Multiscale Porosity Microfluidics to Study Bacterial Transport in Heterogeneous Chemical Landscapes.

Microfluidic models are proving to be powerful systems to study fundamental processes in porous media, due to their ability to replicate topologically complex environments while allowing detailed, quantitative observations at the pore scale. Yet, while porous media such as living tissues, geological substrates, or industrial systems typically display a porosity that spans multiple scales, most microfluidic models to date are limited to a single porosity or a small range of pore sizes. Here, a novel microfluidic system with multiscale porosity is presented. By embedding polyacrylamide (PAAm) hydrogel structures through in-situ photopolymerization in a landscape of microfabricated polydimethylsiloxane (PDMS) pillars with varying spacing, micromodels with porosity spanning several orders of magnitude, from nanometers to millimeters are created. Experiments conducted at different porosity patterns demonstrate the potential of this approach to characterize fundamental and ubiquitous biological and geochemical transport processes in porous media. Accounting for multiscale porosity allows studies of the resulting heterogeneous fluid flow and concentration fields of transported chemicals, as well as the biological behaviors associated with this heterogeneity, such as bacterial chemotaxis. This approach brings laboratory studies of transport in porous media a step closer to their natural counterparts in the environment, industry, and medicine.

Immiscible Rayleigh-Taylor turbulence: Implications for bacterial degradation in oil spills.

An unstable density stratification between two fluids mixes spontaneously under the effect of gravity, a phenomenon known as Rayleigh-Taylor (RT) turbulence. If the two fluids are immiscible, for example, oil and water, surface tension prevents intermixing at the molecular level. However, turbulence fragments one fluid into the other, generating an emulsion in which the typical droplet size decreases over time as a result of the competition between the rising kinetic energy and the surface energy density. Even though the first phenomenological theory describing this emulsification process was derived many years ago, it has remained elusive to experimental verification, hampering our ability to predict the fate of oil in applications such as deep-water spills. Here, we provide the first experimental and numerical verification of the immiscible RT turbulence theory, unveiling a unique turbulent state that originates at the oil-water interface due to the interaction of multiple capillary waves. We show that a single, non-dimensional, and time-independent parameter controls the range of validity of the theory. Our findings have wide-ranging implications for the understanding of the mixing of immiscible fluids. This includes in particular oil spills, where our work enables the prediction of the oil-water interface dynamics that ultimately determine the rate of oil biodegradation by marine bacteria.

Microfluidic approaches in microbial ecology.

Microbial life is at the heart of many diverse environments and regulates most natural processes, from the functioning of animal organs to the cycling of global carbon. Yet, the study of microbial ecology is often limited by challenges in visualizing microbial processes and replicating the environmental conditions under which they unfold. Microfluidics operates at the characteristic scale at which microorganisms live and perform their functions, thus allowing for the observation and quantification of behaviors such as growth, motility, and responses to external cues, often with greater detail than classical techniques. By enabling a high degree of control in space and time of environmental conditions such as nutrient gradients, pH levels, and fluid flow patterns, microfluidics further provides the opportunity to study microbial processes in conditions that mimic the natural settings harboring microbial life. In this review, we describe how recent applications of microfluidic systems to microbial ecology have enriched our understanding of microbial life and microbial communities. We highlight discoveries enabled by microfluidic approaches ranging from single-cell behaviors to the functioning of multi-cellular communities, and we indicate potential future opportunities to use microfluidics to further advance our understanding of microbial processes and their implications.

Swimming towards each other: the role of chemotaxis in bacterial interactions.

Chemotaxis allows microorganisms to direct movement in response to chemical stimuli. Bacteria use this behaviour to develop spatial associations with animals and plants, and even larger microbes. However, current theory suggests that constraints imposed by the limits of chemotactic sensory systems will prevent sensing of chemical gradients emanating from cells smaller than a few micrometres, precluding the utility of chemotaxis in interactions between individual bacteria. Yet, recent evidence has revealed surprising levels of bacterial chemotactic precision, as well as a role for chemotaxis in metabolite exchange between bacterial cells. If indeed widespread, chemotactic sensing between bacteria could represent an important, but largely overlooked, phenotype within interbacterial interactions, and play a significant role in shaping cooperative and competitive relationships.

Strong chemotaxis by marine bacteria towards polysaccharides is enhanced by the abundant organosulfur compound DMSP.

The ability of marine bacteria to direct their movement in response to chemical gradients influences inter-species interactions, nutrient turnover, and ecosystem productivity. While many bacteria are chemotactic towards small metabolites, marine organic matter is predominantly composed of large molecules and polymers. Yet, the signalling role of these large molecules is largely unknown. Using in situ and laboratory-based chemotaxis assays, we show that marine bacteria are strongly attracted to the abundant algal polysaccharides laminarin and alginate. Unexpectedly, these polysaccharides elicited stronger chemoattraction than their oligo- and monosaccharide constituents. Furthermore, chemotaxis towards laminarin was strongly enhanced by dimethylsulfoniopropionate (DMSP), another ubiquitous algal-derived metabolite. Our results indicate that DMSP acts as a methyl donor for marine bacteria, increasing their gradient detection capacity and facilitating their access to polysaccharide patches. We demonstrate that marine bacteria are capable of strong chemotaxis towards large soluble polysaccharides and uncover a new ecological role for DMSP in enhancing this attraction. These navigation behaviours may contribute to the rapid turnover of polymers in the ocean, with important consequences for marine carbon cycling.

Identification of inulin-responsive bacteria in the gut microbiota via multi-modal activity-based sorting.

Prebiotics are defined as non-digestible dietary components that promote the growth of beneficial gut microorganisms. In many cases, however, this capability is not systematically evaluated. Here, we develop a methodology for determining prebiotic-responsive bacteria using the popular dietary supplement inulin. We first identify microbes with a capacity to bind inulin using mesoporous silica nanoparticles functionalized with inulin. 16S rRNA gene amplicon sequencing of sorted cells revealed that the ability to bind inulin was widespread in the microbiota. We further evaluate which taxa are metabolically stimulated by inulin and find that diverse taxa from the phyla Firmicutes and Actinobacteria respond to inulin, and several isolates of these taxa can degrade inulin. Incubation with another prebiotic, xylooligosaccharides (XOS), in contrast, shows a more robust bifidogenic effect. Interestingly, the Coriobacteriia Eggerthella lenta and Gordonibacter urolithinfaciens are indirectly stimulated by the inulin degradation process, expanding our knowledge of inulin-responsive bacteria.

Interspecies interactions determine growth dynamics of biopolymer-degrading populations in microbial communities.

Microbial communities perform essential ecosystem functions such as the remineralization of organic carbon that exists as biopolymers. The first step in mineralization is performed by biopolymer degraders, which harbor enzymes that can break down polymers into constituent oligo- or monomeric forms. The released nutrients not only allow degraders to grow, but also promote growth of cells that either consume the degradation products, i.e., exploiters, or consume metabolites released by the degraders or exploiters, i.e., scavengers. It is currently not clear how such remineralizing communities assemble at the microscale-how interactions between the different guilds influence their growth and spatial distribution, and hence the development and dynamics of the community. Here, we address this knowledge gap by studying marine microbial communities that grow on the abundant marine biopolymer alginate. We used batch growth assays and microfluidics coupled to time-lapse microscopy to quantitatively investigate growth and spatial distribution of single cells. We found that the presence of exploiters or scavengers alters the spatial distribution of degrader cells. In general, exploiters and scavengers-which we collectively refer to as cross-feeder cells-slowed down the growth of degrader cells. In addition, coexistence with cross-feeders altered the production of the extracellular enzymes that break down polymers by degrader cells. Our findings reveal that ecological interactions by nondegrading community members have a profound impact on the functions of microbial communities that remineralize carbon biopolymers in nature.

Controlled motility in the cyanobacterium Trichodesmium regulates aggregate architecture.

The ocean's nitrogen is largely fixed by cyanobacteria, including Trichodesmium, which forms aggregates comprising hundreds of filaments arranged in organized architectures. Aggregates often form upon exposure to stress and have ecological and biophysical characteristics that differ from those of single filaments. Here, we report that Trichodesmium aggregates can rapidly modulate their shape, responding within minutes to changes in environmental conditions. Combining video microscopy and mathematical modeling, we discovered that this reorganization is mediated by "smart reversals" wherein gliding filaments reverse when their overlap with other filaments diminishes. By regulating smart reversals, filaments control aggregate architecture without central coordination. We propose that the modulation of gliding motility at the single-filament level is a determinant of Trichodesmium's aggregation behavior and ultimately of its biogeochemical role in the ocean.

Morphogenesis of Biofilms in Porous Media and Control on Hydrodynamics.

The functioning of natural and engineered porous media, like soils and filters, depends in many cases on the interplay between biochemical processes and hydrodynamics. In such complex environments, microorganisms often form surface-attached communities known as biofilms. Biofilms can take the shape of clusters, which alter the distribution of fluid flow velocities within the porous medium, subsequently influencing biofilm growth. Despite numerous experimental and numerical efforts, the control of the biofilm clustering process and the resulting heterogeneity in biofilm permeability is not well understood, limiting our predictive abilities for biofilm-porous medium systems. Here, we use a quasi-2D experimental model of a porous medium to characterize biofilm growth dynamics for different pore sizes and flow rates. We present a method to obtain the time-resolved biofilm permeability field from experimental images and use the obtained permeability field to compute the flow field through a numerical model. We observe a biofilm cluster size distribution characterized by a spectrum slope evolving in time between -2 and -1, a fundamental measure that can be used to create spatio-temporal distributions of biofilm clusters for upscaled models. We find a previously undescribed biofilm permeability distribution, which can be used to stochastically generate permeability fields within biofilms. An increase in velocity variance for a decrease in physical heterogeneity shows that the bioclogged porous medium behaves differently than expected from studies on heterogeneity in abiotic porous media.

Chemotaxis increases metabolic exchanges between marine picophytoplankton and heterotrophic bacteria.

Behaviours such as chemotaxis can facilitate metabolic exchanges between phytoplankton and heterotrophic bacteria, which ultimately regulate oceanic productivity and biogeochemistry. However, numerically dominant picophytoplankton have been considered too small to be detected by chemotactic bacteria, implying that cell-cell interactions might not be possible between some of the most abundant organisms in the ocean. Here we examined how bacterial behaviour influences metabolic exchanges at the single-cell level between the ubiquitous picophytoplankton Synechococcus and the heterotrophic bacterium Marinobacter adhaerens, using bacterial mutants deficient in motility and chemotaxis. Stable-isotope tracking revealed that chemotaxis increased nitrogen and carbon uptake of both partners by up to 4.4-fold. A mathematical model following thousands of cells confirmed that short periods of exposure to small but nutrient-rich microenvironments surrounding Synechococcus cells provide a considerable competitive advantage to chemotactic bacteria. These findings reveal that transient interactions mediated by chemotaxis can underpin metabolic relationships among the ocean's most abundant microorganisms.

Encounter rates prime interactions between microorganisms.

Properties of microbial communities emerge from the interactions between microorganisms and between microorganisms and their environment. At the scale of the organisms, microbial interactions are multi-step processes that are initiated by cell-cell or cell-resource encounters. Quantification and rational design of microbial interactions thus require quantification of encounter rates. Encounter rates can often be quantified through encounter kernels-mathematical formulae that capture the dependence of encounter rates on cell phenotypes, such as cell size, shape, density or motility, and environmental conditions, such as turbulence intensity or viscosity. While encounter kernels have been studied for over a century, they are often not sufficiently considered in descriptions of microbial populations. Furthermore, formulae for kernels are known only in a small number of canonical encounter scenarios. Yet, encounter kernels can guide experimental efforts to control microbial interactions by elucidating how encounter rates depend on key phenotypic and environmental variables. Encounter kernels also provide physically grounded estimates for parameters that are used in ecological models of microbial populations. We illustrate this encounter-oriented perspective on microbial interactions by reviewing traditional and recently identified kernels describing encounters between microorganisms and between microorganisms and resources in aquatic systems.

Cell aggregation is associated with enzyme secretion strategies in marine polysaccharide-degrading bacteria.

Polysaccharide breakdown by bacteria requires the activity of enzymes that degrade polymers either intra- or extra-cellularly. The latter mechanism generates a localized pool of breakdown products that are accessible to the enzyme producers themselves as well as to other organisms. Marine bacterial taxa often show marked differences in the production and secretion of degradative enzymes that break down polysaccharides. These differences can have profound effects on the pool of diffusible breakdown products and hence on the ecological dynamics. However, the consequences of differences in enzymatic secretions on cellular growth dynamics and interactions are unclear. Here we study growth dynamics of single cells within populations of marine Vibrionaceae strains that grow on the abundant marine polymer alginate, using microfluidics coupled to quantitative single-cell analysis and mathematical modelling. We find that strains that have low extracellular secretions of alginate lyases aggregate more strongly than strains that secrete high levels of enzymes. One plausible reason for this observation is that low secretors require a higher cellular density to achieve maximal growth rates in comparison with high secretors. Our findings indicate that increased aggregation increases intercellular synergy amongst cells of low-secreting strains. By mathematically modelling the impact of the level of degradative enzyme secretion on the rate of diffusive oligomer loss, we find that enzymatic secretion capability modulates the propensity of cells within clonal populations to cooperate or compete with each other. Our experiments and models demonstrate that enzymatic secretion capabilities can be linked with the propensity of cell aggregation in marine bacteria that extracellularly catabolize polysaccharides.

Multiple roads lead to Rome: unique morphology and chemistry of endospores, exospores, myxospores, cysts and akinetes in bacteria.

The production of specialized resting cells is a remarkable survival strategy developed by many organisms to withstand unfavourable environmental factors such as nutrient depletion or other changes in abiotic and/or biotic conditions. Five bacterial taxa are recognized to form specialized resting cells: Firmicutes, forming endospores; Actinobacteria, forming exospores; Cyanobacteria, forming akinetes; the δ-Proteobacterial order Myxococcales, forming myxospores; and Azotobacteraceae, forming cysts. All these specialized resting cells are characterized by low-to-absent metabolic activity and higher resistance to environmental stress (desiccation, heat, starvation, etc.) when compared to vegetative cells. Given their similarity in function, we tested the potential existence of a universal morpho-chemical marker for identifying these specialized resting cells. After the production of endospores, exospores, akinetes and cysts in model organisms, we performed the first cross-species morphological and chemical comparison of bacterial sporulation. Cryo-electron microscopy of vitreous sections (CEMOVIS) was used to describe near-native morphology of the resting cells in comparison to the morphology of their respective vegetative cells. Resting cells shared a thicker cell envelope as their only common morphological feature. The chemical composition of the different specialized resting cells at the single-cell level was investigated using confocal Raman microspectroscopy. Our results show that the different specialized cells do not share a common chemical signature, but rather each group has a unique signature with a variable conservation of the signature of the vegetative cells. Additionally, we present the validation of Raman signatures associated with calcium dipicolinic acid (CaDPA) and their variation across individual cells to develop specific sorting thresholds for the isolation of endospores. This provides a proof of concept of the feasibility of isolating bacterial spores using a Raman-activated cell-sorting platform. This cross-species comparison and the current knowledge of genetic pathways inducing the formation of the resting cells highlights the complexity of this convergent evolutionary strategy promoting bacterial survival.

Intraoperative Patterns of Gastric Microperfusion During Laparoscopic Roux-en-Y Gastric Bypass.

INTRODUCTION: Visible light spectroscopy (VLS) represents a sensitive, non-invasive method to quantify tissue oxygen levels and detect hypoxemia. The aim of this study was to assess the microperfusion patterns of the gastric pouch during laparoscopic Roux-en-Y gastric bypass (LRYGB) using the VLS technique. METHODS: Twenty patients were enrolled. Tissue oxygenation (StO2%) measurements were performed at three different localizations of the gastric wall, prior and after the creation of the gastric pouch, and after the creation of the gastro-jejunostomy. RESULTS: Prior to the creation of the gastric pouch, the lowest StO2% levels were observed at the level of the distal esophagus with a median StO2% of 43 (IQR 40.8-49.5). After the creation of the gastric pouch and after the creation of the gastro-jejunostomy, the lowest StO2% levels were recorded at the level of the His angle with median values of 29% (IQR 20-38.5) and 34.5% (IQR 19-39), respectively. The highest mean StO2 reduction was recorded at the level of the His angle after the creation of the gastric pouch, and it was 18.3% (SD ± 18.1%, p < 0.001). A reduction of StO2% was recorded at all localizations after the formation of the gastro-jejunostomy compared to the beginning of the operation, but the mean differences of the StO2% levels were statistically significant only at the resection line of the pouch and at the His angle (p = 0.044 and p < 0.001, respectively). CONCLUSION: Gastric pouch demonstrates reduction of StO2% during LRYGB. VLS is a useful technique to assess microperfusion patterns of the stomach during LRYGB.

A Microfluidic Platform to Study Bioclogging in Porous Media.

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

Chemotaxis may assist marine heterotrophic bacterial diazotrophs to find microzones suitable for N2 fixation in the pelagic ocean.

Heterotrophic bacterial diazotrophs (HBDs) are ubiquitous in the pelagic ocean, where they have been predicted to carry out the anaerobic process of nitrogen fixation within low-oxygen microenvironments associated with marine pelagic particles. However, the mechanisms enabling particle colonization by HBDs are unknown. We hypothesized that HBDs use chemotaxis to locate and colonize suitable microenvironments, and showed that a cultivated marine HBD is chemotactic toward amino acids and phytoplankton-derived DOM. Using an in situ chemotaxis assay, we also discovered that diverse HBDs at a coastal site are motile and chemotactic toward DOM from various phytoplankton taxa and, indeed, that the proportion of diazotrophs was up to seven times higher among the motile fraction of the bacterial community compared to the bulk seawater community. Finally, three of four HBD isolates and 16 of 17 HBD metagenome assembled genomes, recovered from major ocean basins and locations along the Australian coast, each encoded >85% of proteins affiliated with the bacterial chemotaxis pathway. These results document the widespread capacity for chemotaxis in diverse and globally relevant marine HBDs. We suggest that HBDs could use chemotaxis to seek out and colonize low-oxygen microenvironments suitable for nitrogen fixation, such as those formed on marine particles. Chemotaxis in HBDs could therefore affect marine nitrogen and carbon biogeochemistry by facilitating nitrogen fixation within otherwise oxic waters, while also altering particle degradation and the efficiency of the biological pump.

Random encounters and amoeba locomotion drive the predation of Listeria monocytogenes by Acanthamoeba castellanii.

Predatory protozoa play an essential role in shaping microbial populations. Among these protozoa, Acanthamoeba are ubiquitous in the soil and aqueous environments inhabited by Listeria monocytogenes. Observations of predator-prey interactions between these two microorganisms revealed a predation strategy in which Acanthamoeba castellanii assemble L. monocytogenes in aggregates, termed backpacks, on their posterior. The rapid formation and specific location of backpacks led to the assumption that A. castellanii may recruit L. monocytogenes by releasing an attractant. However, this hypothesis has not been validated, and the mechanisms driving this process remained unknown. Here, we combined video microscopy, microfluidics, single-cell image analyses, and theoretical modeling to characterize predator-prey interactions of A. castellanii and L. monocytogenes and determined whether bacterial chemotaxis contributes to the backpack formation. Our results indicate that L. monocytogenes captures are not driven by chemotaxis. Instead, random encounters of bacteria with amoebae initialize bacterial capture and aggregation. This is supported by the strong correlation between experimentally derived capture rates and theoretical encounter models at the single-cell level. Observations of the spatial rearrangement of L. monocytogenes trapped by A. castellanii revealed that bacterial aggregation into backpacks is mainly driven by amoeboid locomotion. Overall, we show that two nonspecific, independent mechanisms, namely random encounters enhanced by bacterial motility and predator surface-bound locomotion, drive backpack formation, resulting in a bacterial aggregate on the amoeba ready for phagocytosis. Due to the prevalence of these two processes in the environment, we expect this strategy to be widespread among amoebae, contributing to their effectiveness as predators.

Elongation enhances encounter rates between phytoplankton in turbulence.

Phytoplankton come in a stunning variety of shapes but elongated morphologies dominate-typically 50% of species have aspect ratio above 5, and bloom-forming species often form chains whose aspect ratios can exceed 100. How elongation affects encounter rates between phytoplankton in turbulence has remained unknown, yet encounters control the formation of marine snow in the ocean. Here, we present simulations of encounters among elongated phytoplankton in turbulence, showing that encounter rates between neutrally buoyant elongated cells are up to 10-fold higher than for spherical cells and even higher when cells sink. Consequently, we predict that elongation can significantly speed up the formation of marine snow compared to spherical cells. This unexpectedly large effect of morphology in driving encounter rates among plankton provides a potential mechanistic explanation for the rapid clearance of many phytoplankton blooms.

Competition between growth and shear stress drives intermittency in preferential flow paths in porous medium biofilms.

Bacteria in porous media, such as soils, aquifers, and filters, often form surface-attached communities known as biofilms. Biofilms are affected by fluid flow through the porous medium, for example, for nutrient supply, and they, in turn, affect the flow. A striking example of this interplay is the strong intermittency in flow that can occur when biofilms nearly clog the porous medium. Intermittency manifests itself as the rapid opening and slow closing of individual preferential flow paths (PFPs) through the biofilm-porous medium structure, leading to continual spatiotemporal rearrangement. The drastic changes to the flow and mass transport induced by intermittency can affect the functioning and efficiency of natural and industrial systems. Yet, the mechanistic origin of intermittency remains unexplained. Here, we show that the mechanism driving PFP intermittency is the competition between microbial growth and shear stress. We combined microfluidic experiments quantifying Bacillus subtilis biofilm formation and behavior in synthetic porous media for different pore sizes and flow rates with a mathematical model accounting for flow through the biofilm and biofilm poroelasticity to reveal the underlying mechanisms. We show that the closing of PFPs is driven by microbial growth, controlled by nutrient mass flow. Opposing this, we find that the opening of PFPs is driven by flow-induced shear stress, which increases as a PFP becomes narrower due to microbial growth, causing biofilm compression and rupture. Our results demonstrate that microbial growth and its competition with shear stresses can lead to strong temporal variability in flow and transport conditions in bioclogged porous media.

A microfluidic platform for characterizing the structure and rheology of biofilm streamers.

Biofilm formation is the most successful survival strategy for bacterial communities. In the biofilm lifestyle, bacteria embed themselves in a self-secreted matrix of extracellular polymeric substances (EPS), which acts as a shield against mechanical and chemical insults. When ambient flow is present, this viscoelastic scaffold can take a streamlined shape, forming biofilm filaments suspended in flow, called streamers. Streamers significantly disrupt the fluid flow by causing rapid clogging and affect transport in aquatic environments. Despite their relevance, the structural and rheological characterization of biofilm streamers is still at an early stage. In this work, we present a microfluidic platform that allows the reproducible growth of biofilm streamers in controlled physico-chemical conditions and the characterization of their biochemical composition, morphology, and rheology in situ. We employed isolated micropillars as nucleation sites for the growth of single biofilm streamers under the continuous flow of a diluted bacterial suspension. By combining fluorescent staining of the EPS components and epifluorescence microscopy, we were able to characterize the biochemical composition and morphology of the streamers. Additionally, we optimized a protocol to perform hydrodynamic stress tests in situ, by inducing controlled variations of the fluid shear stress exerted on the streamers by the flow. Thus, the reproducibility of the formation process and the testing protocol make it possible to perform several consistent experimental replicates that provide statistically significant information. By allowing the systematic investigation of the role of biochemical composition on the structure and rheology of streamers, this platform will advance our understanding of biofilm formation.