Millifluidic magnetophoresis-based chip for age-specific fractionation: evaluating the impact of age on metabolomics and gene expression in yeast.

A novel millifluidic process introduces age-based fractionation of S. pastorianus var. carlsbergensis yeast culture through magnetophoresis. Saccharomyces yeast is a model organism for aging research used in various industries. Traditional age-based cell separation methods were labor-intensive, but techniques like magnetic labeling have eased the process by being non-invasive and scalable. Our approach introduces an age-specific fractionation using a 3D-printed millfluidic chip in a two-step process, ensuring efficient cell deflection in the magnetic field and counteracting magnetic induced convection. Among various channel designs, the pinch-shaped channel proved most effective for age differentiation based on magnetically labeled bud scar numbers. Metabolomic analyses revealed changes in certain amino acids and increased NAD+ levels, suggesting metabolic shifts in aging cells. Gene expression studies further underlined these age-related metabolic changes. This innovative platform offers a high-throughput, non-invasive method for age-specific yeast cell fractionation, with potential applications in industries ranging from food and beverages to pharmaceuticals.

Membrane-assisted extraction of monoterpenes: from in silico solvent screening towards biotechnological process application.

This work focuses on the process development of membrane-assisted solvent extraction of hydrophobic compounds such as monoterpenes. Beginning with the choice of suitable solvents, quantum chemical calculations with the simulation tool COSMO-RS were carried out to predict the partition coefficient (logP) of (S)-(+)-carvone and terpinen-4-ol in various solvent-water systems and validated afterwards with experimental data. COSMO-RS results show good prediction accuracy for non-polar solvents such as n-hexane, ethyl acetate and n-heptane even in the presence of salts and glycerol in an aqueous medium. Based on the high logP value, n-heptane was chosen for the extraction of (S)-(+)-carvone in a lab-scale hollow-fibre membrane contactor. Two operation modes are investigated where experimental and theoretical mass transfer values, based on their related partition coefficients, were compared. In addition, the process is evaluated in terms of extraction efficiency and overall product recovery, and its biotechnological application potential is discussed. Our work demonstrates that the combination of in silico prediction by COSMO-RS with membrane-assisted extraction is a promising approach for the recovery of hydrophobic compounds from aqueous solutions.

Peptide binding to metal oxide nanoparticles.

Magnetic metal oxide nanoparticles demonstrate great applicability in several fields such as biotechnology, medicine and catalysis. A stable, magnetic and low-cost material, nanoscale magnetite, is an interesting adsorbent for protein purification. Downstream processing can account for up to 80% of the total production costs in biotechnological production. As such, the development of new innovative separation methods can be regarded as highly profitable. While short peptide sequences can be used as specific affinity tags for functionalised adsorber surfaces, they need expensive affinity ligands on the particle surface for adsorption. In order to identify peptide tags for several non-functionalised inorganic surfaces, different binding conditions to iron oxide nanoparticles are evaluated. Therefore, magnetite nanoparticles in a range of 5-20 nm were synthesised with a co-precipitation method. Zeta potential measurements indicated an amphiphilic surface with an isoelectric point in the neutral pH region. Glutamic acid-based homo-peptides were used as affinity peptides for the magnetite nanoparticles. We demonstrate a dependence of the binding affinity of the peptides on pH and buffer ions in two different experimental set-ups. The nature of surface coordination for glutamic acid-based peptides can be demonstrated with different spectroscopic approaches such as infrared spectroscopy (IR), Raman spectroscopy and circular dichroism spectroscopy (CD). We want to emphasise the importance of physicochemical properties such as surface energy, polarity, morphology and charge. These parameters, which are dependent on the environmental conditions, play a crucial role in peptide interactions with iron oxide surfaces. The understanding of the adsorption of simple biomolecules on nanoscale metal oxide surfaces also represents the key to the even more complex interactions of proteins at the bio-nano interface. From the identification of interaction patterns and an understanding of the adsorption of these peptides, the up-scaling to tagged model proteins facilitates the possibility of an industrial magnetic separation process and might therefore reduce time and costs in purification processes.

Impact of nanoparticle aggregation on protein recovery through a pentadentate chelate ligand on magnetic carriers.

The growing need for more efficient separation techniques still dominates downstream processing of biomolecules, thus encouraging the continuous development of advanced nanomaterials. In this paper we present an improved process for recovering recombinant histidine tagged green fluorescent protein from an E. coli cell lysate. Superparamagnetic core-shell nanocarriers are functionalized with a pentadentate chelate affinity ligand and then loaded with metal ions (Cu(2+), Ni(2+), or Zn(2+)). The separation process yields high binding capacity (250 mg/g), good selectivity, purity >98%, good recyclability with 90% capacity after 9 cycles, and long-term stability. We determined the main physical properties of the magnetite-based nanoparticles such as saturation magnetization (59 A m(2)/kg), primary particle diameter (22 ± 4 nm), and specific surface area (89 m(2)/g). Our results show that this material is a promising tool for bioseparation applications. One special focus of the work includes analyzing the changes in the hydrodynamic size distribution using dynamic light scattering and transmission electron microscopy. We relate these effects to different interaction levels in the system and discuss how the stronger aggregation of the magnetite core is the main limiting factor for the separation yield, leading to a considerable decrease in the number of metal ions available for biomolecular capture. Otherwise weaker interactions lead instead to agglomeration effects that have no impact on the binding capacity of the system. The simple relation between the size of the aggregated units and the size of the primary particles corresponds approximately to the relation between the number of existing binding sites and the actual protein binding in the separation process. Compared with that, the effect of steric hindrance among proteins is of less significance.

Continuous rhamnolipid production with integrated product removal by foam fractionation and magnetic separation of immobilized Pseudomonas aeruginosa.

Increasing interest in biological surfactants has led to intensified research directed at more cost-efficient production of biosurfactants, relative to traditional surface-active components based on petrochemical feedstocks. This publication will focus on a new integrated process for continuous rhamnolipid (RL) production. RL was synthesized by Pseudomonas aeruginosa DSM 2874 and was continuously removed in situ by foam fractionation. To prevent loss of the biocatalyst through foaming, bacteria were entrapped in magnetic alginate beads. Immobilizates were retained from the foam by high-gradient magnetic separation and back-flushed in the bioreactor at constant intervals. It was demonstrated that continuous RL production in a 10-L bioreactor over several cycles with intermediate growth periods is feasible. Complete separation of RLs from the production medium with an average enrichment ratio of 15 in the collapsed foam was demonstrated, yielding a final RL amount of 70 g after four production cycles.

Influence of different magnetites on properties of magnetic Pseudomonas aeruginosa immobilizates used for biosurfactant production.

During the last decades, whole-cell immobilization has been used successfully in many bioprocesses. In particular, it is aimed at implementing continuous production processes, reaching higher production rates, and reusing the biocatalyst. In some cases, effective retention of immobilizates in the bioprocess is not feasible by membranes or sieves due to pore plugging or undesired losses of immobilizates. In the present publication, it is reported about the investigation of magnetic immobilizates of Pseudomonas aeruginosa for application in continuous biosurfactant production of rhamnolipids by foam fractionation and retention of entrained immobilizates by high-gradient magnetic separation from foam. Different materials and methods were tested with respect to important parameters, such as stability, diffusion properties or magnetic separation. Good magnetic separation of immobilizates was achieved at 5% (w/w) magnetite loading. Best results in terms of homogeneous embedding, good diffusion properties, and stability enhancement vis-à-vis pure alginate beads was achieved with alginate beads with embedded Bayoxide magnetite or MagPrep silica particles. Although polyurethane immobilizates showed higher stabilities compared with alginate beads, rhamnolipid diffusion in immobilizates was superior in magnetic alginate beads. Regarding bead production, smaller immobilizates were achieved with suspension polymerization compared to droplet extrusion by the JetCutting technology. In total, magnetic immobilizates are a promising tool for an easier handling of biocatalysts in a continuous biological production process, but they have to be adapted to the current production task.

Development and trends of biosurfactant analysis and purification using rhamnolipids as an example.

During the last few decades, increasing interest in biological surfactants led to an intensification of research for the cost-efficient production of biosurfactants compared with traditional petrochemical surface-active components. The quest for alternative production strains also is associated with new demands on biosurfactant analysis. The present paper gives an overview of existing analytical methods, based on the example of rhamnolipids. The methods reviewed range from simple colorimetric testing to sophisticated chromatographic separation coupled with detection systems like mass spectrometry, by means of which detailed structural information is obtained. High-performance liquid chromatography (HPLC) coupled with mass spectrometry currently presents the most precise method for rhamnolipid identification and quantification. Suitable approaches to accelerate rhamnolipid quantification for better control of biosurfactant production are HPLC analysis directly from culture broth by adding an internal standard or Fourier transform infrared attenuated total reflectance spectroscopy measurements of culture broth as a possible quasi-online quantification method in the future. The search for alternative rhamnolipid-producing strains makes a structure analysis and constant adaptation of the existing quantification methods necessary. Therefore, simple colorimetric tests based on whole rhamnolipid content can be useful for strain and medium screening. Furthermore, rhamnolipid purification from a fermentation broth will be considered depending on the following application.

Design of immobilised dextransucrase for fluidised bed application.

Immobilisation of dextransucrase from Leuconostoc mesenteroides NRRL B-512F in alginate is optimised for applications in a fluidised bed reactor with high concentrated sugar solutions, in order to allow a continuous formation of defined oligosaccharides as prebiotic isomalto-oligosaccharides. Efficient design of fluidised bed immobilised biocatalyst in high density solutions requires particles with elevated density, high effectiveness and both thermal and mechanical stability. Inert silica flour/sand (Mikrosil 300) as supplement turned out to be best suited for increasing the density up to 1400 kg m(-3) of the alginate beads and generating a stable expanded bed without diffusional restrictions. Kinetic investigations demonstrate that low effectiveness of immobilised enzyme due to close association to dextranpolymers (dextran content of enzyme preparation >90%) is compensated by reducing the particle size and/or by decreasing the dextran content. A low dextran content (5%) is sufficient to immobilise and stabilise the enzyme, thus diffusional limitation is reduced essentially while operational stability is maintained. Fluidisation behaviour and bed expansion proved to be appropriate for the intended application. Both calculated and measured expansion coefficients showed good agreement for different conditions.

Cloning of the pelA gene from Bacillus licheniformis 14A and biochemical characterization of recombinant, thermostable, high-alkaline pectate lyase.

The pectate lyase gene pelA from alkaliphilic Bacillus licheniformis strain 14A was cloned and sequenced. The nucleotide sequence corresponded to an open reading frame of 1,026 bp that codes for a 39 amino acid signal peptide and a mature protein with a molecular mass of 33,451 Da. The mature PelA showed significant homology to other pectate lyases belonging to polysaccharide lyase family 1, such as enzymes from different Bacillus spp. and Erwinia chrysanthemi. The pelA gene was expressed in Escherichia coli as a recombinant fusion protein containing a C-terminal His-tag, allowing purification to near homogeneity in a one-step procedure. The values for the kinetic parameters K(m) and Vmax of the fusion protein were 0.56 g/l and 51 micromol/min, respectively. The activity of purified PelAHis was inhibited in the presence of excess substrate. Characterization of product formation revealed unsaturated trigalacturonate as the main product. The yields of unsaturated trigalacturonic acids were further examined for the substrates polygalacturonic acid, citrus pectin and sugar-beet pectin.