Liquid Mixing on Falling Films: Marker-Free, Molecule-Sensitive 3D Mapping Using Raman Imaging.

Following up on a proof of concept, this publication presents a new method for mixing mapping on falling liquid films. On falling liquid films, different surfaces, plain or structured, are common. Regarding mixing of different components, the surface has a significant effect on its capabilities and performance. The presented approach combines marker-free and molecule-sensitive measurements with cross-section mapping to emphasize the mixing capabilities of different surfaces. As an example of the mixing capabilities on falling films, the mixing of sodium sulfate with tap water is presented, followed by a comparison between a plain surface and a pillow plate. The method relies upon point-by-point Raman imaging with a custom-built high-working-distance, low-depth-of-focus probe. To compensate for the long-time measurements, the continuous plant is in its steady state, which means the local mixing state is constant, and the differences are based on the liquids' position on the falling film, not on time. Starting with two separate streams, the mixing progresses by falling down the surface. In conclusion, Raman imaging is capable of monitoring mixing without any film disturbance and provides detailed information on liquid flow in falling films.

Film Thickness and Glycerol Concentration Mapping of Falling Films Based on Fluorescence and Near-Infrared Technique.

Falling film evaporation processes involve high fluid velocities with continuous variations in local film thickness, fluid composition, and viscosity. This contribution presents a parallel and complementary film thickness and concentration mapping distribution in falling films using a non-invasive fluorescence and near-infrared imaging technique. The experiments were performed with a mixture of glycerol/water with a mass fraction from 0 to 0.65 gglycgtotal-1 and operating ranges similar to evaporation processes. The measurement system was designed by integrating two optical measurement methods for experimental image analysis. The film thickness was evaluated using a VIS camera and high-power LEDs at 470 nm. The local glycerol concentration gglycgtotal-1 was determined using a NIR camera and high-power LEDs at 1050, 1300, 1450 and 1550 nm. A multiwavelength analysis with all NIR wavelengths was implemented with a better correlation for falling films at low flow velocity. The results show an improvement in the analysis of falling films with high flow velocities up to almost 500 mm/s by using only the 1450 nm wavelength and the fluorescence measurement. Simultaneous imaging analysis of film thickness and concentration in falling films provides further insight into understanding mass and heat transport and thus supports the optimization of falling film evaporators.

Marker-Free, Molecule Sensitive Mapping of Disturbed Falling Fluid Films Using Raman Imaging.

Technical liquid flow films are the basic arrangement for gas fluid transitions of all kinds and are the basis of many chemical processes, such as columns, evaporators, dryers, and different other kinds of fluid/fluid separation units. This publication presents a new method for molecule sensitive, non-contact, and marker-free localized concentration mapping in vertical falling films. Using Raman spectroscopy, no label or marker is needed for the detection of the local composition in liquid mixtures. In the presented cases, the film mapping of sodium sulfate in water on a plain surface as well as an added artificial streaming disruptor with the shape of a small pyramid is scanned in three dimensions. The results show, as a prove of concept, a clear detectable spectroscopic difference between air, back plate, and sodium sulfate for every local point in all three dimensions. In conclusion, contactless Raman scanning on falling films for liquid mapping is realizable without any mechanical film interaction caused by the measuring probe. Surface gloss or optical reflections from a metallic back plate are suppressed by using only inelastic light scattering and the mathematical removal of background noise.

Visualization of Local Concentration and Viscosity Distribution during Glycerol-Water Mixing in a Y-Shape Minichannel: A Proof-of-Concept-Study.

The work presents an efficient and non-invasive method to visualize the local concentration and viscosity distribution of two miscible and non-reacting substances with a significant viscosity difference in a microchannel with a Y-shape cell. The proof-of-concept setup consists of a near-infrared (NIR) camera and cost-effective dome lighting with NIR light-emitting diodes (LED) covering the wavelength range of 1050 to 1650 nm. Absorption differences of glycerol and water and their mixtures with a mass fraction of glycerol from 0 to 0.95 gGlycgtotal-1 were analyzed in the NIR spectral area. The resulting measurement images were converted in a concentration profile by using absorbance calculated with Lambert-Beer law. A linear behavior between the concentration and the absorption coefficient is demonstrated. The result of local concentration in mass fraction was used to determine the local viscosity and illustrated as distribution images. By variating the fluid parameters, the influences of the highly different original viscosities in the mixing procedure were investigated and visualized.

Expression of protein kinase HIPK2 is subject to a quality control mechanism that acts during translation and requires its kinase activity to prevent degradation of nascent HIPK2.

HIPK2 is a highly conserved, constitutively active Ser/Thr protein kinase that is involved in a broad spectrum of biological processes. We have previously reported that the expression of HIPK2 is auto-regulated by a mechanism that depends on the activity of its kinase domain, leading to decreased expression of kinase-dead versus wild-type HIPK2. We have now explored this mechanism in more detail. Differential expression of wild-type and kinase-dead HIPK2 is dependent on sequences located in the C-terminal part of HIPK2, but is only observed when this part of HIPK2 is translated together with the defective kinase domain. On their own, both the defective kinase domain and the C-terminal amino acid sequences are expressed at normal levels and independently of kinase activity. Insertion of a 2A-ribosomal skipping sequence into the HIPK2 coding sequence revealed that the differential expression of wild-type and kinase-dead HIPK2 is caused by degradation of nascent kinase-dead HIPK2. Because HIPK2 is constitutively active and auto-activates its kinase domain already during its translation we speculate that the regulatory mechanism discovered here serves as a quality control mechanism that leads to degradation of nascent kinase molecules with defective kinase domains. Overall our work provides insight into a novel auto-regulatory mechanism of HIPK2 expression, thereby adding a new layer of control to the regulation of HIPK2.

Studying the Impact of Wall Shear Stress on the Development and Performance of Electrochemically Active Biofilms.

A laminar flow reactor was designed that provides constant and reproducible growth conditions for the bioelectrochemical observation of electroactive bacteria (EAB). Experiments were performed using four reactors in parallel to enable the comparison of EAB growth behavior and bioelectrochemical performance under different hydrodynamic conditions while simultaneously keeping biological conditions identical. With regard to the moderate flow conditions found in wastewater treatment applications, the wall shear stress was adjusted to a range between 0.4 mPa to 2.9 mPa. Chronoamperometric data indicate that early stage current densities are improved by a moderate increase of the wall shear stress. In the same way, current onset times were increasing slightly towards higher values of the applied wall shear stress. Long-term observations of EAB performance showed a decrease in current density and a leveling of the trend observed for the early stages of biofilm growth.

Optimal Geometric Parameters for 3D Electrodes in Bioelectrochemical Systems: A Systematic Approach.

In this study, the performance of electroactive bacteria (EAB), cultivated inside tubular electrode ducts, is systematically investigated to derive predictions on the behavior of EAB under conditions limited by electrochemical losses. A modeling approach is applied to assess the influence of the electrochemical losses on the electrochemical performance and scaling characteristics of complex 3D structures, such as sponges and foams. A modular flow reactor is designed that provides laminar and reproducible flow conditions as a platform for the systematic electrochemical and bioelectrochemical characterization of 3D electrodes in bioelectrochemical systems (BES). The bioelectrochemical experiments are carried out in a set of reactors incorporating cylindrical electrodes exhibiting ducts of 1 cm length and different diameters ranging from 0.1 cm up to 1 cm. Single duct calculations are extrapolated to three dimensions through geometrical considerations; trends in 3D bioanode performance are demonstrated using the resulting simplified 3D structure. The combined experimental and modeling approach constitutes a framework for future studies on systematic electrode design.

Limited life cycle and cost assessment for the bioconversion of lignin-derived aromatics into adipic acid.

Lignin is an abundant and heterogeneous waste byproduct of the cellulosic industry, which has the potential of being transformed into valuable biochemicals via microbial fermentation. In this study, we applied a fast-pyrolysis process using softwood lignin resulting in a two-phase bio-oil containing monomeric and oligomeric aromatics without syringol. We demonstrated that an additional hydrodeoxygenation step within the process leads to an enhanced thermochemical conversion of guaiacol into catechol and phenol. After steam bath distillation, Pseudomonas putida KT2440-BN6 achieved a percent yield of cis, cis-muconic acid of up to 95 mol% from catechol derived from the aqueous phase. We next established a downstream process for purifying cis, cis-muconic acid (39.9 g/L) produced in a 42.5 L fermenter using glucose and benzoate as carbon substrates. On the basis of the obtained values for each unit operation of the empirical processes, we next performed a limited life cycle and cost analysis of an integrated biotechnological and chemical process for producing adipic acid and then compared it with the conventional petrochemical route. The simulated scenarios estimate that by attaining a mixture of catechol, phenol, cresol, and guaiacol (1:0.34:0.18:0, mol ratio), a titer of 62.5 (g/L) cis, cis-muconic acid in the bioreactor, and a controlled cooling of pyrolysis gases to concentrate monomeric aromatics in the aqueous phase, the bio-based route results in a reduction of CO2 -eq emission by 58% and energy demand by 23% with a contribution margin for the aqueous phase of up to 88.05 euro/ton. We conclude that the bio-based production of adipic acid from softwood lignins brings environmental benefits over the petrochemical procedure and is cost-effective at an industrial scale. Further research is essential to achieve the proposed cis, cis-muconic acid yield from true lignin-derived aromatics using whole-cell biocatalysts.

Continuous enzymatic production and adsorption of laminaribiose in packed bed reactors.

Bienzymatic production of laminaribiose from sucrose and glucose was combined with adsorption on zeolite BEA to introduce a first capture and purification step. Downstream processing including washing and desorption steps was characterized and optimized on a milliliter scale in batch mode. Results were then transferred to a packed bed system for enzymatic production and adsorption where the influence of adsorbent particle diameter on purity and productivity was evaluated. Finally, a continuous enzymatic production of laminaribiose was conducted over 10 days. The subsequent downstream processing of the loaded zeolites led to purities of over 0.5 gLaminaribiose gsugar -1 in the desorbate with a total productivity of 5.6 mgLaminaribiose Lenzyme bed -1 h-1 without the use of recycles.

Early process development of API applied to poorly water-soluble TBID.

Finding and optimising of synthesis processes for active pharmaceutical ingredients (API) is time consuming. In the finding phase, established methods for synthesis, purification and formulation are used to achieve a high purity API for biological studies. For promising API candidates, this is followed by pre-clinical and clinical studies requiring sufficient quantities of the active component. Ideally, these should be produced with a process representative for a later production process and suitable for scaling to production capacity. This work presents an overview of different approaches for process synthesis based on an existing lab protocol. This is demonstrated for the production of the model drug 4,5,6,7-tetrabromo-2-(1H-imidazol-2-yl) isoindolin-1,3-dione (TBID). Early batch synthesis and purification procedures typically suffer from low and fluctuating yields and purities due to poor process control. In a first step the literature synthesis and purification procedure was modified and optimized using solubility measurements, targeting easier and safer processing for consecutive studies.

Biotechnological production of hyperforin for pharmaceutical formulation.

Hyperforin is a major active constituent of Hypericum perforatum (St. John's wort). It has amazing pharmacological activities, such as antidepressant properties, but it is labile and difficult to synthesize. Its sensitivity and lipophilicity are challenges for processing and formulation. Its chemical complexity provokes approaches of biotechnological production and modification. Dedifferentiated H. perforatum cell cultures lack appropriate storage sites and hence appreciable hyperforin levels. Shoot cultures are capable of forming hyperforin but less suitable for biomass up-scaling in bioreactors. Roots commonly lack hyperforin but a recently established adventitious root line has been demonstrated to produce hyperforin and derivatives at promising levels. The roots also contained lupulones, the typical constituents of hop (Humulus lupulus). Although shear-sensitive, these root cultures provide a potential production platform for both individual compounds and extracts with novel combinations of constituents and pharmacological activities. Besides in vitro cultivation techniques, the reconstruction of hyperforin biosynthesis in microorganisms is a promising alternative for biotechnological production. The biosynthetic pathway is under study, with omics-technologies being increasingly implemented. These biotechnological approaches may not only yield hyperforin at reasonable productivity but also allow for modifications of its chemical structure and pharmacological profile.

Fast and calibration free determination of first order reaction kinetics in API synthesis using in-situ ATR-FTIR.

In early stages of drug development only sparse amounts of the key substances are available, which is problematic for the determination of important process data like reaction kinetics. Therefore, it is important to perform experiments as economically as possible, especially in regards to limiting compounds. Here we demonstrate the use of a temperature step experiment enabling the determination of complete reaction kinetics in a single non-isothermal experiment. In contrast to the traditionally used HPLC, the method takes advantage of the high measuring rate and the low amount of labor involved in using in-situ ATR-FTIR to determine time-dependent concentration-equivalent data.

Downstream processing of hyperforin from Hypericum perforatum root cultures.

Hyperforin is a major metabolite of the medicinal plant Hypericum perforatum (St. John's Wort) and has recently been found in hormone induced root cultures. The objective of this study is to identify a downstream process for the production of a hyperforin-rich extract with maximum extraction efficiency and minimal decomposition. The maximum extraction time was found to be 60min. The comparison of two equipment concepts for the extraction and solvent evaporation was performed employing two different solvents. While the rotary mixer showed better results for the extraction efficiency than a stirred vessel, the latter set-up was able to handle larger volumes but did not meet all process requirements. For the evaporation the prompt evaporation of the extraction agent using nitrogen stripping led to minor decomposition. In a 5L stirred vessel, the highest specific extraction of hyperforin was 4.3mg hyperforin/g dry weight bio material. Parameters for the equipment design for extraction and solvent evaporation were determined based on the experimental data.

Chitosan-based hybrid immobilization in bienzymatic reactions and its application to the production of laminaribiose.

A hybrid-immobilization method was developed to improve the long-term stability of laminaribiose phosphorylase immobilized on epoxy supports Sepabeads EC-EP/S. Entrapment in chitosan retained all of the enzyme activity depending on the amount of entrapped solid materials and increased half-life by a factor of 10-94.4 h. No enzyme activity loss was determined during 12 times reuse. The immobilization method is also applicable to sucrose phosphorylase immobilized on Sepabeads EC-EP/S. Up to 31.9 g/L laminaribiose were produced in bienzymatic batch experiments with reaction-integrated product separation by adsorption on zeolites.

Immobilization and Characterization of E. gracilis Extract with Enriched Laminaribiose Phosphorylase Activity for Bienzymatic Production of Laminaribiose.

Immobilization methods and carriers were screened for immobilization of Euglena gracilis extract with laminaribiose phosphorylase activity. The extract was successfully immobilized on three different carriers via covalent linkage. Suitable immobilization carriers were Sepabeads EC-EP/S and ECR 8209M with epoxy groups and ECR 8309M with amino groups as functional units. Immobilization on Sepabeads EC-EP/S resulted in highest retained activity (65%). The immobilizates were characterized for pH, temperature, and buffer molarity preferences. The immobilized enzyme lost 48% of its activity when used seven times. Together with sucrose phosphorylase, laminaribiose phosphorylase was successfully applied for bienzymatic production of laminaribiose from sucrose and glucose with a final laminaribiose concentration of 14.3 ± 2.1 g/L (20% yield).

Hyperforin production in Hypericum perforatum root cultures.

Extracts of the medicinal plant Hypericum perforatum are used to treat depression and skin irritation. A major API is hyperforin, characterized by sensitivity to light, oxygen and temperature. Total synthesis of hyperforin is challenging and its content in field-grown plants is variable. We have established in vitro cultures of auxin-induced roots, which are capable of producing hyperforin, as indicated by HPLC-DAD and ESI-MS analyses. The extraction yield and the productivity upon use of petroleum ether after solvent screening were ∼5 mg/g DW and ∼50 mg/L culture after six weeks of cultivation. The root cultures also contained secohyperforin and lupulones, which were not yet detected in intact plants. In contrast, they lacked another class of typical H. perforatum constituents, hypericins, as indicated by the analysis of methanolic extracts. Hyperforins and lupulones were stabilized and enriched as dicyclohexylammonium salts. Upon up-scaling of biomass production and downstream processing, H. perforatum root cultures may provide an alternative platform for the preparation of medicinal extracts and the isolation of APIs.

Expanding the elution by characteristic point method to columns with a finite number of theoretical plates.

The elution by characteristic point (ECP) method provides a rapid approach to determine whole isotherm data with small material usage. It is especially desired wherever the adsorbent or the adsorbate is expensive, toxic or only available in small amounts. However, the ECP method is limited to adsorbents that are well optimized for chromatographic use and therefore provide a high number of theoretical plates when packed into columns (2000 or more for Langmuir type isotherms are suggested). Here we present a novel approach that uses a new profile correction to apply the ECP method to poorly optimized adsorbents with less than 200 theoretical plates. Non-ideality effects are determined using a dead volume marker injection and the resulting marker profile is used to compensate the named effects considering their dependency from the actual concentration instead of assuming rectangular profiles. Experimental and literature data are used to compare the new ECP approach with batch method results.

Stability of the Metastable α-Polymorph in Solid Triglyceride Drug-Carrier Nanoparticles.

Colloidal dispersions of crystalline nonpolar lipids are under intensive investigation as carrier systems in pharmaceutics and nutrition. In this context, the controlled preparation of particles in a metastable polymorphic state is of some interest for the delivery of active substances. In the present study, tristearin particles stabilized with three α-polymorph-preserving emulsifier regimes ((I) sodium glycocholate/saturated long-chain phospholipids, (II) sodium glycocholate, and (III) poly(vinyl alcohol) (PVA)) were investigated concerning the stability of the metastable α-polymorph after controlled crystallization of the particles from the melt. Upon long-term storage, the α-polymorph was preserved best in PVA-stabilized dispersions, followed by those stabilized with the glycocholate/phospholipid mixture and finally those stabilized solely with the bile salt. In particular for rapidly crystallized nanoparticles, the formation of an α-polymorph with highly reduced lamellarity was observed. According to time-/temperature-resolved synchrotron X-ray diffraction analysis with simultaneous DSC (differential scanning calorimetry) studies, this less-ordered α-polymorph transformed into the common, lamellar α-form upon heating. Although the presence of the less-ordered form is probably related to the extraordinarily high stability of the metastable α-polymorph observed in some of the dispersions, it could not completely prevent the transition into the stable ß-polymorph. The higher the transition temperature of the less-ordered α-form to the ordered one, the slower was the polymorphic transition to the stable ß-polymorph. To estimate the polymorphic stability of the differently stabilized particles upon isothermal long-term storage, standard DSC measurements on samples stored at 23 °C for 4 weeks seem to be of predictive value.

Preparation of calibration gas mixtures for the measurement of breath alcohol concentration.

Calibration gas mixtures for breath alcohol measuring techniques need to have a defined ethanol concentration in water-saturated air. For the calibration of evidential breath analysers in Germany, these gases are prepared at the Physikalisch-Technische Bundesanstalt according to the saturation method. The ethanol concentration is calculated via the air-liquid partition ratios of the air-water-ethanol substance system. This article deals with the selection and critical consideration of different preparation methods for test gases and their applicability to the specific gas mixture required for breath alcohol measurements.

Structure and shear strength of microbial biofilms as determined with confocal laser scanning microscopy and fluid dynamic gauging using a novel rotating disc biofilm reactor.

The cohesive strength of microbial biofilms cultivated on a rotating disc has been measured using fluid dynamic gauging (FDG). The thickness of heterotrophic mixed culture biofilms was found to depend on substrate concentration and shear force at the biofilm surface during the cultivation. For high substrate concentrations and low shear forces the biofilm thickness increased to several 100 microm within 7 days. Low substrate concentration and higher shear forces yielded thin biofilms of about 100 microm thickness. Independent from cultivation conditions and thickness of the biofilms their cohesive strength ranged between 6.0 and 7.7 N m(-2). The ratio between cohesive strength measured with FDG and shear forces applied during biofilm cultivation have ranged from 200 to 1,100. Higher concentrations of iron in the cultivation media has a positive effect on the stability of the biofilms cultivated. By using the CLSM technique a stable base biofilm with a high amount of stained EPS glycoconjugates could be visualized after gauging. The thickness of the base biofilm was about 100 microm for all biofilms cultivated and was not removable under the applied shear conditions used during FDG.