Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha

Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.United States. Dept. of EnergyUnited States. Advanced Research Projects Agency-Energ

Back-analysis of a failed rock wedge using a 3D numerical

In the present paper, a rock wedge failure that occurred in the Rosandra valley (Trieste, NE Italy) has been back-analysed to compare the results of a traditional rock wedge analysis with those obtained using a 3D finite difference model (FDM). The simulations performed considering a purely frictional model of the rock wedge show that the values of the factor of safety (FOS) are in good agreement with those of the strength reduction factor (SRF). For the failure condition, the calculated values of FOS and SRF are very similar and both <1 (FOS = 0.92 and SRF = 0.93), suggesting that another type of localised strength has to be considered to guarantee block stability, such as that provided by a rock bridge. A second numerical model considers this localised rock bridge, whose presence, location and size have been determined through a detailed field investigation of the detachment surface. The parametric approach indicates that mean values of the shear stress acting on the rock bridge range from 1 to 2 MPa. The shear strength contribution given by the rock bridge is about 1015% of the whole resisting system. Calculated block displacements before the collapse vary from 0.1 -0.5 mm to 2-3 cm, depending on different characteristic stiffness assumed for the basal rock joints and the rock bridge. Finally, the rock bridge cohesion at failure ranges from 0.8 to 1.2 Mpa, depending on different friction angles assumed for the rock bridge.