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- microbial ecology (3)
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Properly designed (randomized and/or balanced) experiments are standard in ecological research. Molecular methods are increasingly used in ecology, but studies generally do not report the detailed design of sample processing in the laboratory. This may strongly influence the interpretability of results if the laboratory procedures do not account for the confounding effects of unexpected laboratory events. We demonstrate this with a simple experiment where unexpected differences in laboratory processing of samples would have biased results if randomization in DNA extraction and PCR steps do not provide safeguards. We emphasize the need for proper experimental design and reporting of the laboratory phase of molecular ecology research to ensure the reliability and interpretability of results.
Retrieval of water constituents from hyperspectral in-situ measurements under variable cloud cover
(2018)
Remote sensing and field spectroscopy of natural waters is typically performed under clear skies, low wind speeds and low solar zenith angles. Such measurements can also be made, in principle, under clouds and mixed skies using airborne or in-situ measurements; however, variable illumination conditions pose a challenge to data analysis. In the present case study, we evaluated the inversion of hyperspectral in-situ measurements for water constituent retrieval acquired under variable cloud cover. First, we studied the retrieval of Chlorophyll-a (Chl-a) concentration and colored dissolved organic matter (CDOM) absorption from in-water irradiance measurements. Then, we evaluated the errors in the retrievals of the concentration of total suspended matter (TSM), Chl-a and the absorption coefficient of CDOM from above-water reflectance measurements due to highly variable reflections at the water surface. In order to approximate cloud reflections, we extended a recent three-component surface reflectance model for cloudless atmospheres by a constant offset and compared different surface reflectance correction procedures. Our findings suggest that in-water irradiance measurements may be used for the analysis of absorbing compounds even under highly variable weather conditions. The extended surface reflectance model proved to contribute to the analysis of above-water reflectance measurements with respect to Chl-a and TSM. Results indicate the potential of this approach for all-weather monitoring.
Recovering genomics clusters of secondary metabolites from lakes using genome-resolved metagenomics
(2018)
Metagenomic approaches became increasingly popular in the past decades due to decreasing costs of DNA sequencing and bioinformatics development. So far, however, the recovery of long genes coding for secondary metabolites still represents a big challenge. Often, the quality of metagenome assemblies is poor, especially in environments with a high microbial diversity where sequence coverage is low and complexity of natural communities high. Recently, new and improved algorithms for binning environmental reads and contigs have been developed to overcome such limitations. Some of these algorithms use a similarity detection approach to classify the obtained reads into taxonomical units and to assemble draft genomes. This approach, however, is quite limited since it can classify exclusively sequences similar to those available (and well classified) in the databases. In this work, we used draft genomes from Lake Stechlin, north-eastern Germany, recovered by MetaBat, an efficient binning tool that integrates empirical probabilistic distances of genome abundance, and tetranucleotide frequency for accurate metagenome binning. These genomes were screened for secondary metabolism genes, such as polyketide synthases (PKS) and non-ribosomal peptide synthases (NRPS), using the Anti-SMASH and NAPDOS workflows. With this approach we were able to identify 243 secondary metabolite clusters from 121 genomes recovered from our lake samples. A total of 18 NRPS, 19 PKS, and 3 hybrid PKS/NRPS clusters were found. In addition, it was possible to predict the partial structure of several secondary metabolite clusters allowing for taxonomical classifications and phylogenetic inferences. Our approach revealed a high potential to recover and study secondary metabolites genes from any aquatic ecosystem.
The vertical distribution of chlorophyll in stratified lakes and reservoirs frequently exhibits a maximum peak deep in the water column, referred to as the deep chlorophyll maximum (DCM). DCMs are ecologically important hot spots of primary production and nutrient cycling, and their location can determine vertical habitat gradients for primary consumers. Consequently, the drivers of DCM structure regulate many characteristics of aquatic food webs and biogeochemistry. Previous studies have identified light and thermal stratification as important drivers of summer DCM depth, but their relative importance across a broad range of lakes is not well resolved. We analyzed profiles of chlorophyll fluorescence, temperature, and light during summer stratification from 100 lakes in the Global Lake Ecological Observatory Network (GLEON) and quantified two characteristics of DCM structure: depth and thickness. While DCMs do form in oligotrophic lakes, we found that they can also form in eutrophic to dystrophic lakes. Using a random forest algorithm, we assessed the relative importance of variables associated with light attenuation vs. thermal stratification for predicting DCM structure in lakes that spanned broad gradients of morphometry and transparency. Our analyses revealed that light attenuation was a more important predictor of DCM depth than thermal stratification and that DCMs deepen with increasing lake clarity. DCM thickness was best predicted by lake size with larger lakes having thicker DCMs. Additionally, our analysis demonstrates that the relative importance of light and thermal stratification on DCM structure is not uniform across a diversity of lake types.
Insight into how environmental change determines the production and distribution of cyanobacterial toxins is necessary for risk assessment. Management guidelines currently focus on hepatotoxins (microcystins). Increasing attention is given to other classes, such as neurotoxins (e.g., anatoxin-a) and cytotoxins (e.g., cylindrospermopsin) due to their potency. Most studies examine the relationship between individual toxin variants and environmental factors, such as nutrients, temperature and light. In summer 2015, we collected samples across Europe to investigate the effect of nutrient and temperature gradients on the variability of toxin production at a continental scale. Direct and indirect effects of temperature were the main drivers of the spatial distribution in the toxins produced by the cyanobacterial community, the toxin concentrations and toxin quota. Generalized linear models showed that a Toxin Diversity Index (TDI) increased with latitude, while it decreased with water stability. Increases in TDI were explained through a significant increase in toxin variants such as MC-YR, anatoxin and cylindrospermopsin, accompanied by a decreasing presence of MC-LR. While global warming continues, the direct and indirect effects of increased lake temperatures will drive changes in the distribution of cyanobacterial toxins in Europe, potentially promoting selection of a few highly toxic species or strains.
A fast and sensitive method for the continuous determination of methane (CH4) and its stable carbon isotopic values (delta C-13-CH4) in surface waters was developed by applying a vacuum to a gas/liquid exchange membrane and measuring the extracted gases by a portable cavity ring-down spectroscopy analyser (M-CRDS). The M-CRDS was calibrated and characterized for CH4 concentration and delta C-13-CH4 with synthetic water standards. The detection limit of the M-CRDS for the simultaneous determination of CH4 and delta C-13-CH4 is 3.6 nmol L-1 CH4. A measurement precision of CH4 concentrations and delta C-13-CH4 in the range of 1.1%, respectively, 1.7 parts per thousand (1 sigma) and accuracy (1.3%, respectively, 0.8 parts per thousand [1 sigma]) was achieved for single measurements and averaging times of 10 min. The response time tau of 57 +/- 5 s allow determination of delta C-13-CH4 values more than twice as fast than other methods. The demonstrated M-CRDS method was applied and tested for Lake Stechlin (Germany) and compared with the headspace-gas chromatography and fast membrane CH4 concentration methods. Maximum CH4 concentrations (577 nmol L-1) and lightest delta C-13-CH4 (-35.2 parts per thousand) were found around the thermocline in depth profile measurements. The M-CRDS-method was in good agreement with other methods. Temporal variations in CH4 concentration and delta C-13-CH4 obtained in 24 h measurements indicate either local methane production/oxidation or physical variations in the thermocline. Therefore, these results illustrate the need of fast and sensitive analyses to achieve a better understanding of different mechanisms and pathways of CH4 formation in aquatic environments.
Genome streamlining is frequently observed in free-living aquatic microorganisms and results in physiological dependencies between microorganisms. However, we know little about the specificity of these microbial associations. In order to examine the specificity and extent of these associations, we established mixed cultures from three different freshwater environments and analyzed the cooccurrence of organisms using a metagenomic time series. Free-living microorganisms with streamlined genomes lacking multiple biosynthetic pathways showed no clear recurring pattern in their interaction partners. Free-living freshwater bacteria form promiscuous cooperative associations. This notion contrasts with the well-documented high specificities of interaction partners in host-associated bacteria. Considering all data together, we suggest that highly abundant free-living bacterial lineages are functionally versatile in their interactions despite their distinct streamlining tendencies at the single-cell level. This metabolic versatility facilitates interactions with a variable set of community members.
Pollution by microplastics in aquatic ecosystems is accumulating at an unprecedented scale, emerging as a new surface for biofilm formation and gene exchange. In this study, we determined the permissiveness of aquatic bacteria towards a model antibiotic resistance plasmid, comparing communities that form biofilms on microplastics vs. those that are free-living. We used an exogenous and red-fluorescent E. coli donor strain to introduce the green-fluorescent broad-host-range plasmid pKJKS which encodes for trimethoprim resistance. We demonstrate an increased frequency of plasmid transfer in bacteria associated with microplastics compared to bacteria that are free-living or in natural aggregates. Moreover, comparison of communities grown on polycarbonate filters showed that increased gene exchange occurs in a broad range of phylogenetically-diverse bacteria. Our results indicate horizontal gene transfer in this habitat could distinctly affect the ecology of aquatic microbial communities on a global scale. The spread of antibiotic resistance through microplastics could also have profound consequences for the evolution of aquatic bacteria and poses a neglected hazard for human health.
In aquatic ecosystems, light availability can significantly influence microbial turnover of terrestrial organic matter through associated metabolic interactions between phototrophic and heterotrophic communities. However, particularly in streams, microbial functions vary significantly with the structure of the streambed, that is the distribution and spatial arrangement of sediment grains in the streambed. It is therefore essential to elucidate how environmental factors synergistically define the microbial turnover of terrestrial organic matter in order to better understand the ecological role of photoheterotrophic interactions in stream ecosystem processes. In outdoor experimental streams, we examined how the structure of streambeds modifies the influence of light availability on microbial turnover of leaf carbon (C). Furthermore, we investigated whether the studied relationships of microbial leaf C turnover to environmental conditions are affected by flow intermittency commonly occurring in streams. We applied leaves enriched with a C-13-stable isotope tracer and combined quantitative and isotope analyses. We thereby elucidated whether treatment induced changes in C turnover were associated with altered use of leaf C within the microbial food web. Moreover, isotope analyses were combined with measurements of microbial community composition to determine whether changes in community function were associated with a change in community composition. In this study, we present evidence, that environmental factors interactively determine how phototrophs and heterotrophs contribute to leaf C turnover. Light availability promoted the utilization of leaf C within the microbial food web, which was likely associated with a promoted availability of highly bioavailable metabolites of phototrophic origin. However, our results additionally confirm that the structure of the streambed modifies light-related changes in microbial C turnover. From our observations, we conclude that the streambed structure influences the strength of photo-heterotrophic interactions by defining the spatial availability of algal metabolites in the streambed and the composition of microbial communities. Collectively, our multifactorial approach provides valuable insights into environmental controls on the functioning of stream ecosystems.