding of the drivers of belowground microbial communities is relatively lacking, despite their importance for ecosystem functioning. In this study, we fitted statistical models to explain landscape‐scale variation in soil microbial community composition using data from 180 sites covering a broad range of grassland types, soil and climatic conditions in England. We found that variation in soil microbial communities was explained by abiotic factors like climate, pH and soil properties. Biotic factors, namely community‐weighted means (CWM) of plant functional traits, also explained variation in soil microbial communities. In particular, more bacterial‐dominated microbial communities were associated with exploitative plant traits versus fungal‐dominated communities with resource‐conservative traits, showing that plant functional traits and soil microbial communities are closely related at the landscape scale.     

Microbial community composition in sediments resists perturbation by nutrient enrichment

Functional redundancy in bacterial communities is expected to allow microbial assemblages to survive perturbation by allowing continuity in function despite compositional changes in communities. Recent evidence suggests, however, that microbial communities change both composition and function as a result of disturbance. We present evidence for a third response: resistance. We examined microbial community response to perturbation caused by nutrient enrichment in salt marsh sediments using deep pyrosequencing of 16S rRNA and functional gene microarrays targeting the nirS gene. Composition of the microbial community, as demonstrated by both genes, was unaffected by significant variations in external nutrient supply in our sampling locations, despite demonstrable and diverse nutrient-induced changes in many aspects of marsh ecology. The lack of response to external forcing demonstrates a remarkable uncoupling between microbial composition and ecosystem-level biogeochemical processes and suggests that sediment microbial communities are able to resist some forms of perturbation.     

Biomarkers at the microscopic range: ToF-SIMS molecular imaging of Archaea-derived lipids in a microbial mat

Time‐of‐Flight Secondary Ion Mass Spectrometry (ToF‐SIMS) with a bismuth cluster primary ion source was used for analysing microbial lipid biomarkers in 10‐µm‐thick microscopic cryosections of methanotrophic microbial mats from the Black Sea. Without further sample preparation, archaeal isopranyl glycerol di‐ and tetraether core lipids, together with their intact diglycoside (gentiobiosyl‐) derivatives, were simultaneously identified by exact mass determination. Utilizing the imaging capability of ToF‐SIMS, the spatial distributions of these biomarkers were mapped at a lateral resolution of < 5 µm in 500 × 500 µm² areas on the mat sections. Using cluster projectiles in the burst alignment mode, it was possible to reach a lateral resolution of 1 µm on an area of 233 × 233 µm, thus approaching the typical size of microbial cells. The mappings showed different ‘provenances’ within the sections that are distinguished by individual lipid fingerprints, namely (A) the diethers archaeol and hydroxyarchaeol co‐occurring with glycerol dialkyl glycerol tetraethers (GDGT), (B) hydroxyarchaeol and dihydroxyarchaeol, and (C) GDGT and gentiobiosyl‐GDGT. Because ToF‐SIMS is a virtually nondestructive technique affecting only the outermost layers of the sample surface (typically 10–100 nm), it was possible to further examine the studied areas using conventional microscopy, and associate the individual lipid patterns with specific morphological traits. This showed that provenance (B) was frequently associated with irregular, methane‐derived CaCO₃ crystallites, whereas provenance (C) revealed a population of fluorescent, filamentous microorganisms showing the morphology of known methanotrophic ANME‐1 archaea. The direct coupling of imaging mass spectrometry with microscopic techniques reveals interesting perspectives for the in‐situ study of lipids in geobiology, microbial ecology, and organic geochemistry. After further developing protocols for handling different kinds of environmental samples, ToF‐SIMS could be used as a tool to attack many challenging problems in these fields, such as the attribution of biological source(s) to particular biomarkers in question, or the high‐resolution tracking of biogeochemical processes in modern and ancient natural environments.     

基于16S rRNA基因测序分析微生物群落多样性

微生物群落多样性的研究对于挖掘微生物资源,探索微生物群落功能,阐明微生物群落与生境间的关系具有重要意义。随着宏基因组概念的提出以及测序技术的快速发展,16S rRNA基因测序在微生物群落多样性的研究中已被广泛应用。文中系统地介绍了16S rRNA基因测序分析流程中的四个重要环节,包括测序平台与扩增区的选择、测序数据预处理以及多样性分析方法,就其面临的问题与挑战进行了探讨并对未来的研究方向进行了展望,以期为微生物群落多样性相关研究提供参考。