Scale-up Procedure Research Articles

HSCCC Straightforward Fast Preparative Method for Isolation of Two Major Cytotoxic Withanolides from Athenaea fasciculata (Vell.) I.M.C. Rodrigues & Stehmann.

Athenaea fasciculata belongs to the Solanaceae family and is a promising source of cytotoxic withanolides known as aurelianolides A and B. In the last years, the pharmacological studies of these aurelianolides on different leukemia cell lines have stimulated new studies on their potential as alternative candidates for new lead anticancer drugs. However, the obtention of these two pure compounds by traditional preparative is a costly and long time-consuming process, which is performed in several steps. This study aimed to propose a straightforward approach for isolating aurelianolides A and B using high-speed countercurrent chromatography (HSCCC). In this study, among 10 different solvent systems, the system composed of n-hexane/ethyl acetate/methanol/water 3:6:2:1 (v/v/v/v) was chosen for optimization. This HEMWat system was optimized to 4:8:2:4 (v/v/v/v) and chosen for HSCCC separation in a tail-to-head elution mode. After the HSCCC scale-up procedure, a withanolides mixture (200.0 mg) was separated within 160 min in a single-step purification process. In total, 78.9 mg of aurelianolide A (up to 95.0% purity) and 54.3 mg of aurelianolide B (up to 88.5% purity) was obtained by this fast sequential liquid-liquid partition process. The isolated withanolides were identified by 1H and 13C NMR spectroscopy (this method has proven to be faster and more efficient than classical procedures (CC and Prep-TLC)).

An industrial perspective on metabolic responses of Penicillium chrysogenum to periodic dissolved oxygen feast-famine cycles in a scale-down system.

While traveling through different zones in large-scale bioreactors, microbes are most likely subjected to fluctuating dissolved oxygen (DO) conditions at the timescales of global circulation time. In this study, to mimic industrial-scale spatial DO gradients, we present a scale-down setup based on dynamic feast/famine regime (150 s) that leads to repetitive cycles with rapid changes in DO availability in glucose-limited chemostat cultures of Penicillium chrysogenum. Such DO feast/famine regime induced a stable and repetitive pattern with a reproducible metabolic response in time, and the dynamic response of intracellular metabolites featured specific differences in terms of both coverage and magnitude in comparison to other dynamic conditions, for example, substrate feast/famine cycles. Remarkably, intracellular sugar polyols were considerably increased as the hallmark metabolites along with a dynamic and higher redox state (NADH/NAD+) of the cytosol. Despite the increased availability of NADPH for penicillin production under the oscillatory DO conditions, this positive effect may be counteracted by the decreased ATP supply. Moreover, it is interesting to note that not only the penicillin productivity was reduced under such oscillating DO conditions, but also that of the unrecyclable byproduct ortho-hydroxyphenyl acetic acidand degeneration of penicillin productivity. Furthermore, dynamic flux profiles showed the most pronounced variations in central carbon metabolism, amino acid (AA) metabolism, energy metabolism and fatty acid metabolism upon the DO oscillation. Taken together, the metabolic responses of P. chrysogenum to DO gradients reported here are important for elucidating metabolic regulation mechanisms, improving bioreactor design and scale-up procedures as well as for constructing robust cell strains to cope with heterogenous industrial culture conditions.

Developments in scale-up procedures for industrial dryers

The aim of dryer scale-up is to increase batch size, or throughput of a continuous process, while maintaining final moisture content, product quality, end-use properties, safety and reliability. This article reviews and updates previously published material on scale-up processes of dryers in general and individual dryer types, particularly material from a Drying Technology special issue on scale-up in 1994. This is adapted to give a basic generic theory for scale-up, applying this to various dryers and noting specific exceptions. It is demonstrated that simplifying assumptions can often be reliably made for scale-up which reduce the level of complexity for modeling compared to equipment design and performance calculations. For example, prediction of drying rate and drying time in spray and flash dryers needs a kinetic model, but for scale-up a heat balance and scaling calculation usually suffices. Approximations such as a one-third power law for drying time are often effective. It is shown that there is some theoretical basis for empirical scale-up rules that have been used by dryer manufacturers. Scale-down is also important, selecting the most appropriate conditions for small-scale tests that will correspond to the desired performance at commercial scale. The individual concepts are not new, but they have not previously been unified into a simply stated general basis for scale-up.

Statistical optimization and sequential scale-up of α-galactosidase production by Actinoplanes utahensis B1 from shake flask to pilot scale

α-Galactosidase (α-GAL) is a class of hydrolase that releases galactose from galacto-oligosaccharides and synthetic substrates such as pNPG. In this study, the production of α-GAL by Actinoplanes utahensis B1 in submerged fermentation was enhanced by using statistical methods. The effects of temperature, pH, and inoculum percentage on enzyme secretion were optimized using BBD of RSM. The optimized process was scaled up from the shake flask to the laboratory scale (5 L) and to pilot scale (30 L) using KLa based scale-up strategy. By using BBD, a maximum yield of 62.5 U/mL was obtained at a temperature of 28 °C, a pH of 6.9, and an inoculum of 6.4%. Scale-up was performed successfully and achieved a yield of 74.4 U/mL and 76.8 U/mL in laboratory scale and pilot scale fermenters. The TOST was performed to validate the scale-up strategy and the results showed a confidence level of 95% for both scales indicating the perfect execution of scale-up procedure. Through the implementation of BBD and scale-up strategy, the overall enzyme yield has been significantly increased to 76%. This is the first article to explore the scale-up of α-GAL from the A. utahensis B1 strain and provide valuable insights for industrial applications.

On the Scalability of a Membrane Unit for Ultrapure Hydrogen Separation

Hydrogen permeation sparked a renewed interest in the second half of the 20th century due to the favorable features of this element as an energy factor. Furthermore, niche applications such as nuclear fusion gained attention for the highest selectivity ensured by self-supported dense metallic membranes, especially those consisting of Pd-based alloys. In this framework, the ENEA Frascati laboratories have decades of experience in the manufacturing, integration, and operation of Pd-Ag permeators. Most of the experimental investigations were performed on single-tube membranes, proving their performance under relevant operational conditions. Nowadays, once the applicability of this technology has been demonstrated, the scalability of the single-tube experience over medium- and large-scale units must be verified. To do this, ENEA Frascati laboratories have designed and constructed a multi-tube permeator, namely the Medium-Scaled Membrane Reactor (MeSMeR), focused on scalability assessment. In this work, the results obtained with the MeSMeR facility have been compared with previous experimental campaigns conducted on single-tube units, and the scalability of the permeation results has been proven. Moreover, post-test simulations have been performed based on single-tube finite element modeling, proving the scalability of the numerical outcomes and the possibility of using this tool for scale-up design procedures.

Nanoparticle depressants in froth flotation – The effect of colloidal silica with different size and surface modifications on the selective separation of semi-soluble salt type minerals

Colloidal silica has been presented as a selective depressant in froth flotation separation of scheelite and calcite by our group in 2020. Here we analyze the effects of colloidal silica dosage, specific surface area (i.e. size), and modification with microflotation on pure scheelite, calcite, fluorite, and apatite minerals. In a reagents scale-up procedure batch flotation experiments were conducted on a low-grade scheelite ore using statistical experimental design methodology. Microflotation experiments showed colloidal silica selectively depressing calcite flotation with increasing intensity with reduced size of colloidal silica. The nanoparticulate depressant did not affect the recoveries of scheelite, apatite, and fluorite – typical ore minerals that occur associated with calcite. Batch flotation of a low-grade scheelite ore confirmed the observations made at the microscale: colloidal silica significantly improves the scheelite-calcite selectivity index. Here, we observe a series of relations between colloidal silica specific surface area and modification on different process indicators such as scheelite-calcite selectivity index, recoveries, and froth properties. These relations are assumed to be due to the different aggregation mechanisms of the colloidal silica dispersions. The aluminate-modified colloidal silica, which tends to form a coherent gel network in the presence of Ca2+, shows the strongest effect on reducing calcite recovery accompanied by a significant reduction in scheelite recovery compared to the non-functionalised and silane-modified colloidal silica.

The impact of batch flotation tests on the industrial plant prediction

To predict the metallurgical results of industrial plants, laboratory batch flotation tests are carried out, relating both operations through scale-up factors. However, robust scale-up procedures and well-defined laboratory protocols are necessary to reach reliable results at industrial scale. In this paper, results from different flotation batch tests are presented, analysing the effect of batch protocol in terms of operating conditions, operator, ore type, water quality, and others on the metallurgical response. Additionally, the ability of batch responses to describe industrial operations and determine scale-up factors was analysed. Then, a sensitivity analysis was carried out, considering the effect of batch and industrial conditions on the estimation of scale-up factors. Results showed that the batch response significantly changes, depending on the operating conditions, flotation operator, and batch flotation machine, even for the same cell design. Additionally, it was observed that the batch recovery significantly changes when modifying ore type and water quality, which can cause changes in batch and/or industrial operation, affecting the scale-up factors. In addition, results showed that the scale-up factors varied significantly by changing operating conditions in a batch cell. This also occurs in plants when metallurgical performance changes, for example, due to a modification in launder design and/or operating condition, to increase recovery, or due to control limitations that prevent efficient metallurgical performance.

Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor

BackgroundThe omnipresence of population heterogeneity in industrial bioprocesses originates from prevailing dynamic bioprocess conditions, which promote differences in the expression of cellular characteristics. Despite the awareness, the concrete consequences of this phenomenon remain poorly understood.ResultsTherefore, for the first time, a L-phenylalanine overproducing Escherichia coli quadruple reporter strain was established for monitoring of general stress response, growth behavior, oxygen limitation and product formation of single cells based on mTagBFP2, mEmerald, CyOFP1, and mCardinal2 expression measured by flow cytometry. This strain was applied for the fed-batch production of L-phenylalanine from glycerol and ammonia in a stirred-tank bioreactor at homogeneous conditions compared to the same process in a novel two-compartment bioreactor. This two-compartment bioreactor consists of a stirred-tank bioreactor with an initial volume of 0.9 L (homogeneous zone) with a coiled flow inverter with a fixed working volume of 0.45 L as a bypass (limitation zone) operated at a mean hydraulic residence time of 102 s. The product formation was similar in both bioreactor setups with maximum L-phenylalanine concentrations of 21.1 ± 0.6 g L−1 demonstrating the consistency of this study’s microbial L-phenylalanine production. However, cell growth was vulnerable to repetitive exposure to the dynamically changing conditions in the two-compartment bioreactor with maximum biomass yields reduced by 21%. The functionality of reporter molecules was approved in the stirred-tank bioreactor cultivation, in which expressed fluorescence levels of all four markers were in accordance with respective process state variables. Additional evaluation of the distributions on single-cell level revealed the presence of population heterogeneity in both bioprocesses. Especially for the marker of the general stress response and the product formation, the corresponding histograms were characterized by bimodal shapes and broad distributions. These phenomena were pronounced particularly at the beginning and the end of the fed-batch process.ConclusionsThe here shown findings confirm multiple reporter strains to be a noninvasive tool for monitoring cellular characteristics and identifying potential subpopulations in bioprocesses. In combination with experiments in scale-down setups, these can be utilized for a better physiological understanding of bioprocesses and support future scale-up procedures.

Plasmids for Efficient Production of Recombinant Proteins in E. coli

Plasmids play a crucial role in the transmission of genetic information across different types of bacteria. Plasmids transfer pathogenicity and survival genes to the host bacterium, allowing the bacteria to adapt to new environments and grow. Several plasmids of differing sizes have been recovered from numerous bacterial species. Plasmids may be used to genetically modify bacteria for a variety of reasons, including the generation of recombinant proteins. Escherichia coli is the most often utilised bacterium for the production of recombinant proteins owing to its quick growth rate, low cost, high yield of recombinant proteins, and simple scale-up procedure. To boost the synthesis of heterologous proteins in E. coli, a number of plasmids have been used. Diverse plasmids have been devised and built to address challenges such as protein refolding, E. coli codon use, a lack of post-translational modifications such as glycosylation, and insufficient recovery of functionally viable recombinant proteins. Recent technical advancements that have made it possible for the E. coli expression system to create more complex proteins, such as glycosylated recombinant proteins and therapeutic antibodies.

Experimental and Numerical Analysis of a Pd-Ag Membrane Unit for Hydrogen Isotope Recovery in a Solid Blanket.

The interest of the fusion community in Pd-Ag membranes has grown in the last decades due to the high value of hydrogen permeability and the possibility of continuous operation, making it a promising technology when a gaseous stream of hydrogen isotopes must be recovered and separated from other impurities. This is the case of the Tritium Conditioning System (TCS) of the European fusion power plant demonstrator, called DEMO. This paper presents an experimental and numerical activity aimed at (i) assessing the Pd-Ag permeator performance under TCS-relevant conditions, (ii) validating a numerical tool for scale-up purposes, and (iii) carrying out a preliminary design of a TCS based on Pd-Ag membranes. Experiments were performed by feeding the membrane with a He-H2 gas mixture in a specific feed flow rate ranging from 85.4 to 427.2 mol h-1 m-2. A satisfactory agreement between experiments and simulations was obtained over a wide range of compositions, showing a root mean squared relative error of 2.3%. The experiments also recognized the Pd-Ag permeator as a promising technology for the DEMO TCS under the identified conditions. The scale-up procedure ended with a preliminary sizing of the system, relying on multi-tube permeators with an overall number ranging between 150 and 80 membranes in lengths of 500 and 1000 mm each.

Norfloxacin Cocrystals: Mechanochemical Synthesis and Scale-up Viability Through Solubility Studies

Cocrystals are recognized as one of the most efficient approaches to improve aqueous solubility of Biopharmaceutical Classification System, BCS, classes II and IV drugs. Cocrystal discovery and the establishment of experimental conditions suitable for scale-up purposes are some of the main challenges in cocrystal investigation. In this work, the investigation of mechanochemical synthesis of norfloxacin cocrystals with picolinic and isonicotinic acids is performed, leading to the discovery of two new cocrystals of this important BCS class IV antibiotic, which were characterized through thermal, spectral and diffractometric analysis. Norfloxacin apparent aqueous solubility using the cocrystals is also presented, with higher values being obtained for all the investigated systems when compared to the pure drug. Norfloxacin has 3 polymorphs and several solvents/hydrates, which represents a challenge for obtaining pure cocrystal forms from solvent crystallization. This challenge was successfully overcome in this work, as experimental conditions to obtain the pure cocrystals (the new ones and also norfloxacin-nicotinic acid and norfloxacin-saccharin) were established using Crystal16 equipment. This is a crucial step to envisage future scale-up procedures and therefore a valuable information for the pharmaceutical industry.

Dialkyl Carbonates as Green Solvents for Polyvinylidene Difluoride Membrane Preparation

Membrane processes are employed in a wide variety of industrial applications such as separation of complex mixtures, hydrogen isolation, CO2 removal, wastewater treatment, etc. Their use allows energy savings on the production cost compared to other traditional separation technologies. Nevertheless, the preparation of membranes not always fulfills sustainability obligations, especially when considering the commonly employed solvents, i.e., N-methyl-2-pyrrolidone and N,N-dimethylformamide, to mention just a few. Dialkyl carbonates (DACs) are well-known green solvents and reagents that have been extensively investigated as safe alternatives to chlorine-based compounds and media such as alkyl halides, phosgene, and chlorinated solvents. Following our recent study on a scale-up procedure to non-commercially available or expensive DACs, herein we report for the first time the application of organic carbonates as green media for membrane preparation. Theoretical thermodynamic studies were first carried out to predict the solubilities in DACs of different polymers commonly employed for membranes preparation. As a result, the use of selected organic carbonates as media for polyvinylidene difluoride membrane preparation was investigated by nonsolvent-induced phase separation (NIPS) and a combination of vapor-induced phase separation (VIPS)-NIPS techniques. Membranes obtained with custom-made DACs displayed greater structural resistances and smaller pore sizes compared to the ones achieved using commercially available cyclic organic carbonates. Data collected showed that it was possible to achieve a wide variety of dense and porous membranes by using a single family of compounds, highlighting once again the great versatility of DACs as green solvents.

Sensitivity of seismic attenuation and dispersion to dynamic elastic interactions of connected fractures: Quasi-static finite element modeling study

Prediction of seismic attenuation and dispersion that are inherently sensitive to hydraulic and elastic properties of the medium of interest in the presence of mesoscopic fractures and pores, is of great interest in the characterization of fractured formations. This has been very difficult, however, considering that stress interactions between fractures and pores, related to their spatial distributions, tend to play a crucial role on affecting overall dynamic elastic properties that are largely unexplored. We thus choose to quantitatively investigate frequency-dependent P-wave characteristics in fractured porous rocks at the scale of a representative sample using a numerical scale-up procedure via performing finite element modelling. Based on 2-D numerical quasi-static experiments, effects of fracture and fluid properties on energy dissipation in response to wave-induced fluid flow at the mesoscopic scale are quantified via solving Biot's equations of consolidation. We show that numerical results are sensitive to some key characteristics of probed synthetic rocks containing unconnected and connected fractures, demonstrating that connectivity, aperture and inclination of fractures as well as fracture infills exhibit strong impacts on the two manifestations of WIFF mechanisms in the connected scenario, and on resulting total wave attenuation and phase velocity. This, in turn, illustrates the importance of these two WIFF mechanisms in fractured rocks and thus, a deeper understanding of them may eventually allow for a better characterization of fracture systems using seismic methods. Moreover, this presented work combines rock physics predictions with seismic numerical simulations in frequency domain to illustrate the sensitivity of seismic signatures on the monitoring of an idealized geologic CO2 sequestration in fractured reservoirs. The simulation demonstrates that these two WIFF mechanisms can strongly modify seismic records and hence, indicating that incorporating the two energy dissipation mechanisms in the geophysical interpretation can potentially improving the monitoring and surveying of fluid variations in fractured formations.

High cell density cultivation of Bacillus subtilis NCIM 2063: Modeling, optimization and a scale-up procedure

Bacillus subtilis is a non-pathogenic, sporulating, Gram-positive bacteria with pronounced antimicrobial and metabolic activity and great potential for wide application in various fields. The aim of this paper was to determine the optimum B. subtilis NCIM 2063 growth conditions and to scale up biomass production from shake flasks to a bioreactor level. The critical growth para-meters and their interaction effects were studied using Box?Behnken experimental design and response surface methodology. Developed model equations were statistically significant with good prediction capability. It was found that during shake flask cultivation glucose should be added in concentration up to 5 g L-1 in DSM medium, OTR at 10 mol m-3 h-1 and temperature of 33?C, to achieve the maximum number of viable cells and spores. To scale up the process from shake flasks to the bioreactor level kLa was used as a main criterion. Scale up effect was evaluated by comparing the growth kinetics in the shake flasks and in a laboratory bioreactor. The total number of cells obtained in the bioreactor was 4.57x109 CFU mL-1 which is 1.41 times higher than the number of cells in the shake flasks (3.24x109 CFU mL-1), proving that the scale-up procedure was conducted successfully.

Direct substitution of the hydroxy group of alcohols with N-nucleophiles mediated by the substoichiometric amount of Selectfluor™ F-TEDA-BF4 as a precatalyst under mild reaction conditions

Selectfluor™ F-TEDA-BF4 was introduced, tested, and applied as an efficient, metal-free, and environmentally benign precatalyst for direct cross-coupling of various alcohols with N-nucleophiles under mild reaction conditions enhancing the green chemical profiles of these transformations. The scale-up procedure was achieved with an almost quantitative yield, confirming the presented method's synthetic applicability and potential for industrial application.

Power draw characteristics and comparison of scaling criteria for sawtooth impellers used in high shear mixing of shear thinning paint slurry

Power draw characteristics and scale-up procedures for high-speed sawtooth impellers were introduced by investigating various production sizes, fluids, impellers, and scale-up criteria to obtain the same product properties between different scales. The relationship between dimensionless Power number, Reynolds number, and Froude numbers were found for each impeller type at transitional flow regimes. The effect of Froude number was found to be very little, even though there was a deep vortex formation and no baffles in the mixing tank. Metzner – Otto constant which gives the relationship between rotational speed and shear rate was found as 20.7 and 12.4 for sawtooth impeller with big and small teeth, respectively. Productions were made by equalizing tip speed, shear rate, and power per volume between the commercial production and laboratory scales. In the result of rheological behavior measurement, stability test, and optical color evaluation, it was observed that the equal tip speed method gives a more intense dispersion in smaller batch sizes while the power per volume and shear rate method results in an insufficient particle size reduction performance.

N-Iodosuccinimide as a Precatalyst for C–N Bond-Forming Reactions from Alcohols under Mild Reaction Conditions

We report an efficient and selective methodology for the direct cross-coupling of alcohols with N-nucleophiles mediated by N-iodosuccinimide (NIS) as the non-metal, commercially available, low-cost, and most effective precatalyst among the N-halosuccinimides (NXSs) under mild reaction conditions enhancing the green chemical profiles of these reactions. The scale-up procedure was accomplished with almost quantitative yield, verifying the presented method’s synthetic applicability and potential for industrial application.

Multicomponent Direct Assembly of N-Heterospirocycles Facilitated by Visible-Light-Driven Photocatalysis.

N-heterospirocycles are interesting structural units found in both natural products and medicinal compounds but have relatively few reliable methods for their synthesis. Here, we enlist the photocatalytic generation of N-centered radicals to construct β-spirocyclic pyrrolidines from N-allylsulfonamides and alkenes. A variety of β-spirocyclic pyrrolidines have been constructed, including drug derivatives, in moderate to very good yields. Further derivatization of the products has also been demonstrated as has a viable scale-up procedure, making use of flow chemistry techniques.

Off-Gas System Scale-Up of HIsarna Iron-Making Process: A CFD-Based Approach

For all industrial applications, predicting system characteristics and behavior plays a vital role before constructing costly and complex multi-physic systems. Correct and reliable predictions become even more important once the aim is to go from small- to large-scale processes to establish an industrial demonstrations. In this study, a CFD-based scale-up of HIsarna off-gas system based on the Eulerian–Lagrangian approach is investigated and detailed step in scale-up procedure is discussed. A three-dimensional CFD model is developed and validated based on the available pilot scale data and used to design and scale up the post-combustion chamber (also known as reflux chamber). Detailed kinetics for volumetric and gas–solid reactions are incorporated in validated CFD model with a special attention to the wall boundary condition and modeling. The effect of reflux chamber geometry, oxygen injection ports, oxygen injection flowrate, isolation wall thickness, and inlet flue gas composition on different system characteristics such as heat loss through the wall, CO–H2–carbon mixture conversion, flue gas, and wall temperature are investigated. The aim of the scaled up geometry, like pilot scale, is to achieve full combustion of unwanted species inside the reflux chamber to assure zero emissions from the off-gas system. Compared to the pilot scale, the scaled up reflux chamber is capable of handling and removing higher amount of unwanted species coming from the main reactor and therefore lower CO–H2 and carbon particle emissions, mainly due to a larger size which provides larger volume and residence time for volumetric and gas–solid reaction to proceed.

Tuning Ice Nucleation by Mussel-Adhesive Inspired Polyelectrolytes: The Role of Hydrogen Bonding

Tuning Ice Nucleation by Mussel-Adhesive Inspired Polyelectrolytes: The Role of Hydrogen Bonding