automated-3d-optical-coherence-tomography-to-elucidate-biofilm

Automated 3D Optical Coherence Tomography to Elucidate Biofilm Morphogenesis Over Large Spatial Scales

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm ...

combining-fluidic-devices-with-microscopy-flow-cytometry-to-study

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...

Ecole Polytechnique Fédérale de Lausanne

École Polytechnique Fédérale de Lausanne

Resources structure ecological communities and potentially link biodiversity to energy flow. It is commonly believed that functional traits (generalists versus specialists) involved in the exploitation of resources depend on resource availability and environmental fluctuations. The longitudinal nature of stream ecosystems provides changing resources to stream biota with yet unknown effects on microbial functional traits and community structure. We investigated the impact of autochthonous (algal extract) and allochthonous (spruce extract) resources, as they change along alpine streams from above to below the treeline, on microbial diversity, community composition and functions of benthic biofilms. Combining bromodeoxyuridine labelling and 454 pyrosequencing, we showed that diversity was lower upstream than downstream of the treeline and that community composition changed along the altitudinal gradient. We also found that, especially for allochthonous resources, specialisation by biofilm bacteria increased along that same gradient. Our results suggest that in streams below the treeline biofilm diversity, specialisation and functioning are associated with increasing niche differentiation as potentially modulated by divers allochthonous and autochthonous constituents contributing to resources. These findings expand our current understanding on biofilm structure and function in alpine streams. 

Altitudinal patterns of diversity and functional traits of metabolically active microorganisms in stream biofilms.

Global climate change is causing a wastage of glaciers and threatening biodiversity in glacier-fed ecosystems. The high turbidity typically found in those ecosystems, which is caused by inorganic particles and result of the erosive activity of glaciers is a key environmental factor influencing temperature and light availability, as well as other factors in the water column. Once these lakes loose hydrological connectivity to glaciers and turn clear, the accompanying environmental changes could represent a potential bottleneck for the established local diversity with yet unknown functional consequences. Here, we study three lakes situated along a turbidity gradient as well as one clear unconnected lake and evaluate seasonal changes in their bacterial community composition and diversity. Further, we assess potential consequences for community functioning. Glacier runoff represented a diverse source community for the lakes and several taxa were able to colonize downstream turbid habitats, although they were not found in the clear lake. Operational taxonomic unit-based alpha diversity and phylogenetic diversity decreased along the turbidity gradient, but metabolic functional diversity was negatively related to turbidity. No evidence for multifunctional redundancy, which may allow communities to maintain functioning upon alterations in diversity, was found. Our study gives a first view on how glacier-fed lake bacterial communities are affected by the melting of glaciers and indicates that diversity and community composition significantly change when hydrological connectivity to the glacier is lost and lakes turn clear.

Shifts in diversity and function of lake bacterial communities upon glacier retreat.

Small-scale hydraulics affects microbial behaviour at the cell level, trophic interactions in marine aggregates and the physical structure and function of stream biofilms. However, it remains unclear how hydraulics, predictably changing from small streams to large rivers, impacts the structure and biodiversity of complex microbial communities in these ecosystems. Here, we present experimental evidence unveiling hydraulics as a hitherto poorly recognized control of microbial lifestyle differentiation in fluvial ecosystems. Exposing planktonic source communities from stream and floodplain ecosystems to different hydraulic environments revealed strong selective hydraulic pressures but only minor founder effects on the differentiation of attached biofilms and suspended aggregates and their biodiversity dynamics. Key taxa with a coherent phylogenetic underpinning drove this differentiation. Only a few resident and phylogenetically related taxa formed the backbone of biofilm communities, whereas numerous resident taxa characterized aggregate communities. Our findings unveil fundamental differences between biofilms and aggregates and build the basis for a mechanistic understanding of how hydraulics drives the distribution of microbial diversity along the fluvial continuum.

Attached biofilms and suspended aggregates are distinct microbial lifestyles emanating from differing hydraulics.

Glacier retreat as a consequence of climate change influences freshwater ecosystems in manifold ways, yet the physical and chemical bases of these effects are poorly studied. Here, we characterize how water temperature differs between alpine lakes with and without direct glacier influence on seasonal and diurnal timescales. Using high temporal resolution monitoring of temperature in 4 lakes located in a catchment influenced by glacier retreat, we reported unexpectedly high surface temperatures, even in proglacial lakes located 2600 m a.s.l. Cold glacier meltwater and low nighttime air temperatures caused a distinct diurnal pattern of water temperature in the water column of glacier-influenced lakes. Precipitation onto glacier surfaces apparently leads to rapid cooling of the glacier-fed lakes and disrupts the thermal stratification with several mixing events during the summer. Taken together, these mechanisms contribute to the unique seasonal and diurnal dynamics of glacier-influenced lakes that contrast with the typical dimictic pattern of clear alpine lakes and represent an example of discontinuous cold polymictic lake type. This work contributes to the basic description of how climate and meteorology affect the physical properties of an increasingly common lake type.

Alpine glacier-fed turbid lakes are discontinuous cold polymictic rather than dimictic.

Microorganisms aggregated into matrix-enclosed biofilms dominate microbial life in most natural, engineered, and medical systems. Despite this, the ecological adaptations and metabolic trade-offs of the formation of complex biofilms are currently poorly understood. Here, exploring the dynamics of bacterial ribosomal RNA operon (rrn) copy numbers, we unravel the genomic underpinning of the formation and success of stream biofilms that contain hundreds of bacterial taxa. Experimenting with stream biofilms, we found that nascent biofilms in eutrophic systems had reduced lag phases and higher growth rates, and more taxa with higher rrn copy number than biofilms from oligotrophic systems. Based on these growth-related traits, our findings suggest that biofilm succession was dominated by slow-but-efficient bacteria likely with leaky functions, such as the production of extracellular polymeric substances at the cost of rapid growth. Expanding our experimental findings to biofilms from 140 streams, we found that rrn copy number distribution reflects functional trait allocation and ecological strategies of biofilms to be able to thrive in fluctuating environments. These findings suggest that alternative trade-offs dominating over rate-yield trade-offs contribute to the evolutionary success of stream biofilms.

Ecological strategies and metabolic trade-offs of complex environmental biofilms.

The loss of environmental heterogeneity threatens biodiversity and ecosystem functioning. It is therefore important to understand the relationship between environmental heterogeneity and spatial resilience as the capacity of ecological communities embedded in a landscape matrix to reorganize following disturbance. We experimented with phototrophic biofilms colonizing streambed landscapes differing in spatial heterogeneity and exposed to flow-induced disturbance. We show how streambed roughness and related features promote growth-related trait diversity and the recovery of biofilms towards carrying capacity (CC) and spatial resilience. At the scale of streambed landscapes, roughness and exposure to water flow promoted biofilm CC and growth trait diversity. Structural equation modelling identified roughness, post-disturbance biomass and a 'neighbourhood effect' to drive biofilm CC. Our findings suggest that the environment selecting for adaptive capacities prior to disturbance (that is, memory effects) and biofilm connectivity into spatial networks (that is, mobile links) contribute to the spatial resilience of biofilms in streambed landscapes. These findings are critical given the key functions biofilms fulfil in streams, now increasingly experiencing shifts in sedimentary and hydrological regimes.

Environmental heterogeneity promotes spatial resilience of phototrophic biofilms in streambeds.

Biofilms regulate critical processes in porous ecosystems. However, the biophysical underpinnings of the ecological success of these biofilms are poorly understood. Combining experiments with fluidic devices, sequencing and modeling, we reveal that architectural plasticity enhances space exploitation by multispecies biofilms in porous environments. Biofilms consistently differentiated into an annular base biofilm coating the grains and into streamers protruding from the grains into the pore space. Although different flow-related processes governed the differentiation of these architectures, both BB and streamers were composed of similar bacterial assemblages. This is evidence for architectural plasticity. Architectural plasticity allowed for complementary use of the space provided by the grain-pore complexes, which increased biofilm carrying capacity at the larger scale of the porous system. This increase comes potentially at the cost of a tradeoff. Contrasting time scales of oxygen replenishment and consumption, we show that streamers locally inhibit the growth of the BB downstream from the grains. Our study provides first insights into the biophysical underpinnings to the success of multispecies biofilms in porous environments.

Unraveling the biophysical underpinnings to the success of multispecies biofilms in porous environments.

Mixotrophy seems to be widespread among phytoplankton, but whether this strategy is more relevant in oligotrophic lakes remains unclear. Here, we tested the hypothesis that the relative abundance of mixotrophic flagellates in lakes increases along an elevational gradient paralleling increasingly oligotrophic conditions. For this purpose, 12 lakes located between 575 and 2796 m above sea level were sampled in summer and fall to include two different seasonal windows in phytoplankton dynamics and environmental conditions. The degree of mixotrophy in phytoplankton was estimated in tracer experiments using fluorescently-labeled bacteria and done with composite samples collected in the euphotic zone and in samples obtained from the chlorophyll- maximum. The results indicated the existence of a positive trend particularly in summer in the relative abundance of mixotrophic flagellates with elevation, however, this trend was not linear, and exceptions along the elevational gradient were found. Changes in the relative abundance of mixotrophic flagellates were related with significant changes in water transparency, DOC and phosphorus concentrations, as well as in bacterial and flagellate abundance. Overall, our results reveal that the harsh growth conditions found in oligotrophic high mountain lakes favor a mixotrophic trophic strategy among phytoplankton.

Importance of mixotrophic flagellates during the ice-free season in lakes located along an elevational gradient.

Viruses drive microbial diversity, function and evolution and influence important biogeochemical cycles in aquatic ecosystems. Despite their relevance, we currently lack an understanding of their potential impacts on stream biofilm structure and function. This is surprising given the critical role of biofilms for stream ecosystem processes. Currently, the study of viruses in stream biofilms is hindered by the lack of an optimized protocol for their extraction, concentration and purification. Here, we evaluate a range of methods to separate viral particles from stream biofilms, and to concentrate and purify them prior to DNA extraction and metagenome sequencing. Based on epifluorescence microscopy counts of viral-like particles (VLP) and DNA yields, we optimize a protocol including treatment with tetrasodium pyrophosphate and ultra-sonication to disintegrate biofilms, tangential-flow filtration to extract and concentrate VLP, followed by ultracentrifugation in a sucrose density gradient to isolate VLP from the biofilm slurry. Viromes derived from biofilms sampled from three different streams were dominated by ,  and  and provide first insights into the viral diversity of stream biofilms. Our protocol optimization provides an important step towards a better understanding of the ecological role of viruses in stream biofilms.

Benchmarking protocols for the metagenomic analysis of stream biofilm viromes.

The dispersal of organisms controls the structure and dynamics of populations and communities, and can regulate ecosystem functioning. Predicting dispersal patterns across scales is important to understand microbial life in heterogeneous porous environments such as soils and sediments. We developed a multi-scale approach, combining experiments with microfluidic devices and time-lapse microscopy to track individual bacterial trajectories and measure the overall breakthrough curves and bacterial deposition profiles: we, then, linked the two scales with a novel stochastic model. We show that motile cells of  disperse more efficiently than non-motile mutants through a designed heterogeneous porous system. Motile cells can evade flow-imposed trajectories, enabling them to explore larger pore areas than non-motile cells. While transported cells exhibited a rotation in response to hydrodynamic shear, motile cells were less susceptible to the torque, maintaining their body oriented towards the flow direction and thus changing the population velocity distribution with a significant impact on the overall transport properties. We also found, in a separate set of experiments, that if the suspension flows through a porous system already colonized by a biofilm,  cells are channelled into preferential flow paths and the cell attachment rate is increased. These two effects were more pronounced for non-motile than for motile cells. Our findings suggest that motility coupled with heterogeneous flows can be beneficial to motile bacteria in confined environments as it enables them to actively explore the space for resources or evade regions with unfavourable conditions. Our study also underlines the benefit of a multi-scale approach to the study of bacterial dispersal in porous systems.

Trait-specific dispersal of bacteria in heterogeneous porous environments: from pore to porous medium scale.

Despite the recognition of streams and rivers as sources of methane (CH) to the atmosphere, the role of CH oxidation (MOX) in these ecosystems remains poorly understood to date. Here, we measured the kinetics of MOX in stream sediments of 14 sites to resolve the ecophysiology of CH oxidizing bacteria (MOB) communities. The streams cover a gradient of land cover and associated physicochemical parameter and differed in stream- and porewater CH concentrations. Michealis-Menten kinetic parameter of MOX, maximum reaction velocity ( ), and CH concentration at half  ( ) increased with CH supply.  values in the micromolar range matched the CH concentrations measured in shallow stream sediments and indicate that MOX is mostly driven by low-affinity MOB. 16S rRNA gene sequencing identified MOB classified as  and particularly . Their relative abundance correlated with  gene counts and MOX rates, underscoring their pivotal role as CH oxidizers in stream sediments. Building on the concept of enterotypes, we identify two distinct groups of co-occurring MOB. While there was no taxonomic difference among the members of each cluster, one cluster contained abundant and common MOB, whereas the other cluster contained rare operational taxonomic units (OTUs) specific to a subset of streams. These integrated analyses of changes in MOB community structure, gene abundance, and the corresponding ecosystem process contribute to a better understanding of the distal controls on MOX in streams.

Microbial Ecology of Methanotrophy in Streams Along a Gradient of CH Availability.

Glacier-fed streams (GFS) are harsh ecosystems dominated by microbial life organized in benthic biofilms, yet the biodiversity and ecosystem functions provided by these communities remain under-appreciated. To better understand the microbial processes and communities contributing to GFS ecosystems, it is necessary to leverage high throughput sequencing. Low biomass and high inorganic particle load in GFS sediment samples may affect nucleic acid extraction efficiency using extraction methods tailored to other extreme environments such as deep-sea sediments. Here, we benchmarked the utility and efficacy of four extraction protocols, including an up-scaled phenol-chloroform protocol. We found that established protocols for comparable sample types consistently failed to yield sufficient high-quality DNA, delineating the extreme character of GFS. The methods differed in the success of downstream applications such as library preparation and sequencing. An adapted phenol-chloroform-based extraction method resulted in higher yields and better recovered the expected taxonomic profile and abundance of reconstructed genomes when compared to commercially-available methods. Affordable and straight-forward, this method consistently recapitulated the abundance and genomes of a mock community, including eukaryotes. Moreover, by increasing the amount of input sediment, the protocol is readily adjustable to the microbial load of the processed samples without compromising protocol efficiency. Our study provides a first systematic and extensive analysis of the different options for extraction of nucleic acids from glacier-fed streams for high-throughput sequencing applications, which may be applied to other extreme environments.

ArticleTotal : 2	Year
Publication title Cited by 7	2019
Publication title Cited by 2	2020

Article	Year
Altitudinal patterns of diversity and functional traits of metabolically active microorganisms in stream biofilms. The ISME journal\| PubMed ID: 25978543	2015
Shifts in diversity and function of lake bacterial communities upon glacier retreat. The ISME journal\| PubMed ID: 26771929	2016
Attached biofilms and suspended aggregates are distinct microbial lifestyles emanating from differing hydraulics. Nature microbiology\| PubMed ID: 27694939	2016
Alpine glacier-fed turbid lakes are discontinuous cold polymictic rather than dimictic. Inland waters : journal of the International Society of Limnology\| PubMed ID: 28690780	2017
Ecological strategies and metabolic trade-offs of complex environmental biofilms. NPJ biofilms and microbiomes\| PubMed ID: 28955480	2017
Environmental heterogeneity promotes spatial resilience of phototrophic biofilms in streambeds. Biology letters\| PubMed ID: 30305460	2018
Unraveling the biophysical underpinnings to the success of multispecies biofilms in porous environments. The ISME journal\| PubMed ID: 30833685	2019
Importance of mixotrophic flagellates during the ice-free season in lakes located along an elevational gradient. Aquatic sciences\| PubMed ID: 31057304	2019
Benchmarking protocols for the metagenomic analysis of stream biofilm viromes. PeerJ\| PubMed ID: 31879573	2019
Trait-specific dispersal of bacteria in heterogeneous porous environments: from pore to porous medium scale. Journal of the Royal Society, Interface\| PubMed ID: 32208823	2020
Microbial Ecology of Methanotrophy in Streams Along a Gradient of CH Availability. Frontiers in microbiology\| PubMed ID: 32477286	2020
Optimised biomolecular extraction for metagenomic analysis of microbial biofilms from high-mountain streams. PeerJ\| PubMed ID: 33194372	2020

Hannes Peter

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