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Nacre-mimetic nanocomposites based on high fractions of synthetic high-aspect-ratio nanoclays in combination with polymers are continuously pushing boundaries for advanced material properties, such as high barrier against oxygen, extraordinary mechanical behavior, fire shielding, and glass-like transparency. Additionally, they provide interesting model systems to study polymers under nanoconfinement due to the well-defined layered nanocomposite arrangement. Although the general behavior in terms of forming such layered nanocomposite materials using evaporative self-assembly and controlling the nanoclay gallery spacing by the nanoclay/polymer ratio is understood, some combinations of polymer matrices and nanoclay reinforcement do not comply with the established models. Here, we demonstrate a thorough characterization and analysis of such an unusual polymer/nanoclay pair that falls outside of the general behavior. Poly(ethylene oxide) (PEO) and sodium fluorohectorite form nacre-mimetic, lamellar nanocomposites that are completely transparent and show high mechanical stiffness and high gas barrier, but there is only limited expansion of the nanoclay gallery spacing when adding increasing amounts of polymer. This behavior is maintained for molecular weights of PEO varied over four orders of magnitude and can be traced back to depletion forces. By careful investigation via X-ray diffraction and proton low-resolution solid-state NMR, we are able to quantify the amount of mobile and immobilized polymer species in between the nanoclay galleries and around proposed tactoid stacks embedded in a PEO matrix. We further elucidate the unusual confined polymer dynamics, indicating a relevant role of specific surface interactions.
In traditional microbial biobutanol production, the solvent must be recovered during fermentation process for a sufficient space-time yield. Thermal separation is not feasible due to the boiling point of n-butanol. As an integrated and selective solid-liquid separation alternative, solvent impregnated resins (SIRs) were applied. Two polymeric resins were evaluated and an extractant screening was conducted. Vacuum application with vapor collection in fixed-bed column as bioreactor bypass was successfully implemented as butanol desorption step. In course of further increasing process economics, fermentation with renewable lignocellulosic substrates was conducted using Clostridium acetobutylicum. Utilization of SIR was shown to be a potential strategy for solvent removal from fermentation broth, while application of a bypass column allows for product removal and recovery at once.
An acetoin biosensor based on a capacitive electrolyte–insulator–semiconductor (EIS) structure modified with the enzyme acetoin reductase, also known as butane-2,3-diol dehydrogenase (Bacillus clausii DSM 8716ᵀ), is applied for acetoin detection in beer, red wine, and fermentation broth samples for the first time. The EIS sensor consists of an Al/p-Si/SiO₂/Ta₂O₅ layer structure with immobilized acetoin reductase on top of the Ta₂O₅ transducer layer by means of crosslinking via glutaraldehyde. The unmodified and enzyme-modified sensors are electrochemically characterized by means of leakage current, capacitance–voltage, and constant capacitance methods, respectively.
Quantitative nuclear magnetic resonance (qNMR) is routinely performed by the internal or external standardization. The manuscript describes a simple alternative to these common workflows by using NMR signal of another active nuclei of calibration compound. For example, for any arbitrary compound quantification by NMR can be based on the use of an indirect concentration referencing that relies on a solvent having both 1H and 2H signals. To perform high-quality quantification, the deuteration level of the utilized deuterated solvent has to be estimated.
In this contribution the new method was applied to the determination of deuteration levels in different deuterated solvents (MeOD, ACN, CDCl3, acetone, benzene, DMSO-d6). Isopropanol-d6, which contains a defined number of deuterons and protons, was used for standardization. Validation characteristics (precision, accuracy, robustness) were calculated and the results showed that the method can be used in routine practice. Uncertainty budget was also evaluated. In general, this novel approach, using standardization by 2H integral, benefits from reduced sample preparation steps and uncertainties, and can be applied in different application areas (purity determination, forensics, pharmaceutical analysis, etc.).
Quantitative nuclear magnetic resonance (qNMR) is considered as a powerful tool for multicomponent mixture analysis as well as for the purity determination of single compounds. Special attention is currently paid to the training of operators and study directors involved in qNMR testing. To assure that only qualified personnel are used for sample preparation at our GxP-accredited laboratory, weighing test was proposed. Sixteen participants performed six-fold weighing of the binary mixture of dibutylated hydroxytoluene (BHT) and 1,2,4,5-tetrachloro-3-nitrobenzene (TCNB). To evaluate the quality of data analysis, all spectra were evaluated manually by a qNMR expert and using in-house developed automated routine. The results revealed that mean values are comparable and both evaluation approaches are free of systematic error. However, automated evaluation resulted in an approximately 20% increase in precision. The same findings were revealed for qNMR analysis of 32 compounds used in pharmaceutical industry. Weighing test by six-fold determination in binary mixtures and automated qNMR methodology can be recommended as efficient tools for evaluating staff proficiency. The automated qNMR method significantly increases throughput and precision of qNMR for routine measurements and extends application scope of qNMR.
As a low-input crop, Miscanthus offers numerous advantages that, in addition to agricultural applications, permits its exploitation for energy, fuel, and material production. Depending on the Miscanthus genotype, season, and harvest time as well as plant component (leaf versus stem), correlations between structure and properties of the corresponding isolated lignins differ. Here, a comparative study is presented between lignins isolated from M. x giganteus, M. sinensis, M. robustus and M. nagara using a catalyst-free organosolv pulping process. The lignins from different plant constituents are also compared regarding their similarities and differences regarding monolignol ratio and important linkages. Results showed that the plant genotype has the weakest influence on monolignol content and interunit linkages. In contrast, structural differences are more significant among lignins of different harvest time and/or season. Analyses were performed using fast and simple methods such as nuclear magnetic resonance (NMR) spectroscopy. Data was assigned to four different linkages (A: β-O-4 linkage, B: phenylcoumaran, C: resinol, D: β-unsaturated ester). In conclusion, A content is particularly high in leaf-derived lignins at just under 70% and significantly lower in stem and mixture lignins at around 60% and almost 65%. The second most common linkage pattern is D in all isolated lignins, the proportion of which is also strongly dependent on the crop portion. Both stem and mixture lignins, have a relatively high share of approximately 20% or more (maximum is M. sinensis Sin2 with over 30%). In the leaf-derived lignins, the proportions are significantly lower on average. Stem samples should be chosen if the highest possible lignin content is desired, specifically from the M. x giganteus genotype, which revealed lignin contents up to 27%. Due to the better frost resistance and higher stem stability, M. nagara offers some advantages compared to M. x giganteus. Miscanthus crops are shown to be very attractive lignocellulose feedstock (LCF) for second generation biorefineries and lignin generation in Europe.
The molecular weight properties of lignins are one of the key elements that need to be analyzed for a successful industrial application of these promising biopolymers. In this study, the use of 1H NMR as well as diffusion-ordered spectroscopy (DOSY NMR), combined with multivariate regression methods, was investigated for the determination of the molecular weight (Mw and Mn) and the polydispersity of organosolv lignins (n = 53, Miscanthus x giganteus, Paulownia tomentosa, and Silphium perfoliatum). The suitability of the models was demonstrated by cross validation (CV) as well as by an independent validation set of samples from different biomass origins (beech wood and wheat straw). CV errors of ca. 7–9 and 14–16% were achieved for all parameters with the models from the 1H NMR spectra and the DOSY NMR data, respectively. The prediction errors for the validation samples were in a similar range for the partial least squares model from the 1H NMR data and for a multiple linear regression using the DOSY NMR data. The results indicate the usefulness of NMR measurements combined with multivariate regression methods as a potential alternative to more time-consuming methods such as gel permeation chromatography.
In this study, a recently proposed NMR standardization approach by 2H integral of deuterated solvent for quantitative multicomponent analysis of complex mixtures is presented. As a proof of principle, the existing NMR routine for the analysis of Aloe vera products was modified. Instead of using absolute integrals of targeted compounds and internal standard (nicotinamide) from 1H-NMR spectra, quantification was performed based on the ratio of a particular 1H-NMR compound integral and 2H-NMR signal of deuterated solvent D2O. Validation characteristics (linearity, repeatability, accuracy) were evaluated and the results showed that the method has the same precision as internal standardization in case of multicomponent screening. Moreover, a dehydration process by freeze drying is not necessary for the new routine. Now, our NMR profiling of A. vera products needs only limited sample preparation and data processing. The new standardization methodology provides an appealing alternative for multicomponent NMR screening. In general, this novel approach, using standardization by 2H integral, benefits from reduced sample preparation steps and uncertainties, and is recommended in different application areas (purity determination, forensics, pharmaceutical analysis, etc.).
The investigation of the possibility to determine various characteristics of powder heparin (n = 115) was carried out with infrared spectroscopy. The evaluation of heparin samples included several parameters such as purity grade, distributing company, animal source as well as heparin species (i.e. Na-heparin, Ca-heparin, and heparinoids). Multivariate analysis using principal component analysis (PCA), soft independent modelling of class analogy (SIMCA), and partial least squares – discriminant analysis (PLS-DA) were applied for the modelling of spectral data. Different pre-processing methods were applied to IR spectral data; multiplicative scatter correction (MSC) was chosen as the most relevant.
Obtained results were confirmed by nuclear magnetic resonance (NMR) spectroscopy. Good predictive ability of this approach demonstrates the potential of IR spectroscopy and chemometrics for screening of heparin quality. This approach, however, is designed as a screening tool and is not considered as a replacement for either of the methods required by USP and FDA.
The feasibility of light-addressed detection and manipulation of pH gradients inside an electrochemical microfluidic cell was studied. Local pH changes, induced by a light-addressable electrode (LAE), were detected using a light-addressable potentiometric sensor (LAPS) with different measurement modes representing an actuator-sensor system. Biosensor functionality was examined depending on locally induced pH gradients with the help of the model enzyme penicillinase, which had been immobilized in the microfluidic channel. The surface morphology of the LAE and enzyme-functionalized LAPS was studied by scanning electron microscopy. Furthermore, the penicillin sensitivity of the LAPS inside the microfluidic channel was determined with regard to the analyte’s pH influence on the enzymatic reaction rate. In a final experiment, the LAE-controlled pH inhibition of the enzyme activity was monitored by the LAPS.
Most drugs are no longer produced in their own countries by the pharmaceutical companies, but by contract manufacturers or at manufacturing sites in countries that can produce more cheaply. This not only makes it difficult to trace them back but also leaves room for criminal organizations to fake them unnoticed. For these reasons, it is becoming increasingly difficult to determine the exact origin of drugs. The goal of this work was to investigate how exactly this is possible by using different spectroscopic methods like nuclear magnetic resonance and near- and mid-infrared spectroscopy in combination with multivariate data analysis. As an example, 56 out of 64 different paracetamol preparations, collected from 19 countries around the world, were chosen to investigate whether it is possible to determine the pharmaceutical company, manufacturing site, or country of origin. By means of suitable pre-processing of the spectra and the different information contained in each method, principal component analysis was able to evaluate manufacturing relationships between individual companies and to differentiate between production sites or formulations. Linear discriminant analysis showed different results depending on the spectral method and purpose. For all spectroscopic methods, it was found that the classification of the preparations to their manufacturer achieves better results than the classification to their pharmaceutical company. The best results were obtained with nuclear magnetic resonance and near-infrared data, with 94.6%/99.6% and 98.7/100% of the spectra of the preparations correctly assigned to their pharmaceutical company or manufacturer.
This study investigated the anaerobic digestion of an algal–bacterial biofilm grown in artificial wastewater in an Algal Turf Scrubber (ATS). The ATS system was located in a greenhouse (50°54′19ʺN, 6°24′55ʺE, Germany) and was exposed to seasonal conditions during the experiment period. The methane (CH4) potential of untreated algal–bacterial biofilm (UAB) and thermally pretreated biofilm (PAB) using different microbial inocula was determined by anaerobic batch fermentation. Methane productivity of UAB differed significantly between microbial inocula of digested wastepaper, a mixture of manure and maize silage, anaerobic sewage sludge, and percolated green waste. UAB using sewage sludge as inoculum showed the highest methane productivity. The share of methane in biogas was dependent on inoculum. Using PAB, a strong positive impact on methane productivity was identified for the digested wastepaper (116.4%) and a mixture of manure and maize silage (107.4%) inocula. By contrast, the methane yield was significantly reduced for the digested anaerobic sewage sludge (50.6%) and percolated green waste (43.5%) inocula. To further evaluate the potential of algal–bacterial biofilm for biogas production in wastewater treatment and biogas plants in a circular bioeconomy, scale-up calculations were conducted. It was found that a 0.116 km2 ATS would be required in an average municipal wastewater treatment plant which can be viewed as problematic in terms of space consumption. However, a substantial amount of energy surplus (4.7–12.5 MWh a−1) can be gained through the addition of algal–bacterial biomass to the anaerobic digester of a municipal wastewater treatment plant. Wastewater treatment and subsequent energy production through algae show dominancy over conventional technologies.
Halophilic and halotolerant microorganisms represent a promising source of salt-tolerant enzymes suitable for various biotechnological applications where high salt concentrations would otherwise limit enzymatic activity. Considering the current growing enzyme market and the need for more efficient and new biocatalysts, the present study aimed at the characterization of a high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T. The protease gene was cloned and expressed in Bacillus subtilis DB104. The recombinant protease SPAO with 269 amino acids belongs to the subfamily of high-alkaline subtilisins. The biochemical characteristics of purified SPAO were analyzed in comparison with subtilisin Carlsberg, Savinase, and BPN'. SPAO, a monomer with a molecular mass of 27.1 kDa, was active over a wide range of pH 6.0–12.0 and temperature 20–80 °C, optimally at pH 9.0–9.5 and 55 °C. The protease is highly oxidatively stable to hydrogen peroxide and retained 58% of residual activity when incubated at 10 °C with 5% (v/v) H2O2 for 1 h while stimulated at 1% (v/v) H2O2. Furthermore, SPAO was very stable and active at NaCl concentrations up to 5.0 m. This study demonstrates the potential of SPAO for biotechnological applications in the future.
NMR standardization approach that uses the 2H integral of deuterated solvent for quantitative multinuclear analysis of pharmaceuticals is described. As a proof of principle, the existing NMR procedure for the analysis of heparin products according to US Pharmacopeia monograph is extended to the determination of Na+ and Cl- content in this matrix. Quantification is performed based on the ratio of a 23Na (35Cl) NMR integral and 2H NMR signal of deuterated solvent, D2O, acquired using the specific spectrometer hardware. As an alternative, the possibility of 133Cs standardization using the addition of Cs2CO3 stock solution is shown. Validation characteristics (linearity, repeatability, sensitivity) are evaluated. A holistic NMR profiling of heparin products can now also be used for the quantitative determination of inorganic compounds in a single analytical run using a single sample. In general, the new standardization methodology provides an appealing alternative for the NMR screening of inorganic and organic components in pharmaceutical products.
Utilizing an appropriate enzyme immobilization strategy is crucial for designing enzyme-based biosensors. Plant virus-like particles represent ideal nanoscaffolds for an extremely dense and precise immobilization of enzymes, due to their regular shape, high surface-to-volume ratio and high density of surface binding sites. In the present work, tobacco mosaic virus (TMV) particles were applied for the co-immobilization of penicillinase and urease onto the gate surface of a field-effect electrolyte-insulator-semiconductor capacitor (EISCAP) with a p-Si-SiO₂-Ta₂O₅ layer structure for the sequential detection of penicillin and urea. The TMV-assisted bi-enzyme EISCAP biosensor exhibited a high urea and penicillin sensitivity of 54 and 85 mV/dec, respectively, in the concentration range of 0.1–3 mM. For comparison, the characteristics of single-enzyme EISCAP biosensors modified with TMV particles immobilized with either penicillinase or urease were also investigated. The surface morphology of the TMV-modified Ta₂O₅-gate was analyzed by scanning electron microscopy. Additionally, the bi-enzyme EISCAP was applied to mimic an XOR (Exclusive OR) enzyme logic gate.
The subtilase family (S8), a member of the clan SB of serine proteases are ubiquitous in all kingdoms of life and fulfil different physiological functions. Subtilases are divided in several groups and especially subtilisins are of interest as they are used in various industrial sectors. Therefore, we searched for new subtilisin sequences of the family Bacillaceae using a data mining approach. The obtained 1,400 sequences were phylogenetically classified in the context of the subtilase family. This required an updated comprehensive overview of the different groups within this family. To fill this gap, we conducted a phylogenetic survey of the S8 family with characterised holotypes derived from the MEROPS database. The analysis revealed the presence of eight previously uncharacterised groups and 13 subgroups within the S8 family. The sequences that emerged from the data mining with the set filter parameters were mainly assigned to the subtilisin subgroups of true subtilisins, high-alkaline subtilisins, and phylogenetically intermediate subtilisins and represent an excellent source for new subtilisin candidates.
Acetoin and diacetyl have a major impact on the flavor of alcoholic beverages such as wine or beer. Therefore, their measurement is important during the fermentation process. Until now, gas chromatographic techniques have typically been applied; however, these require expensive laboratory equipment and trained staff, and do not allow for online monitoring. In this work, a capacitive electrolyte–insulator–semiconductor sensor modified with tobacco mosaic virus (TMV) particles as enzyme nanocarriers for the detection of acetoin and diacetyl is presented. The enzyme acetoin reductase from Alkalihalobacillus clausii DSM 8716ᵀ is immobilized via biotin–streptavidin affinity, binding to the surface of the TMV particles. The TMV-assisted biosensor is electrochemically characterized by means of leakage–current, capacitance–voltage, and constant capacitance measurements. In this paper, the novel biosensor is studied regarding its sensitivity and long-term stability in buffer solution. Moreover, the TMV-assisted capacitive field-effect sensor is applied for the detection of diacetyl for the first time. The measurement of acetoin and diacetyl with the same sensor setup is demonstrated. Finally, the successive detection of acetoin and diacetyl in buffer and in diluted beer is studied by tuning the sensitivity of the biosensor using the pH value of the measurement solution.
We present a concise mini overview on the approaches to the disposal of nuclear waste currently used or deployed. The disposal of nuclear waste is the end point of nuclear waste management (NWM) activities and is the emplacement of waste in an appropriate facility without the intention to retrieve it. The IAEA has developed an internationally accepted classification scheme based on the end points of NWM, which is used as guidance. Retention times needed for safe isolation of waste radionuclides are estimated based on the radiotoxicity of nuclear waste. Disposal facilities usually rely on a multi-barrier defence system to isolate the waste from the biosphere, which comprises the natural geological barrier and the engineered barrier system. Disposal facilities could be of a trench type, vaults, tunnels, shafts, boreholes, or mined repositories. A graded approach relates the depth of the disposal facilities’ location with the level of hazard. Disposal practices demonstrate the reliability of nuclear waste disposal with minimal expected impacts on the environment and humans.