Biofilm Streamers Research Articles

Mesoscopic ring element growth and deformation induced biofilm streamer evolution in microfluidic channels.

In a fluid environment, biofilms usually form and grow into streamers attached to solid surfaces. Existing research on single streamers studied their formation and failure modes. In the experiment on biofilm growth in a microfluidic channel, we found that rings composed of bacteria and an extracellular matrix are important elements on a mesoscopic scale. In the fluid environment, the failure of these ring elements causes damage to streamers. We simulated the growth and deformation of the ring structure in the micro-channel using multi-agent simulation and fluid-structure coupling of a porous elastic body. Based on this, we simulated the biofilm evolution involving multi-ring deformation, which provides a new length scale to study the biofilm streamer dynamics in fluid environments.

Genotoxic stress stimulates eDNA release via explosive cell lysis and thereby promotes streamer formation of Burkholderia cenocepacia H111 cultured in a microfluidic device

DNA is a component of biofilms, but the triggers of DNA release during biofilm formation and how DNA contributes to biofilm development are poorly investigated. One key mechanism involved in DNA release is explosive cell lysis, which is a consequence of prophage induction. In this article, the role of explosive cell lysis in biofilm formation was investigated in the opportunistic human pathogen Burkholderia cenocepacia H111 (H111). Biofilm streamers, flow-suspended biofilm filaments, were used as a biofilm model in this study, as DNA is an essential component of their matrix. H111 contains three prophages on chromosome 1 of its genome, and the involvement of each prophage in causing explosive cell lysis of the host and subsequent DNA and membrane vesicle (MV) release, as well as their contribution to streamer formation, were studied in the presence and absence of genotoxic stress. The results show that two of the three prophages of H111 encode functional lytic prophages that can be induced by genotoxic stress and their activation causes DNA and MVs release by explosive cell lysis. Furthermore, it is shown that the released DNA enables the strain to develop biofilm streamers, and streamer formation can be enhanced by genotoxic stress. Overall, this study demonstrates the involvement of prophages in streamer formation and uncovers an often-overlooked problem with the use of antibiotics that trigger the bacterial SOS response for the treatment of bacterial infections.

A microtiter peg lid with ziggurat geometry for medium-throughput antibiotic testing and in situ imaging of biofilms

Bacteria biofilm responses to disinfectants and antibiotics are quantified and observed using multiple methods, though microscopy, particularly confocal laser scanning microscopy (CLSM) is preferred due to speed, a reduction in user error, and in situ analysis. CLSM can resolve biological and spatial heterogeneity of biofilms in 3D with limited throughput. The microplate peg-lid-based assay, described in ASTM E2799-22, is a medium-throughput method for testing biofilms but does not permit in situ imaging. Breaking off the peg, as recommended by the manufacturer, risks sample damage, and is limited to easily accessible pegs. Here we report modifications to the peg optimized for in situ visualization and visualization of all pegs. We report similar antibiotic challenge recovery via colony formation following the ASTM E2799-22 protocol and in situ imaging. We report novel quantifiable effects of antibiotics on biofilm morphologies, specifically biofilm streamers. The new design bridges the MBEC® assays design that selects for biofilm phenotypes with in situ imaging needs.

Rapid formation of bioaggregates and morphology transition to biofilm streamers induced by pore-throat flows

Bioaggregates are condensed porous materials comprising microbes, organic and inorganic matters, and water. They are commonly found in natural and engineered porous media and often cause clogging. Despite their importance, the formation mechanism of bioaggregates in porous media systems is largely unknown. Through microfluidic experiments and direct numerical simulations of fluid flow, we show that the rapid bioaggregation is driven by the interplay of the viscoelastic nature of biomass and hydrodynamic conditions at pore throats. At an early stage, unique flow structures around a pore throat promote the biomass attachment at the throat. Then, the attached biomass fluidizes when the shear stress at the partially clogged pore throat reaches a critical value. After the fluidization, the biomass is displaced and accumulated in the expansion region of throats forming bioaggregates. We further find that such criticality in shear stress triggers morphological changes in bioaggregates from rounded- to streamer-like shapes. This knowledge was used to control the clogging of throats by tuning the flow conditions: When the shear stress at the throat exceeded the critical value, clogging was prevented. The bioaggregation process did not depend on the detailed pore-throat geometry, as we reproduced the same dynamics in various pore-throat geometries. This study demonstrates that pore-throat structures, which are ubiquitous in porous media systems, induce bioaggregation and can lead to abrupt disruptions in flow.

Early biofilm and streamer formation is mediated by wall shear stress and surface wettability: A multifactorial microfluidic study.

Biofilms are intricate communities of microorganisms encapsulated within a self‐produced matrix of extra‐polymeric substances (EPS), creating complex three‐dimensional structures allowing for liquid and nutrient transport through them. These aggregations offer constituent microorganisms enhanced protection from environmental stimuli—like fluid flow—and are also associated with higher resistance to antimicrobial compounds, providing a persistent cause of concern in numerous sectors like the marine (biofouling and aquaculture), medical (infections and antimicrobial resistance), dentistry (plaque on teeth), food safety, as well as causing energy loss and corrosion. Recent studies have demonstrated that biofilms interact with microplastics, often influencing their pathway to higher trophic levels. Previous research has shown that initial bacterial attachment is affected by surface properties. Using a microfluidic flow cell, we have investigated the relationship between both wall shear stress (τw) and surface properties (surface wettability) upon biofilm formation of two species (Cobetia marina and Pseudomonas aeruginosa). We investigated biofilm development on low‐density polyethylene (LDPE) membranes, Permanox® slides, and glass slides, using nucleic acid staining and end‐point confocal laser scanning microscopy. The results show that flow conditions affect biomass, maximum thickness, and surface area of biofilms, with higher τw (5.6 Pa) resulting in thinner biofilms than lower τw (0.2 Pa). In addition, we observed differences in biofilm development across the surfaces tested, with LDPE typically demonstrating more overall biofilm in comparison to Permanox® and glass. Moreover, we demonstrate the formation of biofilm streamers under laminar flow conditions within straight micro‐channels.

The structural role of bacterial eDNA in the formation of biofilm streamers

Across diverse habitats, bacteria are mainly found as biofilms, surface-attached communities embedded in a self-secreted matrix of extracellular polymeric substances (EPS), which enhance bacterial recalcitrance to antimicrobial treatment and mechanical stresses. In the presence of flow and geometric constraints such as corners or constrictions, biofilms can take the form of long, suspended filaments (streamers), which bear important consequences in industrial and clinical settings by causing clogging and fouling. The formation of streamers is thought to be driven by the viscoelastic nature of the biofilm matrix. Yet, little is known about the structural composition of streamers and how it affects their mechanical properties. Here, using a microfluidic platform that allows growing and precisely examining biofilm streamers, we show that extracellular DNA (eDNA) constitutes the backbone and is essential for the mechanical stability of Pseudomonas aeruginosa streamers. This finding is supported by the observations that DNA-degrading enzymes prevent the formation of streamers and clear already formed ones and that the antibiotic ciprofloxacin promotes their formation by increasing the release of eDNA. Furthermore, using mutants for the production of the exopolysaccharide Pel, an important component of P. aeruginosa EPS, we reveal an concurring role of Pel in tuning the mechanical properties of the streamers. Taken together, these results highlight the importance of eDNA and of its interplay with Pel in determining the mechanical properties of P. aeruginosa streamers and suggest that targeting the composition of streamers can be an effective approach to control the formation of these biofilm structures.

Biofilm streamer growth dynamics in various microfluidic channels.

Biofilms are microbial colonies that are encapsulated in extracellular polymers secreted by cells through their proliferation and differentiation. Biofilms exist on solid surfaces, liquid surfaces, or in liquid media, where the growth of the bacterial biofilm is closely related to the velocity of the secondary flow, main flow, and geometry of the channel, which are difficult to measure in a natural fluid environment, making the study of the biofilm streamer growth process difficult. In this study, we used microfluidic channels made of polydimethylsiloxane to study the growth dynamics of Bacillus subtilis biofilm streamers. We observed that the biofilm streamer growth undergoes three stages with different growth characteristics. First, we found that the initial growth of the streamer is located at the position with the maximum value of P = secondary flow velocity × main flow velocity. Second, the biofilm underwent floating growth around the microcolumn obstacle. After the transition stage, the last growth stage includes two types because of the different attachment strengths and mechanical properties of the biofilm. Our research provides new insights into the formation and shedding of biofilm streamers in natural and industrial environments and helps us to better understand biofilm growth in fluid flow.

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.

Late Ediacaran life on land: desiccated microbial mats and large biofilm streamers.

The Ediacaran period witnessed transformational change across the Earth–life system, but life on land during this interval is poorly understood. Non-marine/transitional Ediacaran sediments preserve a variety of probable microbially induced sedimentary structures and fossil matgrounds, and the ecology, biogeochemistry and sedimentological impacts of the organisms responsible are now ripe for investigation. Here, we report well-preserved fossils from emergent siliciclastic depositional environments in the Ediacaran of Newfoundland, Canada. These include exquisite, mouldically preserved microbial mats with desiccation cracks and flip-overs, abundant Arumberia-type fossils and, most notably, assemblages of centimetre-to-metre-scale, subparallel, branching, overlapping, gently curving ribbon-like features preserved by aluminosilicate and phosphate minerals, with associated filamentous microfossils. We present morphological, petrographic and taphonomic evidence that the ribbons are best interpreted as fossilized current-induced biofilm streamers, the earliest record of an important mode of life (macroscopic streamer formation) for terrestrial microbial ecosystems today. Their presence shows that late Ediacaran terrestrial environments could produce substantial biomass, and supports recent interpretations of Arumberia as a current-influenced microbial mat fossil, which we here suggest existed on a ‘streamer–arumberiamorph spectrum’. Finally, the absence of classic Ediacaran macrobiota from these rocks despite evidently favourable conditions for soft tissue preservation upholds the consensus that those organisms were exclusively marine.

Zooming in to acquire micro-reaction: Application of microfluidics on soil microbiome

Microfluidics confers unique advantages in microbiological studies as these devices can accurately replicate the micro- and even nano-scale structures of soil to simulate the habitats of bacteria. It not only helps us understand the spatial distribution of bacterial communities (such as biofilms), but also provides mechanistic insights into microbial behaviors including chemotaxis and horizontal gene transfer (HGT). Microfluidics provides a feasible means for real-time, in situ studies and enables in-depth exploration of the mechanisms of interactions in the soil microbiome. This review aims to introduce the basic principles of microfluidic technology and summarize the recent progress in microfluidic devices to study bacterial spatial distribution and functions, as well as biological processes, such bacterial chemotaxis, biofilm streamers (BS), quorum sensing (QS), and HGT. The challenges in and future development of microfluidics for soil microbiological studies are also discussed.

CFD-DEM modelling of biofilm streamer oscillations and their cohesive failure in fluid flow.

Biofilm streamer motion under different flow conditions is important for a wide range of industries. The existing work has largely focused on experimental characterisations of these streamers, which is often time-consuming and expensive. To better understand the physics of biofilm streamer oscillation and their interactions in fluid flow, a computational fluid dynamics-discrete element methodmodel has been developed. The model was used to study the flow-induced oscillations and cohesive failure of single and multiple biofilm streamers. We have studied the effect of streamer length on the oscillation at varied flow rates. The predicted single biofilm streamer oscillations in various flow rates agreed well with experimental measurements. We have also investigated the effect of the spatial arrangement of streamers on interactions between two oscillating streamers in parallel and tandem arrangements. Furthermore, cohesive failure of streamers was studied in an accelerating fluid flow, which is important for slowing down biofilm-induced clogging.

A mechanistic Individual-based Model of microbial communities.

Accurate predictive modelling of the growth of microbial communities requires the credible representation of the interactions of biological, chemical and mechanical processes. However, although biological and chemical processes are represented in a number of Individual-based Models (IbMs) the interaction of growth and mechanics is limited. Conversely, there are mechanically sophisticated IbMs with only elementary biology and chemistry. This study focuses on addressing these limitations by developing a flexible IbM that can robustly combine the biological, chemical and physical processes that dictate the emergent properties of a wide range of bacterial communities. This IbM is developed by creating a microbiological adaptation of the open source Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS). This innovation should provide the basis for “bottom up” prediction of the emergent behaviour of entire microbial systems. In the model presented here, bacterial growth, division, decay, mechanical contact among bacterial cells, and adhesion between the bacteria and extracellular polymeric substances are incorporated. In addition, fluid-bacteria interaction is implemented to simulate biofilm deformation and erosion. The model predicts that the surface morphology of biofilms becomes smoother with increased nutrient concentration, which agrees well with previous literature. In addition, the results show that increased shear rate results in smoother and more compact biofilms. The model can also predict shear rate dependent biofilm deformation, erosion, streamer formation and breakup.

The Mechanical Analysis of the Biofilm Streamer Nucleation and Geometry Characterization in Microfluidic Channels.

Bacteria can form biofilm streamers in microfluidic channels with various geometries. Experiments show that the streamer geometry, such as its shape or thickness, depends on the fluid velocity and the geometry and curvature of the microfluidic channel. In the paper, a mechanical analysis of the flow field is made in different channels, which shows that the secondary flow in the channel is the reason for streamer nucleation and that the shear stress distribution decides the streamer geometry including shape and thickness. Through a finite elements simulation, we obtain the secondary flow forming positions in both static and rotating channels: positions that are the location of nucleation of the streamer. Thick or wide biofilm streamers occur at the points of minimum shear stress in static channels. Furthermore, in rotating channels, spiral-like streamers form, due to the helical shape of the minimum shear stress distribution. The findings may allow the prevention of biofilm formation and also the removal of bacteria adhered onto certain surfaces in channels with small cross sections. The analysis also indicates how one can obtain desirable biofilm streamers by control of the channel geometry and the loading conditions.

Formation and post-formation dynamics of bacterial biofilm streamers as highly viscous liquid jets.

It has been recently reported that in presence of low Reynolds number (Re ≪ 1) transport, preformed bacterial biofilms, several hours after their formation, may degenerate in form of filamentous structures, known as streamers. In this work, we explain that such streamers form as the highly viscous liquid states of the intrinsically viscoelastic biofilms. Such “viscous liquid” state can be hypothesized by noting that the time of appearance of the streamers is substantially larger than the viscoelastic relaxation time scale of the biofilms, and this appearance is explained by the inability of a viscous liquid to withstand external shear. Further, by identifying the post formation dynamics of the streamers as that of a viscous liquid jet in a surrounding flow field, we can interpret several unexplained issues associated with the post-formation dynamics of streamers, such as the clogging of the flow passage or the exponential time growth of streamer dimensions. Overall our manuscript provides a biophysical basis for understanding the evolution of biofilm streamers in creeping flows.

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Protocol for biofilm streamer formation in a microfluidic device with micro-pillars.

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Filaments in curved streamlines: rapid formation of Staphylococcus aureus biofilm streamers

Biofilms are surface-associated conglomerates of bacteria that are highly resistant to antibiotics. These bacterial communities can cause chronic infections in humans by colonizing, for example, medical implants, heart valves, or lungs. Staphylococcus aureus, a notorious human pathogen, causes some of the most common biofilm-related infections. Despite the clinical importance of S. aureus biofilms, it remains mostly unknown how physical effects, in particular flow, and surface structure influence biofilm dynamics. Here we use model microfluidic systems to investigate how environmental factors, such as surface geometry, surface chemistry, and fluid flow affect biofilm development of S. aureus. We discovered that S. aureus rapidly forms flow-induced, filamentous biofilm streamers, and furthermore if surfaces are coated with human blood plasma, streamers appear within minutes and clog the channels more rapidly than if the channels are uncoated. To understand how biofilm streamer filaments reorient in flows with curved streamlines to bridge the distances between corners, we developed a mathematical model based on resistive force theory of slender filaments. Understanding physical aspects of biofilm formation of S. aureus may lead to new approaches for interrupting biofilm formation of this pathogen.

Assessment and Interpretation of the Internal Structure of Biofilm Streamers by Combination of Imaging Techniques on Two Scales

Assessment and Interpretation of the Internal Structure of Biofilm Streamers by Combination of Imaging Techniques on Two Scales

Rapid Formation and Flow Around Staphylococcus Aureus Biofilm Streamers

Bacterial biofilms are surface-associated conglomerates of bacteria that are highly resistant to antibiotics. These bacterial communities can cause chronic infections in humans by colonizing, for example, medical implants, heart valves, or lungs. Staphylococcus aureus, a notorious human pathogen, causes some of the most common biofilm-related infections. Despite the clinical importance of S. aureus biofilms, it remains mostly unknown how physical effects, and in particular flow, shape the morphology and growth dynamics of biofilms. Here we use model microfluidic systems to investigate how environmental factors, such as surface geometry, surface chemistry, and fluid flow affect the biofilm development in S. aureus. We discovered that S. aureus rapidly forms flow-induced, filamentous biofilm streamers and that if surfaces are coated with human blood plasma, streamers appear within minutes and clog the channels more rapidly than if the channels are uncoated. We document and model the deformation of the flow field generated by the streamers. Understanding physical aspects of biofilm formation in S. aureus may lead to new approaches for interrupting biofilm formation of this pathogen.

Biofilm streamers cause catastrophic disruption of flow with consequences for environmental and medical systems

Biofilms are antibiotic-resistant, sessile bacterial communities that occupy most moist surfaces on Earth and cause chronic and medical device-associated infections. Despite their importance, basic information about biofilm dynamics in common ecological environments is lacking. Here, we demonstrate that flow through soil-like porous materials, industrial filters, and medical stents dramatically modifies the morphology of Pseudomonas aeruginosa biofilms to form 3D streamers, which, over time, bridge the spaces between obstacles and corners in nonuniform environments. We discovered that accumulation of surface-attached biofilm has little effect on flow through such environments, whereas biofilm streamers cause sudden and rapid clogging. We demonstrate that flow-induced shedding of extracellular matrix from surface-attached biofilms generates a sieve-like network that captures cells and other biomass, which add to the existing network, causing exponentially fast clogging independent of growth. These results suggest that biofilm streamers are ubiquitous in nature and strongly affect flow through porous materials in environmental, industrial, and medical systems.