Development and experimental validation of a nifH oligonucleotide microarray to study diazotrophic communities in a glacier forefield

Functional microarrays are powerful tools that allow the parallel detection of multiple strains at the species level and therefore to rapidly obtain information on microbial communities in the environment. However, the design of suitable probes is prone to uncertainties, as it is based so far on in silico predictions including weighted mismatch number and Gibbs free-energy values. This study describes the experimental selection of probes targeting subsequences of the nifH gene to study the community structure of diazotrophic populations present in Damma glacier (Swiss Central Alps) forefield soils. Using the Geniom^® One in situ synthesis technology (Febit, Germany), 2727 in silico designed candidate probes were tested. A total of 946 specific probes were selected and validated. This probe set covered a large diversity of the NifH phylotypes (35 out of the 45) found in the forefield. Hybridization predictors were tested statistically. Gibbs free-energy value for probe-target binding gave the best prediction for hybridization efficiency, while the weighted mismatch number was not significantly associated to probe specificity. In this study, we demonstrate that extensive experimental tests of probe-hybridization behaviour against sequences present in the studied environment remain a prerequisite for meaningful probe selection.     

Potential feedback mediated by soil microbiome response to warming in a glacier forefield

Mountain glaciers are retreating at an unprecedented rate due to global warming. Glacier retreat is widely believed to be driven by the physiochemical characteristics of glacier surfaces; however, the current knowledge of such biological drivers remains limited. An estimated 130 Tg of organic carbon (OC) is stored in mountain glaciers globally. As a result of global warming, the accelerated microbial decomposition of OC may further accelerate the melting process of mountain glaciers by heat production with the release of greenhouse gases, such as carbon dioxide (CO₂) and methane. Here, using short‐term aerobic incubation data from the forefield of Urumqi Glacier No. 1, we assessed the potential climate feedback mediated by soil microbiomes at temperatures of 5°C (control), 6.2°C (RCP 2.6), 11°C (RCP 8.5), and 15°C (extreme temperature). We observed enhanced CO₂‐C release and heat production under warming conditions, which led to an increase in near‐surface (2 m) atmospheric temperatures, ranging from 0.9°C to 3.4°C. Warming significantly changed the structures of the RNA‐derived (active) and DNA‐derived (total) soil microbiomes, and active microbes were more sensitive to increased temperatures than total microbes. Considering the positive effects of temperature and deglaciation age on the CO₂‐C release rate, the alterations in the active microbial community structure had a negative impact on the increased CO₂‐C release rate. Our results revealed that glacial melting could potentially be significantly accelerated by heat production from increased microbial decomposition of OC. This risk might be true for other high‐altitude glaciers under emerging warming, thus improving the predictions of the effects of potential feedback on global warming.     

Vertical distribution of the soil microbiota along a successional gradient in a glacier forefield

Spatial patterns of microbial communities have been extensively surveyed in well‐developed soils, but few studies investigated the vertical distribution of micro‐organisms in newly developed soils after glacier retreat. We used 454‐pyrosequencing to assess whether bacterial and fungal community structures differed between stages of soil development (SSD) characterized by an increasing vegetation cover from barren (vegetation cover: 0%/age: 10 years), sparsely vegetated (13%/60 years), transient (60%/80 years) to vegetated (95%/110 years) and depths (surface, 5 and 20 cm) along the Damma glacier forefield (Switzerland). The SSD significantly influenced the bacterial and fungal communities. Based on indicator species analyses, metabolically versatile bacteria (e.g. Geobacter) and psychrophilic yeasts (e.g. Mrakia) characterized the barren soils. Vegetated soils with higher C, N and root biomass consisted of bacteria able to degrade complex organic compounds (e.g. Candidatus Solibacter), lignocellulolytic Ascomycota (e.g. Geoglossum) and ectomycorrhizal Basidiomycota (e.g. Laccaria). Soil depth only influenced bacterial and fungal communities in barren and sparsely vegetated soils. These changes were partly due to more silt and higher soil moisture in the surface. In both soil ages, the surface was characterized by OTUs affiliated to Phormidium and Sphingobacteriales. In lower depths, however, bacterial and fungal communities differed between SSD. Lower depths of sparsely vegetated soils consisted of OTUs affiliated to Acidobacteria and Geoglossum, whereas depths of barren soils were characterized by OTUs related to Gemmatimonadetes. Overall, plant establishment drives the soil microbiota along the successional gradient but does not influence the vertical distribution of microbiota in recently deglaciated soils.     

Shifts in desulfonating bacterial communities along a soil chronosequence in the forefield of a receding glacier

Forefields of receding glaciers are unique and sensitive environments representing natural soil chronosequences, where sulfate availability is assumed to be a limiting factor. Bacterial mineralization of organosulfur is an important sulfate-providing process in soils. We analyzed the diversity of sulfonate-desulfurizing (desulfonating) bacteria in the Damma glacier forefield on the basis of the key gene asfA by terminal restriction fragment length polymorphism and clone libraries. The community structure and sequence diversity of desulfonating bacteria differed significantly between forefield soils deglaciated in the 1990s and the 1950s. Soil age had a strong effect on the desulfonating rhizosphere communities of Agrostis rupestris , but only a slight impact on the ones from Leucanthemopsis alpina . AsfA affiliated to Polaromonas sp. was predominantly found in the more recent ice-free soils and the corresponding rhizospheres of A. rupestris , while a group of unidentified sequences was found to be dominating the matured soils and the corresponding rhizospheres of A. rupestris . The desulfonating bacterial diversity was not affected by varying levels of sulfate concentrations. The level of asfA diversity in recently deglaciated soils suggests that desulfonating bacteria are a critical factor in sulfur cycling, with defined groups dominating at different stages of soil formation.     

Variations in soil culturable bacteria communities and biochemical characteristics in the Dongkemadi glacier forefield along a chronosequence

The variations in the soil culturable bacterial communities and biochemical parameters of early successional soils from a receding glacier in the Tanggula Mountain were investigated. We examined low organic carbon (C) and nitrogen (N) contents and enzymatic activity, correlated with fewer bacterial groups and numbers in the glacier forefield soils. The soil pH values decreased, but the soil water content, organic C and total N significantly increased, along the chronosequence. The soil C/N ratio decreased in the early development soils and increased in the late development soils and it did not correlate with the soil age since deglaciation. The activities of soil urease, sucrase, protease, polyphenol oxidase, catalase, and dehydrogenase increased along the chronosequence. The numbers of culturable bacteria in the soils increased as cultured at 25°C while decreased at 4°C from younger soils to older soils. Total numbers of culturable bacteria in the soils cultured at 25°C were significantly positively correlated to the soil total N, organic C, and soil water content, as well as the activities of soil urease, sucrase, dehydrogenase, catalase, and polyphenol oxidase. We have obtained 224 isolates from the glacier forefield soils. The isolates were clustered into 28 groups by amplified ribosomal DNA restriction analysis (ARDRA). Among them, 27 groups and 25 groups were obtained from the soils at 25°C and at 4°C incubation temperatures, respectively. These groups are affiliated with 18 genera that belong to six taxa, viz, Actinobacteria, Gammaproteobacteria, Bacteroidetes, Firmicutes, Alphaproteobacteria, and Betaproteobacteria. The dominant taxa were Actinobacteria, Gammaproteobacteria, and Bacteroidetes in all the samples. The abundance and the diversity of the genera isolated at 25°C incubation temperature were greater than that at 4°C.     

Microbial dynamics in soils of the Damma glacier forefield show succession in the functional genetic potential

Glacier retreat is a visible consequence of climate change worldwide. Although taxonomic change of the soil microbiomes in glacier forefields have been widely documented, how microbial genetic potential changes along succession is little known. Here, we used shotgun metagenomics to analyse whether the soil microbial genetic potential differed between four stages of soil development (SSD) sampled along three transects in the Damma glacier forefield (Switzerland). The SSDs were characterized by an increasing vegetation cover, from barren soil, to biological soil crust, to sparsely vegetated soil and finally to vegetated soil. Results suggested that SSD significantly influenced microbial genetic potential, with the lowest functional diversity surprisingly occurring in the vegetated soils. Overall, carbohydrate metabolism and secondary metabolite biosynthesis genes overrepresented in vegetated soils, which could be partly attributed to plant–soil feedbacks. For C degradation, glycoside hydrolase genes enriched in vegetated soils, while auxiliary activity and carbohydrate esterases genes overrepresented in barren soils, suggested high labile C degradation potential in vegetated, and high recalcitrant C degradation potential in barren soils. For N-cycling, organic N degradation and synthesis genes dominated along succession, and gene families involved in nitrification were overrepresented in barren soils. Our study provides new insights into how the microbial genetic potential changes during soil formation along the Damma glacier forefield.     

Use of stochastic models to assess the effect of environmental factors on microbial growth

We present a novel application of a stochastic ecological model to the study and analysis of microbial growth dynamics as influenced by environmental conditions in an extensive experimental data set. The model proved to be useful in bridging the gap between theoretical ideas in ecology and an applied problem in microbiology. The data consisted of recorded growth curves of Escherichia coli grown in triplicate in a base medium with all 32 possible combinations of five supplements: glucose, NH(4)Cl, HCl, EDTA, and NaCl. The potential complexity of 2(5) experimental treatments and their effects was reduced to 2(2) as just the metal chelator EDTA, the presumed osmotic pressure imposed by NaCl, and the interaction between these two factors were enough to explain the variability seen in the data. The statistical analysis showed that the positive and negative effects of the five chemical supplements and their combinations were directly translated into an increase or decrease in time required to attain stationary phase and the population size at which the stationary phase started. The stochastic ecological model proved to be useful, as it effectively explained and summarized the uncertainty seen in the recorded growth curves. Our findings have broad implications for both basic and applied research and illustrate how stochastic mathematical modeling coupled with rigorous statistical methods can be of great assistance in understanding basic processes in microbial ecology.     

Application of a nifH oligonucleotide microarray for profiling diversity of N2-fixing microorganisms in marine microbial mats

Diazotrophic community structure in microbial mats from Guerrero Negro (GN), Baja California, Mexico, was studied using polymerase chain reaction amplification of the nifH gene and a newly developed nifH oligonucleotide microarray. Ninety-six oligonucleotide probes designed for nifH sequences from cultivated isolates and the environment were printed on glass microarrays. Phylogenetic analysis showed that the probes represented all of the main nifH clusters. Specificity was tested by (i) evaluation of cross hybridization using individual targets, and (ii) comparison of the observed hybridization signals and those predicted from the sequences cloned from microbial mats. Signal intensity had a positive relationship with target concentration and the percentage identity between probe and target. Under moderate stringency and high target concentration, specificity of the probes varied from 77% to 100% with the individual targets tested. At the end of a 7-month long nutrient manipulation experiment in GN microbial mats, no expression of nitrogen fixation under nitrogen loading was detected, although a diverse community of diazotrophs was detected. The diversity in diazotrophic population present was higher than in the population expressing the nifH gene, and there were taxa specific differences in response to nutrients. The nifH microarray is a powerful tool for diazotroph community analysis in the marine environment.     

Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.     

Diversity of diazotrophs in the sediments of saline and soda lakes analyzed with the use of the nifH gene as a molecular marker

Phylogenetic analysis of the nifH genes, encoding the Fe protein of the nitrogenase enzymatic complex, was carried out for pure cultures of anoxygenic phototrophic bacteria of diverse origin, as well as for heterotrophic alkaliphilic sulfate reducers isolated from saline and soda lakes. Topology of the nitrogenase tree correlated with that of the 16S rRNA gene tree to a considerable degree, which made it possible to use the nifH gene as a molecular marker for investigation of diazotrophic bacterial communities in sediments of hyper saline and soda lakes. Although diazotrophs were revealed in all environmental samples, their phylogenetic diversity was relatively low. Sulfate-reducing deltaproteobacteria and photo- and chemotrophic gammaproteobacteria were predominant in integrated samples. Analysis of the upper sediment layers revealed predominance of phototrophic diazotrophs of various phyla, including purple sulfur and nonsulfur proteobacteria, green nonsulfur bacteria, heliobacteria, and cyanobacteria. Some phylotypes could not be identified, probably indicating the presence of bacterial groups which have not yet been studied by conventional microbiological techniques.     

Validation of a portable EMG device to assess muscle activity during free-living situations

Portable amplifiers that record electromyograms (EMGs) for longer than four hours are commonly priced over $20,000 USD. This cost, and the technical challenges associated with recording EMGs during free-living situations, typically restrict EMG use to laboratory settings. A low-cost system (μEMG; OT Bioelecttronica, 100€), using specialized concentric bipolar electrodes, has been developed specifically for free-living situations. The purpose of this study was to validate the μEMG system by comparing EMGs from μEMG with a laboratory-based alternative (Telemyo 900; Noraxon USA, Inc.). Surface EMGs from biceps brachii (BB) and tibialis anterior (TA) of ten subjects were recorded simultaneously with both systems as subjects performed maximal voluntary contractions (MVCs), submaximal contractions at 25%, 50%, and 75% MVC, seven simulated activities of daily living (ADLs), and >60 min of simulated free-living inside the laboratory. In general, EMG parameters (e.g., average full-wave rectified EMG amplitude) derived from both systems were not significantly different for all outcome variables, except there were small differences across systems in baseline noise and absolute EMG amplitudes during MVCs. These results suggest that μEMG is a valid approach to the long-term recording of EMG.     

The impact of environmental and social factors on learning abilities: a meta-analysis

Since the 1950s, researchers have examined how differences in the social and asocial environment affect learning in rats, mice, and, more recently, a variety of other species. Despite this large body of research, little has been done to synthesize these findings and to examine if social and asocial environmental factors have consistent effects on cognitive abilities, and if so, what aspects of these factors have greater or lesser impact. Here, we conducted a systematic review and meta-analysis examining how different external environmental features, including the social environment, impact learning (both speed of acquisition and performance). Using 531 mean-differences from 176 published articles across 27 species (with studies on rats and mice being most prominent) we conducted phylogenetically corrected mixed-effects models that reveal: (i) an average absolute effect size |d| = 0.55 and directional effect size d = 0.34; (ii) interventions manipulating the asocial environment result in larger effects than social interventions alone; and (iii) the length of the intervention is a significant predictor of effect size, with longer interventions resulting in larger effects. Additionally, much of the variation in effect size remained unexplained, possibly suggesting that species differ widely in how they are affected by environmental interventions due to varying ecological and evolutionary histories. Overall our results suggest that social and asocial environmental factors do significantly affect learning, but these effects are highly variable and perhaps not always as predicted. Most notably, the type (social or asocial) and length of interventions are important in determining the strength of the effect.     

A method for calculating the dose absorbed by terrestrial animals to assess the environmental impact of radioactive contamination

A method for calculating the exposures of terrestrial animals in areas contaminated with radionuclides using a point source dose function is presented. To take into account scattered γ-radiation, the Berger formula for dose buildup factor in an infinite air medium has been parameterized. In the dosimetric model proposed, an animal phantom is presented as a parallelepiped to estimate external exposures and as a tissue-quivalent sphere to estimate internal doses. Using analytical expressions, dose rate conversion coefficients for external and internal exposures of animals have been estimated for individual radionuclides. For energies of γ-rays above 50 keV, the results are in good agreement with those estimated by the Monte Carlo method for ellipsoidal phantoms of animals.     

Vegetation and environmental factors during primary succession on glacier forelands: Some outlines from the Italian Alps

ABSTRACT

Relationships between plant communities and the physical environment during primary succession on recently deglaciated glacier forelands were studied in 3 areas of the Italian Alps. The aim of the research was to relate traditional phytosociological data with environmental variables. Twenty-eight phytosociological relevés were performed, each associated with twenty-six environmental variables; quantitative parameters of richness and diversity were also calculated. Species/relevés, environmental variables/relevés and species/environmental variables matrices were analyzed by cluster analysis, PCA and Spearman correlation coefficient. Three main stages of succession were identified by floristic composition and confirmed by environmental parameter evaluation. A complex of environmental variables seems to be closely correlated with terrain age and richness/diversity parameters, even though diversity decreases in late successional stages. The phytosociological significance of species is in accordance with their position in the context of succession.     

Design of a rotating disk reactor to assess the colonization of biofilms by free-living amoebae under high shear rates

The present study was aimed at designing and optimizing a rotating disk reactor simulating high hydrodynamic shear rates (γ), which are representative of cooling circuits. The characteristics of the hydrodynamic conditions in the reactor and the complex approach used to engineer it are described. A 60 l tank was filled with freshwater containing free-living amoebae (FLA) and bacteria. Adhesion of the bacteria and formation of a biofilm on the stainless steel coupons were observed. FLA were able to establish in these biofilms under γ as high as 85,000 s^?1. Several physical mechanisms (convection, diffusion, sedimentation) could explain the accumulation of amoeboid cells on surfaces, but further research is required to fully understand and model the fine mechanisms governing such transport under γ similar to those encountered in the industrial environment. This technological advance may enable research into these topics.     

土壤微生物多样性及其环境影响因子研究进展

土壤中存在丰富的微生物资源,不同土壤系统具有不同的微生物群落,微生物多样性既依赖于生态系统又服务于生态系统。本文从物种多样性、遗传多样性、生态类型多样性、功能多样性四个方面对土壤微生物多样性进行了新的诠释,总结论述了土壤微生物多样性与环境因子如土壤、植物群落和气候条件之间的关系,并对目前存在的问题和今后面临的挑战提出几点看法。     

冰川前缘土壤微生物原生演替的生态特征——以乌鲁木齐河源1号冰川为例

为了解冰川前缘土壤微生物在原生演替过程中的生态特征及其影响因素,用空间距离代替时间序列,以乌鲁木齐河源1号冰川终碛堤为起点,沿6个不同演替时期(0,4,15,31,43a和对照)的样带采集土样,以冰川附近发育良好的土壤为对照,测定土壤酶活性、微生物氮矿化与脱氨作用以及微生物生物量。结果表明,土壤脲酶、蛋白酶、酸性磷酸酶、芳基硫酸酯酶、蔗糖酶活性、微生物氮矿化及脱氨作用随演替时间而增加,微生物生物量碳和氮变化呈波动状,趋势不明显。相关分析表明,土壤有机质与酶活、微生物生物量存在极显著正相关(P0.01)。1号冰川前缘微生物多样性指数随着演替时间持续增加,但目前仍未达稳定状态。     

Using Google trends data to assess public understanding on the environmental risks

This study proposes a method to assess the public understanding toward themes related to environment, biodiversity conservation and fishery. Since the Internet traffic is seen as the proxy of the public interest, this article analyzes the related Google Trends data to explore the cognitive map of the public. Patterning changes in the search volume (synchronous increase and decrease) were detected by using the association analysis. Weekly search volumes of the United States for the last 5 years have been gathered and were converted into values ranging between –1 and 1, indicating the gradual level of increase/decrease by the time. Apriori algorithm was used to determine the associations between search volumes of the keywords. As a consequence, several patterns were detected with a significant support indicating synchronous increases/decreases of the volumes. Upon the development of cognitive maps based on the detected patterns, it is found that the terms “Environment” and “Pollution” were the most significant and central terms. The search volume of the terms such as “Conservation,” “Ecology,” “Climate change,” “Invasive species” and “Endangered Species” increases as the search increases in the central terms. Therefore, these terms can be seen as the drivers/triggers of the Google search for some central words. It is apparent that the public has awareness of the central terms and further interest should be provoked and fostered on the environment-related concepts.