A High-Aspect-Ratio Deterministic Lateral Displacement Array for High-Throughput Fractionation.

Future industrial applications of microparticle fractionation with deterministic lateral displacement (DLD) devices are hindered by exceedingly low throughput rates. To enable the necessary high-volume flows, high flow velocities as well as high aspect ratios in DLD devices have to be investigated. However, no experimental studies have yet been conducted on the fractionation of bi-disperse suspensions containing particles below 10 µm with DLD at a Reynolds number (Re) above 60. Furthermore, devices with an aspect ratio of more than 4:1, which require advanced microfabrication, are not known in the DLD literature. Therefore, we developed a suitable process with deep reactive ion etching of silicon and anodic bonding of a glass lid to create pressure-resistant arrays. With a depth of 120 µm and a gap of 23 µm between posts, a high aspect ratio of 6:1 was realized, and devices were investigated using simulations and fractionation experiments. With the two-segmented array of 3° and 7° row shifts, critical diameters of 8 µm and 12 µm were calculated for low Re conditions, but it was already known that vortices behind the posts can shift these values to lower critical diameters. Suspensions with polystyrene particles in different combinations were injected with an overall flow rate of up to 15 mL/min, corresponding to Re values of up to 90. Suspensions containing particle combinations of 2 µm with 10 µm as well as 5 µm with 10 µm were successfully fractionated, even at the highest flow rate. Under these conditions, a slight widening of the displacement position was observed, but there was no further reduction in the critical size as it was for Re = 60. With an unprecedented fractionation throughput of nearly 1 L per hour, entirely new applications are being developed for chemical, pharmaceutical, and recycling technologies.

Quantification and modeling of macroparticle-induced mechanical stress for varying shake flask cultivation conditions.

In biotechnological processes, filamentous microorganisms are known for their broad product spectrum and complex cellular morphology. Product formation and cellular morphology are often closely linked, requiring a well-defined level of mechanical stress to achieve high product concentrations. Macroparticles were added to shake flask cultures of the filamentous actinomycete Lentzea aerocolonigenes to find these optimal cultivation conditions. However, there is currently no model concept for the dependence of the strength and frequency of the bead-induced stress on the process parameters. Therefore, shake flask simulations were performed for combinations of bead size, bead concentration, bead density and shaking frequency. Contact analysis showed that the highest shear stresses were caused by bead-bottom contacts. Based on this, a newly generated characteristic parameter, the stress area ratio (SAR), was defined, which relates the bead wall shear and normal stresses to the total shear area. Comparison of the SAR with previous cultivation results revealed an optimum pattern for product concentration and mean product-to-biomass related yield coefficient. Thus, this model is a suitable tool for future optimization, comparison and scaling up of shear-sensitive microorganism cultivation. Finally, the simulation results were validated using high-speed recordings of the bead motion on the bottom of the shake flask.

Temperature influence on the compression and breakage behaviour of yeast cells.

Industrial biotechnology uses microbial cells to produce a wide range of products. While the genetic and molecular properties of these organisms are well understood, less is known about their mechanical properties. Previous work has established a test procedure for single yeast cells using a nanoindentation instrument equipped with a flat-punch probe, which allows single cells (Saccharomyces cerevisiae) to be compressed between two parallel surfaces. The resulting force-displacement curves clearly showed the bursting of the cells and were used to determine characteristics such as burst force and burst energy. Other studies have investigated the influence of growth conditions and measurement conditions on the mechanical characteristics. The recent study examined the mechanical characteristics according to the temperature during compression. Temperature from 0°C to 25°C has no significant effect on the micromechanical properties. Increasing the temperature up to 35°C causes a reduction in the strength of the cells. At even higher temperatures, up to 50°C, the burst force and burst energy increase significantly. A deformation geometry model was used to calculate the cell wall tensile strength as a function of temperature. The results of these studies may facilitate the identification of efficient conditions for cell disruption and product recovery in downstream biotechnological processes.

Process and formulation parameters influencing the survival of Saccharomyces cerevisiae during spray drying and tableting.

Probiotic microorganisms provide health benefits to the patient when administered in a viable form and in sufficient doses. To ensure this, dry dosage forms are preferred, with tablets in particular being favored due to several advantages. However, the microorganisms must first be dried as gently as possible. Here, the model organism Saccharomyces cerevisiae was dried by spray drying. Various additives were tested for their ability to improve yeast cell survival during drying. In addition, the influence of various process parameters such as inlet temperature, outlet temperature, spray rate, spray pressure and nozzle diameter was investigated. It was possible to dry the yeast cells in such a way that a substantial proportion of living microorganisms was recovered after reconstitution. Systematic variation of formulation and process parameters showed that the use of protective additives is essential and that the outlet temperature determines the survival rate. The subsequent compression of the spray-dried yeast reduced viability and survival could hardly be improved by the addition of excipients, but the tabletability of spray-dried yeast protectant particles was quite good. For the first time, loss of viability during compaction of spray-dried microorganisms was correlated with the specific densification, allowing a deeper understanding of the mechanism of cell inactivation during tableting.

Influence of compression kinetics during tableting of fluidized bed-granulated microorganisms on microbiological and physical-mechanical tablet properties.

As tablets are convenient to administer to patients, ensure safe dosing and allow cost-effective production on a large scale, they are the favored dosage form for numerous active pharmaceutical ingredients but also for the administration of viable probiotic microorganisms. Granules with viable yeast cells (Saccharomyces cerevisiae) formed by fluidized bed granulation with dicalcium phosphate (DCP), lactose (LAC) or microcrystalline cellulose (MCC) as carrier materials were tableted using a compaction simulator. Besides the compression stress, the compression speed was systematically studied by varying consolidation time and dwell time. The microbial survival as well as physical properties of the tablets, e.g., porosity and tensile strength, were determined. Higher compression stresses result in lower porosities. While on the one hand this has a detrimental effect on microbial survival (due to increased pressure and shear stress during particle rearrangement / densification), on the other hand it results in higher tensile strengths. At the same compression stress, a prolonged dwell time resulted in lower porosity and thus in lower survival rates but higher tensile strength. Against that, consolidation time showed no significant influence on the considered tablet quality attributes. Since changes of the tensile strength related survival rate were negligible (due to opposite but balancing dependence on porosity), high production speeds could be used for tableting of these granules without additional loss of viability, as long as tablets with the same tensile strength are produced.

Tableting of fluidized bed granules containing living microorganisms.

Tablets are the favored dosage form for numerous active pharmaceutical ingredients, among others because they are easy to take, ensure safe dosing and allow cost-effective production on a large scale. This dosage form is also frequently chosen for the administration of viable probiotic microorganisms. Saccharomyces cerevisiae cells granulated in a fluidized bed process, with dicalcium phosphate (DCP), lactose (LAC) and microcrystalline cellulose (MCC) as carrier materials, were tableted using a compaction simulator, varying the compression stress. The tablets were analyzed regarding physical properties, e.g., porosity and tensile strength, as well as microbial survival. Carrier material and compression stress showed a significant influence on survival rate and physical tablet properties. The dependencies were related to material specific deformation characteristics and linked to mechanistic approaches to explain the different sensitivities.

Effect of Process Parameters, Protectants and Carrier Materials on the Survival of Yeast Cells during Fluidized Bed Granulation for Tableting.

The administration of living microorganisms is of special interest, with regard to probiotic microorganisms providing health benefits to the patient. Effective dosage forms require the preservation of microbial viability until administration. Storage stability can be improved by drying, and the tablet is an especially attractive final solid dosage form due to its ease of administration and its good patient compliance. In this study, drying of the yeast Saccharomyces cerevisiae via fluidized bed spray granulation is investigated, as the probiotic Saccharomyces boulardii is a variety of it. Fluidized bed granulation enables faster drying than lyophilization on the one hand and lower temperatures than spray drying on the other hand, which are the two predominantly used techniques for life-sustaining drying of microorganisms. Yeast cell suspensions enriched with protective additives were sprayed onto the carrier particles of common tableting excipients, namely, dicalcium phosphate (DCP), lactose (LAC) and microcrystalline cellulose (MCC). Different protectants, such as mono-, di-, oligo- and polysaccharides, but also skimmed milk powder and one alditol, were tested; as they themselves, or chemically similar molecules, are known from other drying technologies to stabilize biological structures such as cell membranes, and thus, improve survival during dehydration. With the combined use of trehalose and skimmed milk powder, survival rates were 300 times higher than without the use of protective additives. In addition to these formulation aspects, the influence of process parameters such as inlet temperature and spray rate were considered. The granulated products were characterized regarding their particle size distribution, moisture content and the viability of the yeast cells. It has been shown that thermal stress on the microorganisms is especially critical, which can be reduced, for example, by reducing the inlet temperature or increasing the spray rate; however, formulation parameters such as cell concentration also influenced survival. The results were used to specify the influencing factors on the survival of microorganisms during fluidized bed granulation and to derive their linkages. Granules based on the three different carrier materials were tableted and the survival of the microorganisms was evaluated and linked to the tablet tensile strength achieved. Using LAC enabled the highest survival of the microorganisms throughout the considered process chain.

Along the Process Chain to Probiotic Tablets: Evaluation of Mechanical Impacts on Microbial Viability.

Today, probiotics are predominantly used in liquid or semi-solid functionalized foods, showing a rapid loss of cell viability. Due to the increasing spread of antibiotic resistance, probiotics are promising in pharmaceutical development because of their antimicrobial effects. This increases the formulation requirements, e.g., the need for an enhanced shelf life that is achieved by drying, mainly by lyophilization. For oral administration, the process chain for production of tablets containing microorganisms is of high interest and, thus, was investigated in this study. Lyophilization as an initial process step showed low cell survival of only 12.8%. However, the addition of cryoprotectants enabled survival rates up to 42.9%. Subsequently, the dried cells were gently milled. This powder was tableted directly or after mixing with excipients microcrystalline cellulose, dicalcium phosphate or lactose. Survival rates during tableting varied between 1.4% and 24.1%, depending on the formulation and the applied compaction stress. More detailed analysis of the tablet properties showed advantages of excipients in respect of cell survival and tablet mechanical strength. Maximum overall survival rate along the complete manufacturing process was >5%, enabling doses of 6 × 108 colony forming units per gram (CFU gtotal-1), including cryoprotectants and excipients.

Accelerated Particle Separation in a DLD Device at Re > 1 Investigated by Means of µPIV.

A pressure resistant and optically accessible deterministic lateral displacement (DLD) device was designed and microfabricated from silicon and glass for high-throughput fractionation of particles between 3.0 and 7.0 µm comprising array segments of varying tilt angles with a post size of 5 µm. The design was supported by computational fluid dynamic (CFD) simulations using OpenFOAM software. Simulations indicated a change in the critical particle diameter for fractionation at higher Reynolds numbers. This was experimentally confirmed by microparticle image velocimetry (µPIV) in the DLD device with tracer particles of 0.86 µm. At Reynolds numbers above 8 an asymmetric flow field pattern between posts could be observed. Furthermore, the new DLD device allowed successful fractionation of 2 µm and 5 µm fluorescent polystyrene particles at Re = 0.5-25.

Characterization and control of fungal morphology for improved production performance in biotechnology.

Filamentous fungi have been widely applied in industrial biotechnology for many decades. In submerged culture processes, they typically exhibit a complex morphological life cycle that is related to production performance--a link that is of high interest for process optimization. The fungal forms can vary from dense spherical pellets to viscous mycelia. The resulting morphology has been shown to be influenced strongly by process parameters, including power input through stirring and aeration, mass transfer characteristics, pH value, osmolality and the presence of solid micro-particles. The surface properties of fungal spores and hyphae also play a role. Due to their high industrial relevance, the past years have seen a substantial development of tools and techniques to characterize the growth of fungi and obtain quantitative estimates on their morphological properties. Based on the novel insights available from such studies, more recent studies have been aimed at the precise control of morphology, i.e., morphology engineering, to produce superior bio-processes with filamentous fungi.

Linking aggregation of Aspergillus niger spores to surface electrostatics: a theoretical approach.

The effect of medium pH on conidial aggregation during submerged cultivation of Aspergillus niger is considered to originate from the electrostatic surface properties of the spores. As previously shown, these properties are greatly influenced by the presence of a melanin-containing surface coating covering the outer spore wall layer. The present study was designed to elucidate the impact of such a coating on the spores' surface potential and their electrostatic repulsion under acidic conditions. A Poisson-Boltzmann model was proposed and potential profiles across the surface coating of noninteracting and interacting spores were calculated. The surface potentials thus obtained were in line with the observed pH dependence of the zeta potential. This dependence was consistent with the outcome of aggregation experiments. Apparently contradictory results regarding the zeta potential and the aggregation behavior of the spores were obtained when the ionic strength was varied. However, both of these observations could be explained by the model.

On the origin of the electrostatic surface potential of Aspergillus niger spores in acidic environments.

The electrostatic surface potential of fungal spores is generally regarded as potentially influencing spore aggregation and pellet formation in submerged cultures of filamentous fungi. Spores of Aspergillus niger are typically characterized by negative zeta potentials over a wide range of pH values. In this study, this particular behavior is ascribed to the presence of an extensive melanin coating. It is proposed on the basis of zeta potential and pigment extraction experiments that this outermost layer affects the pH-dependent surface potential in two manners: (i) by the addition of negative charges to the spore surface and (ii) by the pH-dependent release of melanin pigment. Chemical analyses revealed that deprotonation of melanin-bound carboxyl groups is most probably responsible for pigment release under acidic conditions. These findings were incorporated into a simple model which has the ability to qualitatively explain the results of zeta potential experiments and, moreover, to provide the basis for quantitative investigations on the role of electrostatics in spore aggregation.

Atomic force microscopy studies on the nanomechanical properties of Saccharomyces cerevisiae.

In the past years atomic force microscopy (AFM) techniques have turned out to be a suitable and versatile tool for probing the physical properties of microbial cell surfaces. Besides interaction forces, nanomechanical properties can be obtained from force spectroscopic measurements. Analyzing the recorded force curves by applying appropriate models allows the extraction of cell mechanical parameters, e.g. the Young's modulus or the cellular spring constant. In the present work the nanomechanical properties of the baker's yeast Saccharomyces cerevisiae are extensively studied by force spectroscopy using an AFM. Single cells deform purely elastically so that a cellular spring constant can reliably be determined. It is presented, how this spring constant depends on the probing position on the cell, and how it depends on the extracellular osmotic conditions. Investigations aiming a statistically firm description of the nanomechanical behavior of the yeast cell population are conducted. Finally, the informative value of the cellular spring constant as a cell mechanical parameter is critically discussed.

Morphology of filamentous fungi: linking cellular biology to process engineering using Aspergillus niger.

In various biotechnological processes, filamentous fungi, e.g. Aspergillus niger, are widely applied for the production of high value-added products due to their secretion efficiency. There is, however, a tangled relationship between the morphology of these microorganisms, the transport phenomena and the related productivity. The morphological characteristics vary between freely dispersed mycelia and distinct pellets of aggregated biomass. Hence, advantages and disadvantages for mycel or pellet cultivation have to be balanced out carefully. Due to this inadequate understanding of morphogenesis of filamentous microorganisms, fungal morphology, along with reproducibility of inocula of the same quality, is often a bottleneck of productivity in industrial production. To obtain an optimisation of the production process it is of great importance to gain a better understanding of the molecular and cell biology of these microorganisms as well as the approaches in biochemical engineering and particle technique, in particular to characterise the interactions between the growth conditions, cell morphology, spore-hyphae-interactions and product formation. Advances in particle and image analysis techniques as well as micromechanical devices and their applications to fungal cultivations have made available quantitative morphological data on filamentous cells. This chapter provides the ambitious aspects of this line of action, focussing on the control and characterisation of the morphology, the transport gradients and the approaches to understand the metabolism of filamentous fungi. Based on these data, bottlenecks in the morphogenesis of A. niger within the complex production pathways from gene to product should be identified and this may improve the production yield.