Improving biological detoxification of furfural and acetate in lignocellulosic hydrolysates using metabolic engineering

Cellulosic ethanol biofuel, made from plant waste products or perennial energy crops like switchgrass, offers many advantages over corn starch-derived ethanol, including less land competition and a lower carbon footprint. However, the efficiency of conversion is currently lower and the cost higher due to the recalcitrance of lignocellulosic biomass. A chemical and/or physical pretreatment step is required to overcome recalcitrance, and common pretreatment methods (e.g., acid, steam, and/or depressurization) release microbial inhibitors, including furfural and acetate, into the hydrolysate, lowering the yield of ethanol via fermentation. Furfural is generated via sugar dehydration, and acetate derives from acetylated xylan in hemicellulose. Utilizing a detoxifying strain is one strategy to overcome the inhibitor dilemma. Biological detoxification potentially allows lower process costs compared to chemical or enzymatic detoxification or alternative pretreatment methods aimed at reducing inhibitor generation. One drawback is time. Thus increasing the rate of furfural and acetate detoxification is desirable. While most microbial species can catabolize acetate, most do not possess the furfural catabolic pathway. A novel Pseudomonas putida isolate ALS1267, with a growth rate of 0.25/h in 10 mM minimal furfural medium, was characterized. The genome was sequenced and the furfural pathway cloned into wild-type P. putida KT2400, which cannot metabolize furfural, creating a novel strain with an improved growth rate of 0.34/h in 10 mM minimal furfural medium. Genomic library screening was used to find targets for engineering increased acetate consumption in Escherichia coli. Sixteen plasmid clones were generated, with growth rate increases of 42.6 to 76.9 percent in 10 g/l minimal acetate medium, which is a highly inhibitory concentration for this strain. Clones included an uncharacterized oxidoreductase, transporters and other outer membrane proteins, carbon scavengers, and stress defense mechanisms. Genomic mutants with improved acetate consumption were also generated during selection, with mutations in the gluconeogenesis gene pck promoter, the '5 UTR of the poorly characterized cold-shock gene ynaE, and the global regulator of secondary carbon sources CRP. The second major drawback to biological detoxification is consumption of the sugars to be used to produce ethanol by the detoxifying strain. Elimination of glucose metabolism in E. coli was studied by characterizing fast-growing revertants in strains engineered to be glucose minus. All of the revertants either altered or overproduced the N-acetylglucosamine phosphotransferase system. Deletion of the N-acetylglucosamine transporter stabilized the glucose minus phenotype and prevented the occurrence of fast-growing revertants.Ph.D

The secretome of bone marrow mesenchymal stem cells-conditioned media varies with time and drives a distinct effect on mature neurons and glial cells (primary cultures)

Transplantation of bone marrow mesenchymal stem cells (BM-MSCs) has been shown to ameliorate the injured central nervous system (CNS). Although these effects were initially attributed to the putative differentiation of MSCs towards the neural lineage, it is now known that most of them are mediated by the secretome. Up to now most in vitro reports have dealt with the effects of the secretome on neural stem cells and their differentiation. Consequently, there is a lack of information regarding the role of the secretome on the viability and survival of pre-existent matured differentiated cell populations. Moreover, it is also not known how the time points of conditioned media (CM) collection affect such parameters. In the present study, primary cultures of hippocampal neurons and glial cells were incubated with CM obtained from MSCs. To determine how the temporal profiles of CM collection impact on post-natal neurons and glial cells, we collected MSCs CM at 24, 48, 72 and 96 h of conditioning. MTS test revealed that for the hippocampal cultures the incubation with CM increased cell viability for all time points, with significant increases in the percentage of neurons in culture incubated with CM 24 h. For glial cells only the later time point of CM collection (96 h) increased cell viability. Fluorescence microscopy observations also revealed that CM 48 h and 72 h increased astrocytes percentages, while CM 24 h decreased microglial cell and oligodendrocytes values. These results revealed that post-natal neuronal and glial cells respond differently to MSCs CM; moreover, there are specific temporal variations in the composition of the CM of MSCs collected at different time points that trigger different effects on mature neurons and the distinct glial cell populations (astrocytes, oligodendrocytes and microglial cells).Fundação Calouste de Gulbenkia

Application of Theodorsen's Theory to Propeller Design

A theoretical analysis is presented for obtaining by use of Theodorsen's propeller theory the load distribution along a propeller radius to give the optimum propeller efficiency for any design condition.The efficiencies realized by designing for the optimum load distribution are given in graphs, and the optimum efficiency for any design condition may be read directly from the graph without any laborious calculations. Examples are included to illustrate the method of obtaining the optimum load distributions for both single-rotating and dual-rotating propellers

Application of Theodorsen's theory to propeller design

A theoretical analysis is presented for obtaining, by use of Theodorsen's propeller theory, the load distribution along a propeller radius to give the optimum propeller efficiency for any design condition. The efficiencies realized by designing for the optimum load distribution are given in graphs, and the optimum efficiency for any design condition may be read directly from the graph without any laborious calculations. Examples are included to illustrate the method of obtaining the optimum load distributions for both single-rotating and dual-rotating propellers

Comparison of calculated and experimental load distributions on thin wings at high subsonic and sonic speeds

A method for calculating the aerodynamic loading on a wing in combination with a body is presented. Calculated results are compared with experimentally measured data for two wing-body configurations throughout a range of Mach number up to 1.0. The magnitude and the distribution of spanwise loading of the calculated data are generally in good agreement with the experimental data