Engineering of Saccharomyces cerevisiae for the production of (+)-ambrein

The triterpenoid (+)-ambrein is the major component of ambergris, a coprolite of the sperm whale that can only be rarely found on shores. Upon oxidative degradation of (+)-ambrein, several fragrance molecules are formed, amongst them (−)-ambrox, one of the highest valued compounds in the perfume industry. In order to generate a Saccharomyces cerevisiae whole-cell biocatalyst for the production of (+)-ambrein, intracellular supply of the squalene was enhanced by overexpression of two central enzymes in the mevalonate and sterol biosynthesis pathway, namely the N-terminally truncated 3-hydroxy-3-methylglutaryl-CoA reductase 1 (tHMG) and the squalene synthase (ERG9). In addition, another key enzyme in sterol biosynthesis, squalene epoxidase (ERG1) was inhibited by an experimentally defined amount of the inhibitor terbinafine in order to reduce flux of squalene towards ergosterol biosynthesis while retaining sufficient activity to maintain cell viability and growth. Heterologous expression of a promiscuous variant of Bacillus megaterium tetraprenyl-β-curcumene cyclase (BmeTC-D373C), which has been shown to be able to catalyse the conversion of squalene to 3-deoxyachillol and then further to (+)-ambrein resulted in production of these triterpenoids in S. cerevisiae for the first time. Triterpenoid yields are comparable with the best microbial production chassis described in literature so far, the methylotrophic yeast Pichia pastoris. Consequently, we discuss similarities and differences of these two yeast species when applied for whole-cell (+)-ambrein production.     

Topology optimization for crashworthiness of thin-walled structures under axial impact using hybrid cellular automata

Although topology optimization is well established in most engineering fields, it is still in its infancy concerning highly non-linear structural applications like vehicular crashworthiness. One of the approaches recently proposed and based on Hybrid Cellular Automata is modified here such that it can be applied for the first time to thin-walled structures. Classical methods based on voxel techniques, i.e., on solid three-dimensional volume elements, cannot derive structures made from thin metal sheets where the main energy absorption mode is related to plastic buckling, folding and failure. Because the main components of car structures are made from such thin-walled beams and panels, a special approach using SFE CONCEPT was developed, which is presented in this paper.     

Inelastic Neutron Scattering and Density Functional Theory–Molecular Dynamics Study of Si Dynamics in Ti3SiC2

Observed differences between measured and calculated elastic constants for Ti₃SiC₂ are investigated using Density Functional Theory and Inelastic Neutron Scattering. The agreement between the calculated lattice dynamics and the dynamics measured by inelastic neutron scattering is considered good except at energies below ~20 meV where discrepancies suggest anharmonic potentials. This suggestion is confirmed by Density Functional Theory—Molecular Dynamics simulation which shows multiple site occupancy of the Si atoms within the basal plane at finite temperature and produces a calculated inelastic spectrum in better agreement with the measured spectrum in the low‐energy region. The highly anharmonic potential energy surface of the Si atoms offers an explanation for the failure of elastic constants, calculated based on the harmonic approximation, to agree with initial experimental measurements.     

Viable Mycobacterium avium ssp. paratuberculosis isolated from calf milk replacer

When advising farmers on how to control Johne's disease in an infected herd, one of the main recommendations is to avoid feeding waste milk to calves and instead feed calf milk replacer (CMR). This advice is based on the assumption that CMR is free of viable Mycobacterium avium ssp. paratuberculosis (MAP) cells, an assumption that has not previously been challenged. We tested commercial CMR products (n = 83) obtained from dairy farms around the United States by the peptide-mediated magnetic separation (PMS)-phage assay, PMS followed by liquid culture (PMS-culture), and direct IS900 quantitative PCR (qPCR). Conventional microbiological analyses for total mesophilic bacterial counts, coliforms, Salmonella, coagulase-negative staphylococci, streptococci, nonhemolytic Corynebacterium spp., and Bacillus spp. were also performed to assess the overall microbiological quality of the CMR. Twenty-six (31.3%) of the 83 CMR samples showed evidence of the presence of MAP. Seventeen (20.5%) tested positive for viable MAP by the PMS-phage assay, with plaque counts ranging from 6 to 1,212 pfu/50 mL of reconstituted CMR (average 248.5 pfu/50 mL). Twelve (14.5%) CMR samples tested positive for viable MAP by PMS-culture; isolates from all 12 of these samples were subsequently confirmed by whole-genome sequencing to be different cattle strains of MAP. Seven (8.4%) CMR samples tested positive for MAP DNA by IS900 qPCR. Four CMR samples tested positive by both PMS-based tests and 5 CMR samples tested positive by IS900 qPCR plus one or other of the PMS-based tests, but only one CMR sample tested positive by all 3 MAP detection tests applied. All conventional microbiology results were within current standards for whole milk powders. A significant association existed between higher total bacterial counts and presence of viable MAP indicated by either of the PMS-based assays. This represents the first published report of the isolation of viable MAP from CMR. Our findings raise concerns about the potential ability of MAP to survive manufacture of dried milk-based products.     

Performance optimization of polymer electrolyte membrane fuel cells using the Nelder-Mead algorithm

The direct-search simplex method for function optimization has been adapted to performance optimization of polymer electrolyte membrane fuel cells (PEMFCs). The established method is strongly application oriented and uses only experimentally determined data for optimization. It is not restricted to discrete parameters optimums and does not require the use of third-party software or computational resources. Hence, it is easy to implement in fuel cell testing stations. The optimization consists of finding, for a given fuel cell load, an optimum set of values of the 7 fuel cell operating parameters: the fuel cell temperature, the reactants' stoichiometric ratios, the reactants' inlet relative humidity, and the reactants' outlet pressures, resulting in the highest fuel cell performance. The performance is measured using a scalar function of the operating parameters and the load and can be defined according to needs.Two PEMFC performance functions: the fuel cell voltage and the system-related fuel cell efficiency were optimized using the procedure for practically sized PEMFC stacks of two designs. With respect to the nominal operating conditions defined as optimal for each stack design by its manufacturer, the gains from the optimization procedure were up to over 12% and up to over 7% for the stack voltage and efficiency, respectively. The validation of the procedure involved 5 stack specimens and four laboratories and consistent results were obtained.     

In-situ measurement of higher-order strain derivatives for advanced analysis of forming processes using spatio-temporal optical flow

In-situ full-field measurements became one of the drivers for process understanding, model creation, validation and inverse analysis. Therefore, a novel spatio-temporal optical flow method for the robust measurement of higher-order strain derivatives is proposed. This computer vision approach overcomes inherent restrictions of established DIC methods. For advanced process analysis of shear cutting processes, the deformation curvature (2nd-order displacement derivative) and the respective rate (3rd-order displacement derivative) are of high interest. For the first time, it is possible to quantify experimentally these higher-order derivatives in sufficient quality with the proposed spatio-temporal optical flow approach. In addition, interesting correlations between the microstructure of the material and macroscopic process results are determined. This demonstrates the potential of the novel in-situ measurement approach for the advanced process analysis of metal forming processes in general.     

Identification of the Thermodynamically Stable Fe‐Containing Phase in Aged Cement Pastes

Current developments in cement chemistry increasingly rely on predictive thermodynamic modeling of the phase composition in cementitious composites with the aim of linking the performance of the material with the phase composition of the material. This approach requires identification of the cement phases that form in hydrating cementitious materials using standard techniques, such as X‐ray diffraction (XRD) and thermal analysis (DTA, TGA), but also state‐of‐the‐art synchrotron‐based techniques, in particular for those cases in which the signals of solid solutions overlap in XRD and TGA measurements. In this study, two ordinary Portland cements, with different chemical compositions and subject to different hydration times (~10, ~50 yr), were investigated aiming at identifying the most stable Fe‐containing cement phase in the cement pastes. The Fe‐containing cement phases and their solid solutions with the Al analogues in the complex cement matrix were analyzed with X‐ray absorption spectroscopy, indicating the formation of a mixed Fe–Al siliceous hydrogarnet as the major Fe‐containing phase. The presence of this phase after long hydration periods and upon selective dissolution of the pastes further indicates that, independent of the chemical compositions of cements, formation of the mixed Fe–Al siliceous hydrogarnet is thermodynamically favored in aged pastes, which is supported by published thermodynamic calculations.