Microbioreactors for nutrient-controlled microbial cultures: Bridging the gap between bioprocess development and industrial use.

It is common practice in the development of bioprocesses to genetically modify a microorganism and study a large number of resulting mutants in order to select the ones that perform best for use at the industrial scale. At industrial scale, strict nutrient-controlled growth conditions are imposed to control the metabolic activity and growth rate of the microorganism, thereby enhancing the expression of the product of interest. Although it is known that microorganisms that perform best under these strictly controlled conditions are not the same as the ones that perform best under uncontrolled batch conditions, screening, and selection is predominantly performed under batch conditions. Tools that afford high throughput on the one hand and dynamic control over cultivation conditions on the other hand are not yet available. Microbioreactors offer the potential to address this problem, resolving the gap between bioprocess development and industrial scale use. In this review, we highlight the current state-of-the-art of microbioreactors that offer the potential to screen microorganisms under dynamically controlled conditions. We classify them into: (i) microtiter plate-based platforms, (ii) microfluidic chamber-based platforms, and (iii) microfluidic droplet-based platforms. We conclude this review by discussing the opportunities of nutrient-fed microbioreactors in the field of biotechnology.

Actin networks regulate the cell membrane permeability during electroporation.

Transient physical disruption of cell membranes by electric pulses (or electroporation) has significance in biomedical and biological applications requiring the delivery of exogenous (bio)molecules to living cells. We demonstrate that actin networks regulate the cell membrane permeability during electroporation. Disruption of actin networks increases the uptake of membrane-impermeable molecules such as propidium iodide during electroporation. Our experiments at different temperatures ranging from 11 °C to 37 °C show that molecular uptake during electroporation increases with temperature. Furthermore, by examining the temperature-dependent kinetics of propidium iodide uptake, we infer that the activation energy barrier of electroporation is lowered when the actin networks are disrupted. Our numerical calculations of transmembrane voltage show that the reduced activation energy barrier for the cells with disrupted actin is not a consequence of the changes in transmembrane voltage associated with changes in the cell shape due to the disruption of actin, indicating that this could be due to changes in membrane mechanical properties. Our results suggest that the current theoretical models of electroporation should be advanced further by including the contributions of the cytoskeletal networks on the cell membrane permeability during the delivery of exogenous materials.

Synthesis of a Rationally Designed Multi-Component Photocatalyst Pt:SiO2:TiO2(P25) with Improved Activity for Dye Degradation by Atomic Layer Deposition.

Photocatalysts for water purification typically lack efficiency for practical applications. Here we present a multi-component (Pt:SiO2:TiO2(P25)) material that was designed using knowledge of reaction mechanisms of mono-modified catalysts (SiO2:TiO2, and Pt:TiO2) combined with the potential of atomic layer deposition (ALD). The deposition of ultrathin SiO2 layers on TiO2 nanoparticles, applying ALD in a fluidized bed reactor, demonstrated in earlier studies their beneficial effects for the photocatalytic degradation of organic pollutants due to more acidic surface Si-OH groups which benefit the generation of hydroxyl radicals. Furthermore, our investigation on the role of Pt on TiO2(P25), as an improved photocatalyst, demonstrated that suppression of charge recombination by oxygen adsorbed on the Pt particles, reacting with the separated electrons to superoxide radicals, acts as an important factor for the catalytic improvement. Combining both materials into the resulting Pt:SiO2:TiO2(P25) nanopowder exceeded the dye degradation performance of both the individual SiO2:TiO2(P25) (1.5 fold) and Pt:TiO2(P25) (4-fold) catalysts by 6-fold as compared to TiO2(P25). This approach thus shows that by understanding the individual materials' behavior and using ALD as an appropriate deposition technique enabling control on the nano-scale, new materials can be designed and developed, further improving the photocatalytic activity. Our research demonstrates that ALD is an attractive technology to synthesize multicomponent catalysts in a precise and scalable way.

Scalable microfluidic droplet on-demand generator for non-steady operation of droplet-based assays.

We developed a microfluidic droplet on-demand (DoD) generator that enables the production of droplets with a volume solely governed by the geometry of the generator for a range of operating conditions. The prime reason to develop this novel type of DoD generator is that its robustness in operation enables scale out and operation under non-steady conditions, which are both essential features for the further advancement of droplet-based assays. We first detail the working principle of the DoD generator and study the sensitivity of the volume of the generated droplets with respect to the used fluids and control parameters. We next compare the performance of our DoD generator when scaled out to 8 parallel generators to the performance of a conventional DoD generator in which the droplet volume is not geometry-controlled, showing its superior performance. Further scale out to 64 parallel DoD generators shows that all generators produce droplets with a volume between 91% and 105% of the predesigned volume. We conclude the paper by presenting a simple droplet-based assay in which the DoD generator enables sequential supply of reagent droplets to a droplet stored in the device, illustrating its potential to be used in droplet-based assays for biochemical studies under non-steady operation conditions.

DNA-membrane complex formation during electroporation is DNA size-dependent.

Size of DNA molecules governs their interaction with the cell membrane during electroporation and their subsequent transport inside the cell. In order to investigate the effect of DNA size on DNA-membrane interaction during electroporation, cells are electro-pulsed with DNA molecules; 15 bp, 25 bp, 50 bp, 100 bp and 1000 bp (bp = base pairs). Within the experimental parameter space, DNA-membrane complexes or DNA aggregates are observed at the cell membrane for DNA molecules containing 25 or more base pairs. No aggregates are observed for DNA molecules containing 15 bp. For all DNA sizes, direct access to the cytoplasm is observed, however the amount translocated decays with the size. The observed dependency of DNA aggregate formation on the size of the DNA molecules is consistent with the Onsager's theory of condensation of anisotropic rod-like molecules.

DNA translocation to giant unilamellar vesicles during electroporation is independent of DNA size.

Delivery of naked DNA molecules into living cells via physical disruption of the membrane under electric pulses has potential biomedical applications ranging from gene electro-transfer, electro-chemotherapy, to gene therapy, yet the mechanisms involved in DNA transport remain vague. To investigate the mechanism of DNA translocation across the cell membrane, giant unilamellar vesicles (GUVs) were electroporated in the presence of DNA molecules keeping the size of the DNA molecules as a variable parameter. We experimentally determined the translocation efficiency for each size of the DNA molecule, to compare the results with the existing and conflicting theories of the translocation mechanism i.e. stochastic threading and bulk electrophoresis. We observed that the translocation efficiency is independent of DNA size (ranging from 25-20 000 bp, bp = base pairs), implying that DNA molecules translocate freely across the electro-pores in the lipid membrane in their native polymer conformation, as opposed to unravelling and threading through the electro-pore. Bulk electrophoretic mobility determines the relationship between translocation efficiency and the size of the DNA molecule. This research provides experimental evidence of the mechanistic understanding of DNA translocation across lipid membranes which is essential for devising efficient and predictable protocols for electric field mediated naked DNA delivery.

Response of an actin network in vesicles under electric pulses.

We study the role of a biomimetic actin network during the application of electric pulses that induce electroporation or electropermeabilization, using giant unilamellar vesicles (GUVs) as a model system. The actin cortex, a subjacently attached interconnected network of actin filaments, regulates the shape and mechanical properties of the plasma membrane of mammalian cells, and is a major factor influencing the mechanical response of the cell to external physical cues. We demonstrate that the presence of an actin shell inhibits the formation of macropores in the electroporated GUVs. Additionally, experiments on the uptake of dye molecules after electroporation show that the actin network slows down the resealing process of the permeabilized membrane. We further analyze the stability of the actin network inside the GUVs exposed to high electric pulses. We find disruption of the actin layer that is likely due to the electrophoretic forces acting on the actin filaments during the permeabilization of the GUVs. Our findings on the GUVs containing a biomimetic network provide a step towards understanding the discrepancies between the electroporation mechanism of a living cell and its simplified model of the empty GUV.

Control over the formation of supramolecular material objects using reaction-diffusion.

Controlled diffusion, reaction and assembly of hydrogelator precursors can be used to create soft hydrogel objects of defined shape and size. In this study we show that controlling local reaction kinetics by means of pH, diffusion length and the concentrations of reactants allows control over the dimensions of formed supramolecular structures. By correlating a reaction diffusion model to experimental results, we show that the influence of all these control parameters can be unified using the Damköhler number, thus providing an easy-to-use relation between experimental parameters and structure dimensions. Finally, our study suggests that control over concentration gradients and chemical reactivity in combination with supramolecular chemistry is a promising platform for the design of soft matter objects of defined sizes, a concept that has received little attention up until now.

Oriented Attachment and Nanorod Formation in Atomic Layer Deposition of TiO2 on Graphene Nanoplatelets.

Understanding the spontaneous organization of atoms on well-defined surfaces promises to enable control over the shape and size of supported nanostructures. Atomic layer deposition (ALD) boasts atomic-scale control in the synthesis of thin films and nanoparticles. Yet, the possibility to control the shape of ALD-grown nanostructures remains mostly unexplored. Here, we report on the bottom-up formation of both linear and V-shaped anatase TiO2 nanorods (NRs) on graphene nanoplatelets during TiCl4/H2O ALD carried out at 300 °C. NRs as large as 200 nm form after only five ALD cycles, indicating that diffusional processes rather than layer-by-layer growth are behind the NR formation. In particular, high-resolution transmission electron microscopy reveals that the TiO2 NRs and graphene nanoplatelets are in rotational alignment as a result of lattice matching. Crucially, we also show that individual nanocrystals can undergo in-plane oriented attachment.

From Single Atoms to Nanoparticles: Autocatalysis and Metal Aggregation in Atomic Layer Deposition of Pt on TiO2 Nanopowder.

A fundamental understanding of the interplay between ligand-removal kinetics and metal aggregation during the formation of platinum nanoparticles (NPs) in atomic layer deposition of Pt on TiO2 nanopowder using trimethyl(methylcyclo-pentadienyl)platinum(IV) as the precursor and O2 as the coreactant is presented. The growth follows a pathway from single atoms to NPs as a function of the oxygen exposure (PO2 × time). The growth kinetics is modeled by accounting for the autocatalytic combustion of the precursor ligands via a variant of the Finke-Watzky two-step model. Even at relatively high oxygen exposures (<120 mbar s) little to no Pt is deposited after the first cycle and most of the Pt is atomically dispersed. Increasing the oxygen exposure above 120 mbar s results in a rapid increase in the Pt loading, which saturates at exposures >> 120 mbar s. The deposition of more Pt leads to the formation of NPs that can be as large as 6 nm. Crucially, high PO2 (≥5 mbar) hinders metal aggregation, thus leading to narrow particle size distributions. The results show that ALD of Pt NPs is reproducible across small and large surface areas if the precursor ligands are removed at high PO2 .

The role of gel-phase domains in electroporation of vesicles.

Transient permeabilisation of the cell membrane is a critical step to introduce drugs or DNA into living cells, yet challenging for both biological research and therapeutic applications. To achieve this, electroporation (or electropermeabilisation) has become a widely used method due to its simplicity to deliver almost any biomolecule to any cell type. Although this method demonstrates promise in the field of drug/gene delivery, the underlying physical mechanisms of the response of the heterogeneous cell membrane to strong electric pulses is still unknown. In this study, we have investigated the role of gel-phase lipids in the electroporation of binary giant unilamellar vesicles (GUVs), composed from DPPC (gel-phase) and DPhPC (fluid-phase) lipids (molar ratio 8:2 and 2:8). We have observed that the exposure to electric pulses leads to expel of fluid-phase lipids and concomitant decrease in GUV size, whereas the gel-phase domains become buckled. Based on experiments on pure fluid-phase and gel-phase GUVs, we have found that fluid-phase lipids can be expelled by electrical forces and the highly viscous gel-phase lipids cannot. Moreover, our analyses suggest that pore formation occurs primarily in fluid-phase domains and that the pore size is similar in all GUVs containing fluid-phase lipids, irrespective of the gel-phase percentage.

Polymer conformation during flow in porous media.

Molecular conformations of individual polymers during flow through porous media are directly observed by single-DNA imaging in microfluidics. As the Weissenberg number increases during flow (Wi > 1), we observe two types of elastic instabilities: (a) stationary dead-zone and (b) time-dependant dead-zone washing. When stretched polymer chains enter a dead-zone, they first re-coil and, once inside the dead-zone, they rotate and re-stretch again. The probability distribution of DNA chains under the stretched condition inside the dead-zone is found to be heterogeneous with a broad distribution.

Low-temperature atomic layer deposition delivers more active and stable Pt-based catalysts.

We tailored the size distribution of Pt nanoparticles (NPs) on graphene nanoplatelets at a given metal loading by using low-temperature atomic layer deposition carried out in a fluidized bed reactor operated at atmospheric pressure. The Pt NPs deposited at low temperature (100 °C) after 10 cycles were more active and stable towards the propene oxidation reaction than their high-temperature counterparts. Crucially, the gap in the catalytic performance was retained even after prolonged periods of time (>24 hours) at reaction temperatures as high as 450 °C. After exposure to such harsh conditions the Pt NPs deposited at 100 °C still retained a size distribution that is narrower than the one of the as-synthesized NPs obtained at 250 °C. The difference in performance correlated with the difference in the number of facet sites as estimated after the catalytic test. Our approach provides not only a viable route for the scalable synthesis of stable supported Pt NPs with tailored size distributions but also a tool for studying the structure-function relationship.

Understanding and Controlling the Aggregative Growth of Platinum Nanoparticles in Atomic Layer Deposition: An Avenue to Size Selection.

We present an atomistic understanding of the evolution of the size distribution with temperature and number of cycles in atomic layer deposition (ALD) of Pt nanoparticles (NPs). Atomistic modeling of our experiments teaches us that the NPs grow mostly via NP diffusion and coalescence rather than through single-atom processes such as precursor chemisorption, atom attachment, and Ostwald ripening. In particular, our analysis shows that the NP aggregation takes place during the oxygen half-reaction and that the NP mobility exhibits a size- and temperature-dependent scaling. Finally, we show that contrary to what has been widely reported, in general, one cannot simply control the NP size by the number of cycles alone. Instead, while the amount of Pt deposited can be precisely controlled over a wide range of temperatures, ALD-like precision over the NP size requires low deposition temperatures (e.g., T < 100 °C) when growth is dominated by atom attachment.

Elastic instabilities during the flow of hydrolyzed polyacrylamide solution in porous media: effect of pore-shape and salt.

We experimentally investigate the flow of hydrolyzed polyacrylamide (HPAM) solution with and without salt in model porous media at high Weissenberg numbers (Wi > 1.0). The effect of pore shapes on the flow pattern and pressure drop is explored by using periodic arrays of circular and square pillars in aligned and staggered layouts. In the apparent shear-thinning regime, we observe stationary dead zones upstream of the pillars. In addition, we confirm that the size of stationary dead zones correlates with the level of shear-thinning, by varying the amount of salt in HPAM solution. At higher shear rates (or Wi), these dead zones are periodically washed away. We present the mechanism of this elastic instability and characterize it based on the pressure drop fluctuation spectral density.

Contact mechanics of highly porous oxide nanoparticle agglomerates.

Efficient nanopowder processing requires knowledge of the powder's mechanical properties. Due to the large surface area to volume ratio, nanoparticles experience relatively strong attractive interactions, leading to the formation of micron-size porous structures called agglomerates. Significant effort has been directed towards the development of models and experimental procedures to estimate the elasticity of porous objects such as nanoparticle agglomerates; however, none of the existing models has been validated for solid fractions below 0.1. Here, we measure the elasticity of titania (TiO[Formula: see text], 22 nm), alumina (Al[Formula: see text]O[Formula: see text], 8 nm), and silica (SiO[Formula: see text], 16 nm) nanopowder agglomerates by Atomic Force Microscopy, using a 3.75 [Formula: see text]m glass colloid for the stress-strain experiments. Three sample preparations with varying degree of powder manipulation are assessed. The measured Young's moduli are in the same order of magnitude as those predicted by the model of Kendall et al., thus validating it for the estimation of the Young's modulus of structures with porosity above 90 %.

Gas-Phase Deposition of Ultrathin Aluminium Oxide Films on Nanoparticles at Ambient Conditions.

We have deposited aluminium oxide films by atomic layer deposition on titanium oxide nanoparticles in a fluidized bed reactor at 27 ± 3 °C and atmospheric pressure. Working at room temperature allows coating heat-sensitive materials, while working at atmospheric pressure would simplify the scale-up of this process. We performed 4, 7 and 15 cycles by dosing a predefined amount of precursors, i.e., trimethyl aluminium and water. We obtained a growth per cycle of 0.14-0.15 nm determined by transmission electron microscopy (TEM), similar to atomic layer deposition (ALD) experiments at a few millibars and ~180 °C. We also increased the amount of precursors dosed by a factor of 2, 4 and 6 compared to the base case, maintaining the same purging time. The growth per cycle (GPC) increased, although not linearly, with the dosing time. In addition, we performed an experiment at 170 °C and 1 bar using the dosing times increased by factor 6, and obtained a growth per cycle of 0.16 nm. These results were verified with elemental analysis, which showed a good agreement with the results from TEM pictures. Thermal gravimetric analysis (TGA) showed a negligible amount of unreacted molecules inside the alumina films. Overall, the dosage of the precursors is crucial to control precisely the growth of the alumina films at atmospheric pressure and room temperature. Dosing excess of precursor provokes a chemical vapour deposition type of growth due to the physisorption of molecules on the particles, but this can be avoided by working at high temperatures.

Droplets on inclined plates: local and global hysteresis of pinned capillary surfaces.

Local contact line pinning prevents droplets from rearranging to minimal global energy, and models for droplets without pinning cannot predict their shape. We show that experiments are much better described by a theory, developed herein, that does account for the constrained contact line motion, using as an example droplets on tilted plates. We map out their shapes in suitable phase spaces. For 2D droplets, the critical point of maximum tilt depends on the hysteresis range and Bond number. In 3D, it also depends on the initial width, highlighting the importance of the deposition history.

Scale-up study of a multiphase photocatalytic reactor--degradation of cyanide in water over TiO2.

This paper provides an integrated view on various aspects of reactor design for photocatalytic reactions and presents a scale-up study of photocatalytic reactors. This study focuses on degrading organic pollutants in the effluent of an integrated gasification coal combustion plant over TiO2, with the target of degrading cyanide to below its allowable emission threshold set by European legislation. Here, we show the interplay of different efficiencies that affect the overall apparent photonic efficiency and the reactor volume required to achieve a certain objective in conversion. The chosen reactor configuration is rectangular slurry-bubble-columns-in-series to ensure a good mass transfer rate per photoreactor while approaching plug-flow behavior as a sum, and a high reactor surface-area-to-volume ratio for a good capture of incident photons. We consider a simple 1D photonic description of a photoreactor, in the direction of incident solar light, and implement a bidirectional scattering model for photocatalytic particles and bubbles to calculate the local rate of photon absorption and the photon absorption efficiency in the photoreactor. We show that, implementing the principles of process intensification, the large scale degradation of cyanide to below European emission limits is achievable.

Rapid microfluidic screening of CO2 solubility and diffusion in pure and mixed solvents.

We present a high-throughput method to determine rapidly and simultaneously the solubility and the diffusivity of CO(2) in pure solvents and mixtures using segmented flow in a microchannel. Gas bubbles are injected via a T-junction into the liquid stream and the evolution of the bubbles' lengths are followed visually. We measure both solubility and diffusion coefficient from the shrinkage and expansion of the bubbles. The presented method is used to study the physical absorption of CO(2) in various pure solvents and to screen the complete composition space of binary and ternary mixtures.