Formation of bacterial streamers during filtration in microfluidic systems

Bacterial behavior during filtration is complex and is influenced by numerous factors. The aim of this paper is to report on experiments designed to make progress in the understanding of bacterial transfer in filters and membranes. Polydimethylsiloxane (PDMS) microsystems were built to allow direct dynamic observation of bacterial transfer across different microchannel geometries mimicking filtration processes. When filtering Escherichia coli suspensions in such devices, the bacteria accumulated in the downstream zone of the filter forming long streamers undulating in the flow. Confocal microscopy and 3D reconstruction of streamers showed how the streamers are connected to the filter and how they form in the stream. Streamer development was found to be influenced by the flow configuration and the presence of connections or tortuosity between channels. Experiments showed that streamer formation was greatest in a filtration system composed of staggered arrays of squares 10 μm apart.     

Robust scale-up of dead end filtration: impact of filter fouling mechanisms and flow distribution

Robust design of a dead end filtration step and the resulting performance at manufacturing scale relies on laboratory data collected with small filter units. During process development it is important to characterize and understand the filter fouling mechanisms of the process streams so that an accurate assessment can be made of the filter area required at manufacturing scale. Successful scale-up also requires integration of the lab-scale filtration data with an understanding of flow characteristics in the full-scale filtration equipment. A case study is presented on the development and scale-up of a depth filtration step used in a 2nd generation polysaccharide vaccine manufacturing process. The effect of operating parameters on filter performance was experimentally characterized for a diverse set of process streams. Filter capacity was significantly reduced when operating at low fluxes, caused by both low filtration pressure and high stream viscosity. The effect of flux on filter capacity could be explained for a variety of diverse streams by a single mechanistic model of filter fouling. To complement the laboratory filtration data, the fluid flow and distribution characteristics in manufacturing-scale filtration equipment were carefully evaluated. This analysis identified the need for additional scale-up factors to account for non-uniform filter area usage in large-scale filter housings. This understanding proved critical to the final equipment design and depth filtration step definition, resulting in robust process performance at manufacturing scale.     

Biophysical Controls on Community Succession in Stream Biofilms

Biofilm formation is controlled by an array of coupled physical, chemical, and biotic processes. Despite the ecological relevance of microbial biofilms, their community formation and succession remain poorly understood. We investigated the effect of flow velocity, as the major physical force in stream ecosystems, on biofilm community succession (as continuous shifts in community composition) in microcosms under laminar, intermediate, and turbulent flow. Flow clearly shaped the development of biofilm architecture and community composition, as revealed by microscopic investigation, denaturing gradient gel electrophoresis (DGGE) analysis, and sequencing. While biofilm growth patterns were undirected under laminar flow, they were clearly directed into ridges and conspicuous streamers under turbulent flow. A total of 51 biofilm DGGE bands were detected; the average number ranged from 13 to 16. Successional trajectories diverged from an initial community that was common in all flow treatments and increasingly converged as biofilms matured. We suggest that this developmental pattern was primarily driven by algae, which, as “ecosystem engineers,” modulate their microenvironment to create similar architectures and flow conditions in all treatments and thereby reduce the physical effect of flow on biofilms. Our results thus suggest a shift from a predominantly physical control to coupled biophysical controls on bacterial community succession in stream biofilms.     

Biophysical controls on community succession in stream biofilms

Biofilm formation is controlled by an array of coupled physical, chemical, and biotic processes. Despite the ecological relevance of microbial biofilms, their community formation and succession remain poorly understood. We investigated the effect of flow velocity, as the major physical force in stream ecosystems, on biofilm community succession (as continuous shifts in community composition) in microcosms under laminar, intermediate, and turbulent flow. Flow clearly shaped the development of biofilm architecture and community composition, as revealed by microscopic investigation, denaturing gradient gel electrophoresis (DGGE) analysis, and sequencing. While biofilm growth patterns were undirected under laminar flow, they were clearly directed into ridges and conspicuous streamers under turbulent flow. A total of 51 biofilm DGGE bands were detected; the average number ranged from 13 to 16. Successional trajectories diverged from an initial community that was common in all flow treatments and increasingly converged as biofilms matured. We suggest that this developmental pattern was primarily driven by algae, which, as "ecosystem engineers," modulate their microenvironment to create similar architectures and flow conditions in all treatments and thereby reduce the physical effect of flow on biofilms. Our results thus suggest a shift from a predominantly physical control to coupled biophysical controls on bacterial community succession in stream biofilms.     

Investigation of virus retention by size exclusion membranes under different flow regimes

Virus removal by filter membranes is regarded as a robust and efficient unit operation, which is frequently applied in the downstream processing of biopharmaceuticals. The retention of viruses by virus filtration membranes is predominantly based on size exclusion. However, recent results using model membranes and bacteriophage PP7 point to the fact that virus retention can also significantly be influenced by adsorptive interactions between virus, product molecules, and membranes. Furthermore, the impact of flow rate and flow interruptions on virus retention have been studied and responsible mechanisms discussed. The aim of this investigation was to gain a holistic understanding of the underlying mechanisms for virus retention in size exclusion membranes as a function of membrane structure and membrane surface properties, as well as flow and solution conditions. The results of this study contribute to the differentiation between size exclusion and adsorptive effects during virus filtration and broaden the current understanding of mechanisms related to virus breakthroughs after temporary flow interruptions. Within the frame of a Design of Experiments approach it was found that the level of retention of virus filtration membranes was mostly influenced by the membrane structure during typical process-related flow conditions. The retention performance after a flow interruption was also significantly influenced by membrane surface properties and solution conditions. While size exclusion was confirmed as main retention mechanism, the analysis of all results suggests that especially after a flow interruption virus retention can be influenced by adsorptive effects between the virus and the membrane surface. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2747, 2019.     

Connecting through space and time: catchment-scale distributions of bacteria in soil,stream water and sediment

Terrestrial and aquatic environments are linked through hydrological networks that transport abiotic components from upslope environments into aquatic ecosystems. However, our understanding of how bacteria are transported through these same networks is limited. Here, we applied 16S rRNA gene sequencing to over 500 soil, stream water and stream sediment samples collected within a native forest catchment to determine the extent to which bacterial communities in these habitats are connected. We provide evidence that while the bacterial communities in each habitat were significantly distinct from one another (PERMANOVA pairwise P < 0.001), the bacterial communities in soil and stream samples were weakly connected to each other when stream sediment sample locations were downhill of surface runoff flow paths. This pattern decreased with increasing distance between the soil and sediment samples. The connectivity between soil and stream water samples was less apparent and extremely transient; the greatest similarity between bacterial communities in soil and stream water overall was when comparing stream samples collected 1 week post soil sampling. This study shows how bacterial communities in soil, stream water and stream sediments are connected at small spatial scales and provides rare insights into the temporal dynamics of terrestrial and aquatic bacterial community connectivity.     

Capture of bacteria from fermentation broth by body feed filtration: a solved problem?

The direct capture of bacteria produced in high cell density fermentation by filtration is not possible once the milliliter-scale has been surpassed. Filtration in the presence of a filter aid (body feed filtration) constitutes a putative and scalable alternative, but only if conditions proposed by industry for large-scale filtration processes, namely, flow rates (for aqueous solutions) in the range of 500-1,500 L/(m(2) x h) and a filter aid concentration of 相似文献   

Secondary flow as a mechanism for the formation of biofilm streamers

In most environments, such as natural aquatic systems, bacteria are found predominantly in self-organized sessile communities known as biofilms. In the presence of a significant flow, mature multispecies biofilms often develop into long filamentous structures called streamers, which can greatly influence ecosystem processes by increasing transient storage and cycling of nutrients. However, the interplay between hydrodynamic stresses and streamer formation is still unclear. Here, we show that suspended thread-like biofilms steadily develop in zigzag microchannels with different radii of curvature. Numerical simulations of a low-Reynolds-number flow around these corners indicate the presence of a secondary vortical motion whose intensity is related to the bending angle of the turn. We demonstrate that the formation of streamers is directly proportional to the intensity of the secondary flow around the corners. In addition, we show that a model of an elastic filament in a two-dimensional corner flow is able to explain how the streamers can cross fluid streamlines and connect corners located at the opposite sides of the channel.     

Macroscopic streamer growths in acidic, metal-rich mine waters in north wales consist of novel and remarkably simple bacterial communities

The microbial composition of acid streamers (macroscopic biofilms) in acidic, metal-rich waters in two locations (an abandoned copper mine and a chalybeate spa) in north Wales was studied using cultivation-based and biomolecular techniques. Known chemolithotrophic and heterotrophic acidophiles were readily isolated from disrupted streamers, but they accounted for only <1 to 7% of the total microorganisms present. Fluorescent in situ hybridization (FISH) revealed that 80 to 90% of the microbes in both types of streamers were beta-Proteobacteria. Terminal restriction fragment length polymorphism analysis of the streamers suggested that a single bacterial species was dominant in the copper mine streamers, while two distinct bacteria (one of which was identical to the bacterium found in the copper mine streamers) accounted for about 90% of the streamers in the spa water. 16S rRNA gene clone libraries showed that the beta-proteobacterium found in both locations was closely related to a clone detected previously in acid mine drainage in California and that its closest characterized relatives were neutrophilic ammonium oxidizers. Using a modified isolation technique, this bacterium was isolated from the copper mine streamers and shown to be a novel acidophilic autotrophic iron oxidizer. The beta-proteobacterium found only in the spa streamers was closely related to the neutrophilic iron oxidizer Gallionella ferruginea. FISH analysis using oligonucleotide probes that targeted the two beta-proteobacteria confirmed that the biodiversity of the streamers in both locations was very limited. The microbial compositions of the acid streamers found at the two north Wales sites are very different from the microbial compositions of the previously described acid streamers found at Iron Mountain, California, and the Rio Tinto, Spain.     

Computational Fluid Dynamics of Fish Gill Rakers During Crossflow Filtration

We study crossflow filtration mechanisms in suspension-feeding fishes using computational fluid dynamics to model fluid flow and food particle movement in the vicinity of the gill rakers. During industrial and biological crossflow filtration, particles are retained when they remain suspended in the mainstream flow traveling across the filter surface rather than traveling perpendicularly to the filter. Here we identify physical parameters and hydrodynamic processes that determine food particle movement and retention inside the fish oral cavity. We demonstrate how five variables affect flow patterns and particle trajectories: (1) flow speed inside the fish oral cavity, (2) incident angle of the flow approaching the filter, (3) dimensions of filter structures, (4) particle size, and (5) particle density. Our study indicates that empirical experiments are needed to quantify flow parameters inside the oral cavity, and morphological research is needed to quantify dimensions of the filter apparatus such as gill rakers, the gaps between rakers, and downstream barriers. Ecological studies on suspension-feeding fishes are also needed to quantify food particle size and density, as these variables can affect particle retention due to hydrodynamic processes during crossflow filtration.     

Air Filters for Germ-free Isolators

A germ-free isolator must have a perfect bacterial filter. This paper describes a new, relatively inexpensive, stainless-steel filter frame, which is easily and quickly assembled and protects the enclosed filter material at all times. Resistance to the flow of air was less than 4 inches of water at an airflow of 30 ft³/min through the filter frame with 204 inches² of surface area and four, one-half inch thick pieces of fiberglass filter material. This filter performed satisfactorily in our gnotobiotic laboratory and was found to be consistently 100% efficient in removing an aerosol containing Serratia marcescens from an air stream under a variety of operating conditions.     

Macroscopic Streamer Growths in Acidic, Metal-Rich Mine Waters in North Wales Consist of Novel and Remarkably Simple Bacterial Communities

The microbial composition of acid streamers (macroscopic biofilms) in acidic, metal-rich waters in two locations (an abandoned copper mine and a chalybeate spa) in north Wales was studied using cultivation-based and biomolecular techniques. Known chemolithotrophic and heterotrophic acidophiles were readily isolated from disrupted streamers, but they accounted for only <1 to 7% of the total microorganisms present. Fluorescent in situ hybridization (FISH) revealed that 80 to 90% of the microbes in both types of streamers were β-Proteobacteria. Terminal restriction fragment length polymorphism analysis of the streamers suggested that a single bacterial species was dominant in the copper mine streamers, while two distinct bacteria (one of which was identical to the bacterium found in the copper mine streamers) accounted for about 90% of the streamers in the spa water. 16S rRNA gene clone libraries showed that the β-proteobacterium found in both locations was closely related to a clone detected previously in acid mine drainage in California and that its closest characterized relatives were neutrophilic ammonium oxidizers. Using a modified isolation technique, this bacterium was isolated from the copper mine streamers and shown to be a novel acidophilic autotrophic iron oxidizer. The β-proteobacterium found only in the spa streamers was closely related to the neutrophilic iron oxidizer Gallionella ferruginea. FISH analysis using oligonucleotide probes that targeted the two β-proteobacteria confirmed that the biodiversity of the streamers in both locations was very limited. The microbial compositions of the acid streamers found at the two north Wales sites are very different from the microbial compositions of the previously described acid streamers found at Iron Mountain, California, and the Rio Tinto, Spain.     

Filtration of bacteria and yeast by ultrasound-enhanced sedimentation

Continuous flow filtration of suspensions of eukaryotic cells by ultrasonic standing wave enhanced sedimentation has recently been reported. The filtration efficiency for Escherichia coli in such a filter has been characterized at frequencies of 1 and 3 MHz in the present work and compared with results for Saccharomyces cerevisiae. The yeast can be filtered at greater than 99% efficiency at a flow rate of 5 ml min^-1 at either frequency. The filtration efficiency of the smaller E. coli at 3 MHz is in excess of 80% at concentrations in the region of 10¹⁰ mI^-1 but decreased at lower concentrations. However, E. coli in a mixed suspension with yeast were, because of inter-particle interactions, removed with the filtrate at an efficiency ranging from 80 to 50% over the eight orders of bacterial concentrations tested (down to 10³ mI^-1) at 3 MHz. Quantitative considerations show that poor filtration of pure suspensions of the smaller cells at the lower frequency arises because, at reasonable flow rates, the residence time is not sufficient for the cells to reach the pressure nodal cell concentration regions. The filtration efficiencies of both cell types are comparable at 3 MHz. It is suggested that the more comparable efficiencies arise because concentration regions are narrower at the high frequency and Stokes drag by the filter bulk flow inhibits sedimentation of the concentrated cells.     

Mass transfer enhancement in moving biofilm structures

Biofilms are layers of microbial cells growing on an interface and they can form highly complex structures adapted to a wide variety of environmental conditions. Biofilm streamers have a small immobile base attached to the support and a flexible tail elongated in the flow direction, which can vibrate in fast flows. Herein we report numerical results for the role of the periodical movement of biofilm streamers on the nutrient uptake and in general on the solute mass transfer enhancement due to flow-induced oscillations. We developed what to our knowledge is a novel two-dimensional fluid-structure interaction model coupled to unsteady solute mass transport and solved the model using the finite element method with a moving mesh. Results demonstrate that the oscillatory movement of the biofilm tail significantly increases the substrate uptake. The mass transfer coefficient is the highest in regions close to the streamer tip. The reason for substrate transfer enhancement is the increase in speed of tip movement relative to the surrounding liquid, thereby reducing the thickness of the mass transfer boundary layer. In addition, we show that the relative mass transfer enhancement in unsteady conditions compared with the rigid static structure is larger at higher flow velocities, and this relative increase favors a more flexible structure.     

Influence of dissolved organic matter and inorganic nutrients on the biofilm bacterial community on artificial substrates in a northeastern Ohio, USA, stream

Stream bacteria may be influenced by the composition and availability of dissolved organic matter (DOM) and inorganic nutrients, but knowledge about how individual phylogenetic groups in biofilm are affected is still limited. In this study, the influence of DOM and inorganic nutrients on stream biofilm bacteria was examined. Biofilms were developed on artificial substrates (unglazed ceramic tiles) for 21 days in a northeastern Ohio (USA) stream for five consecutive seasons. Then, the developed biofilm assemblages were exposed, in the laboratory, to DOM (glucose, leaf leachate, and algal exudates) and inorganic nutrients (nitrate, phosphate, and nitrate and phosphate in combination) amendments for 6 days. Bacterial numbers in the biofilms were generally higher in response to the DOM treatments than to the inorganic nutrient treatments. There were also apparent seasonal variations in the response patterns of the individual bacterial taxa to the nutrient treatments; an indication that limiting resources to bacteria in stream biofilms may change over time. Overall, in contrast to the other treatments, bacterial abundance was generally highest in response to the low-molecular-weight DOM (i.e., glucose) treatment. These results further suggest that there are interactions among the different bacterial groups in biofilms that are impacted by the associated nutrient dynamics among seasons in stream ecosystems.     

Investigation of the parameters affecting the separation of bacterial enzymes from cell debris by tangential flow filtration

The effect of organism, enzyme, method of cell breakage and membrane characteristics on the separation of bacterial enzymes from cell debris by tangential flow filtration has been studied. The effectiveness of separation was assessed by process time, enzyme yield and specific activity, and turbidity of the filtrate. For a particular organism and enzyme, method of cell breakage and membrane characteristics significantly influenced separation performance, though results indicate that it is not yet possible to optimize all aspects of performance simultaneously.     

Evaluation of Recovery Methods to Detect Coliforms in Water

Various recovery methods used to detect coliforms in water were evaluated by applying the membrane filter chamber technique. The membrane filter chambers, containing pure-culture suspensions of Escherichia coli or natural suspensions of raw sewage, were immersed in the stream environment. Samples were withdrawn from the chamber at regular time intervals and enumerated by several detection methods. In general, multiple-tube fermentation techniques gave better recovery than plating or membrane filtration procedures. The least efficient method of recovery resulted when using membrane filtration procedures, especially as the exposure period of the organisms to the stream environment increased. A 2-h enrichment on a rich, nonselective medium before exposure to selective media improved the recovery of fecal coliforms with membrane filtration techniques. Substantially enhanced recoveries of E. coli from pure-culture suspensions and of fecal coliforms from raw-sewage suspensions were observed when compared with recoveries obtained by direct primary exposure to selective media. Such an enrichment period appears to provide a nontoxic environment for the gradual adjustment and repair of injured cells.     

Modeling flux in tangential flow filtration using a reverse asymmetric membrane for Chinese hamster ovary cell clarification

Tangential flow filtration is advantageous for bioreactor clarification as the permeate stream could be introduced directly to the subsequent product capture step. However, membrane fouling coupled with high product rejection has limited its use. Here, the performance of a reverse asymmetric hollow fiber membrane where the more open pore structure faces the feed stream and the barrier layer faces the permeate stream has been investigated. The open surface contains pores up to 40 μm in diameter while the tighter barrier layer has an average pore size of 0.4 μm. Filtration of Chinese hamster ovary cell feed streams has been investigated under conditions that could be expected in fed batch operations. The performance of the reverse asymmetric membrane is compared to that of symmetric hollow fiber membranes with nominal pore sizes of 0.2 and 0.65 μm. Laser scanning confocal microscopy was used to observe the locations of particle entrapment. The throughput of the reverse asymmetric membrane is significantly greater than the symmetric membranes. The membrane stabilizes an internal high permeability cake that acts like a depth filter. This stabilized cake can remove particulate matter that would foul the barrier layer if it faced the feed stream. An empirical model has been developed to describe the variation of flux and transmembrane pressure drop during filtration using reverse asymmetric membranes. Our results suggest that using a reverse asymmetric membrane could avoid severe flux decline associated with fouling of the barrier layer during bioreactor clarification.     

A filter assay for polysialyltransferas

Abstract Polysialic acids occur as capsular polysaccharides of several pathogenic bacteria. An understanding of how polysialyltransferase functions in the synthesis of polysialic acid will require enzyme purification and characterization in concert with genetic analysis. A rapid filter assay has been developed for bacterial polysialyltransferase suitable for enzyme purification. The filter assay and the currently used paper chromatography methods are equivalent in parallel experiments. The Escherichia coli K92 polysialyltransferase exhibited the same pH and temperature optima, Mg²⁺ dependence and acceptor specificity in both assays. [¹⁴C]Sialic acid bound in filter assays correlates with polymer formed by gel filtration. Specificity may be increased by the addition of exogenous acceptors.     

Exploitation of the adsorptive properties of depth filters for host cell protein removal during monoclonal antibody purification

Depth filtration has been widely used during process scale clarification of cell culture supernatants for the removal of cells and cell debris. However, in addition to their filtration capabilities, depth filters also possess the ability to adsorb soluble species. This aspect of depth filtration has largely not been exploited in process scale separations and is usually ignored during cell culture harvest development. Here, we report on the ability of depth filters to adsorptively remove host cell protein contaminants from a recombinant monoclonal antibody process stream and characterize some of the underlying interactions behind the binding phenomenon. Following centrifugation, filtration through a depth filter prior to Protein A chromatographic capture was shown to significantly reduce the level of turbidity observed in the Protein A column eluate of the monoclonal antibody. The Protein A eluate turbidity was shown to be linked to host cell protein contaminant levels in the Protein A column load and not to the DNA content. Analogous to flowthrough chromatography in which residence time/bed height and column loading are key parameters, both the number of passes through the depth filter and the amount of centrifuge centrate loaded on the filter were seen to be important operational parameters governing the adsorptive removal of host cell protein contaminants. Adsorption of proteins to the depth filter was shown to be due to a combination of electrostatic and hydrophobic adsorptive interactions. These results demonstrate the ability to employ depth filtration as an integrative unit operation combining filtration for particulate removal with adsorptive binding for contaminant removal.