Optimization of ethanol production by yeasts from lignocellulosic feedstocks

Ethanol from lignocellulosic feedstocks is not currently competitive with corn-based ethanol in terms of yields and commercial feasibility. Through optimization of the pretreatment and fermentation steps this could change. The overall goal of this study was to evaluate, characterize, and optimize ethanol production from lignocellulosic feedstocks by the yeasts Saccharomyces cerevisiae (strain Ethanol Red, ER) and Pichia stipitis CBS 6054. Through a series of fermentations and growth studies, P. stipitis CBS 6054 and S. cerevisiae (ER) were evaluated on their ability to produce ethanol from both single substrate (xylose and glucose) and mixed substrate (five sugars present in hemicellulose) fermentations. The yeasts were also evaluated on their ability to produce ethanol from dilute acid pretreated hydrolysate and enzymatic hydrolysate. Hardwood (aspen), softwood (balsam), and herbaceous (switchgrass) hydrolysates were also tested to determine the effect of the source of the feedstock. P. stipitis produced ethanol from 66-98% of the theoretical yield throughout the fermentation studies completed over the course of this work. S. cerevisiae (ER) was determined to not be ideal for dilute acid pretreated lignocellulose because it was not able to utilize all the sugars found in hemicellulose. S. cerevisiae (ER) was instead used to optimize enzymatic pretreated lignocellulose that contained only glucose monomers. It was able to produce ethanol from enzymatically pretreated hydrolysate but the sugar level was so low (>3 g/L) that it would not be commercially feasible. Two lignocellulosic degradation products, furfural and acetic acid, were evaluated for whether or not they had an inhibitory effect on biomass production, substrate utilization, and ethanol production by P. stipitis and S. cerevisiae (ER). It was determined that inhibition is directly related to the concentration of the inhibitor and the organism. The final phase for this thesis focused on adapting P. stipitis CBS 6054 to toxic compounds present in dilute acid pretreated hydrolysate through directed evolution. Cultures were transferred to increasing concentrations of dilute acid pretreated hydrolysate in the fermentation media. The adapted strains’ fermentation capabilities were tested against the unadapted parent strain at each hydrolysate concentration. The fermentation capabilities of the adapted strain were significantly improved over the unadapted parentstrain. On media containing 60% hydrolysate the adapted strain yielded 0.30 g_ethanol/g_sugar ± 0.033 (g/g) and the unadapted parent strain yielded 0.11 g/g ±0.028. The culture has been successfully adapted to growth on media containing 65%, 70%, 75%, and 80% hydrolysate but with below optimal ethanol yields (0.14-0.19 g/g). Cell recycle could be a viable option for improving ethanol yields in these cases. A study was conducted to determine the optimal media for production of ethanol from xylose and mixed substrate fermentations by P. stipitis. Growth, substrate utilization, and ethanol production were the three factors used to evaluate the media. The three media tested were Yeast Peptone (YP), Yeast Nitrogen Base (YNB), and Corn Steep Liquor (CSL). The ethanol yields (g/g) for each medium are as follows: YP - 0.40-0.42, YNB -0.28-.030, and CSL - 0.44-.051. The results show that media containing CSL result in slightly higher ethanol yields then other fermentation media. P. stipitis was successfully adapted to dilute acid pretreated aspen hydrolysate in increasing concentrations in order to produce higher ethanol yields compared to the unadapted parent strain. S. cerevisiae (ER) produced ethanol from enzymatic pretreated cellulose containing low concentrations of glucose (1-3g/L). These results show that fermentations of lignocellulosic feedstocks can be optimized based on the substrate and organism for increased ethanol yields.

Anti-HLA I antibodies induce VEGF production by endothelial cells, which increases proliferation and paracellular permeability

Anti-human leukocyte antigen class I (HLA I) antibodies were shown to activate several protein kinases in endothelial cells (ECs), which induces proliferation and cell survival. An important phenomenon in antibody-mediated rejection is the occurrence of interstitial edema. We investigated the effect of anti-HLA I antibodies on endothelial proliferation and permeability, as one possible underlying mechanism of edema formation. HLA I antibodies increased the permeability of cultured ECs isolated from umbilical veins. Anti-HLA I antibodies induced the production of vascular endothelial growth factor (VEGF) by ECs, which activated VEGF receptor 2 (VEGFR2) in an autocrine manner. Activated VEGFR2 led to a c-Src-dependent phosphorylation of vascular endothelial (VE)-cadherin and its degradation. Aberrant VE-cadherin expression resulted in impaired adherens junctions, which might lead to increased endothelial permeability. This effect was only observed after cross-linking of HLA I molecules by intact antibodies. Furthermore, our results suggest that increased endothelial proliferation following anti-HLA I treatment occurs via autocrine VEGFR2 activation. Our data indicate the ability of anti-HLA I to induce VEGF production in ECs. Transactivation of VEGFR2 leads to increased EC proliferation and paracellular permeability. The autocrine effect of VEGF on endothelial permeability might be an explanation for the formation of interstitial edema after transplantation.

Protein phosphatase 2A and phosphoprotein SET regulate androgen production by P450c17

Cytochrome P450c17 catalyzes 17 alpha-hydroxylation needed for cortisol synthesis and 17,20 lyase activity needed to produce sex steroids. Serine phosphorylation of P450c17 specifically increases 17,20 lyase activity, but the physiological factors regulating this effect remain unknown. Treating human adrenal NCI-H295A cells with the phosphatase inhibitors okadaic acid, fostriecin, and cantharidin increased 17,20 lyase activity, suggesting involvement of protein phosphatase 2A (PP2A) or 4 (PP4). PP2A but not PP4 inhibited 17,20 lyase activity in microsomes from cultured cells, but neither affected 17 alpha-hydroxylation. Inhibition of 17,20 lyase activity by PP2A was concentration-dependent, could be inhibited by okadaic acid, and was restored by endogenous protein kinases. PP2A but not PP4 coimmunoprecipitated with P450c17, and suppression of PP2A by small interfering RNA increased 17,20 lyase activity. Phosphoprotein SET found in adrenals inhibited PP2A, but not PP4, and fostered 17,20 lyase activity. The identification of PP2A and SET as post-translational regulators of androgen biosynthesis suggests potential additional mechanisms contributing to adrenarche and hyperandrogenic disorders such as polycystic ovary syndrome.

Assessment of ultra-high resolution optical coherence tomography for monitoring tissue effects caused by laser photocoagulation of ex-vivo porcine retina

Retinal laser photocoagulation is an established and successful treatment for a variety of retinal diseases. While being a valuable treatment modality, laser photocoagulation shows the drawback of employing high energy lasers which are capable of physically destroying the neural retina. For reliable therapy, it is therefore crucial to closely monitor the therapy effects caused in the retinal tissue. A depth resolved representation of optical tissue properties as provided by optical coherence tomography may provide valuable information about the treatment effects in the retinal layers if recorded simultaneously to laser coagulation. Therefore, in this work, the use of ultra-high resolution optical coherence tomography to represent tissue changes caused by conventional and selective retinal photocoagulation is investigated. Laser lesions were placed on porcine retina ex-vivo using a 577 nm laser as well as a pulsed laser at 527 nm built for selective treatment of the retinal pigment epithelium. Applied energies were varied to generate lesions best representing the span from under- to overtreatment. The lesions were examined using a custom-designed optical coherence tomography system with an axial resolution of 1.78 μm and 70 kHz Ascan rate. Optical coherence tomography scans included volume scans before and after irradiation, as well as time lapse scans (Mscan) of the lesions. Results show OCT lesion visibility thresholds to be below the thresholds of ophthalmoscopic inspection. With the ultra-high resolution OCT, 42% - 44% of ophthalmoscopically invisible lesions could be detected and lesions that were under- or overexposed could be distinguished using the OCT data.

Mechanisms of recognition and binding of α-TTP to the plasma membrane by multi-scale molecular dynamics simulations

We used multiple sets of simulations both at the atomistic and coarse-grained level of resolution to investigate interaction and binding of α-tochoperol transfer protein (α-TTP) to phosphatidylinositol phosphate lipids (PIPs). Our calculations indicate that enrichment of membranes with such lipids facilitate membrane anchoring. Atomistic models suggest that PIP can be incorporated into the binding cavity of α-TTP and therefore confirm that such protein can work as lipid exchanger between the endosome and the plasma membrane. Comparison of the atomistic models of the α-TTP-PIPs complex with membrane-bound α-TTP revealed different roles for the various basic residues composing the basic patch that is key for the protein/ligand interaction. Such residues are of critical importance as several point mutations at their position lead to severe forms of ataxia with vitamin E deficiency (AVED) phenotypes. Specifically, R221 is main residue responsible for the stabilization of the complex. R68 and R192 exchange strong interactions in the protein or in the membrane complex only, suggesting that the two residues alternate contact formation, thus facilitating lipid flipping from the membrane into the protein cavity during the lipid exchange process. Finally, R59 shows weaker interactions with PIPs anyway with a clear preference for specific phosphorylation positions, hinting a role in early membrane selectivity for the protein. Altogether, our simulations reveal significant aspects at the atomistic scale of interactions of α-TTP with the plasma membrane and with PIP, providing clarifications on the mechanism of intracellular vitamin E trafficking and helping establishing the role of key residue for the functionality of α-TTP.

Post-operative monitoring of free muscle transfers by Laser Doppler Imaging: A prospective study.

PURPOSE Despite different existing methods, monitoring of free muscle transfer is still challenging. In the current study we evaluated our clinical setting regarding monitoring of such tissues, using a recent microcirculation-imaging camera (EasyLDI) as an additional tool for detection of perfusion incompetency. PATIENTS AND METHODS This study was performed on seven patients with soft tissue defect, who underwent reconstruction with free gracilis muscle. Beside standard monitoring protocol (clinical assessment, temperature strips, and surface Doppler), hourly EasyLDI monitoring was performed for 48 hours. Thereby a baseline value (raised flap but connected to its vascular bundle) and an ischaemia perfusion value (completely resected flap) were measured at the same point. RESULTS The mean age of the patients, mean baseline value, ischaemia value perfusion were 48.00 ± 13.42 years, 49.31 ± 17.33 arbitrary perfusion units (APU), 9.87 ± 4.22 APU, respectively. The LDI measured values in six free muscle transfers were compatible with hourly standard monitoring protocol, and normalized LDI values significantly increased during time (P < 0.001, r = 0.412). One of the flaps required a return to theatre 17 hours after the operation, where an unsalvageable flap loss was detected. All normalized LDI values of this flap were under the ischaemia perfusion level and the trend was significantly descending during time (P < 0.001, r = -0.870). CONCLUSION Due to the capability of early detection of perfusion incompetency, LDI may be recommended as an additional post-operative monitoring device for free muscle flaps, for early detection of suspected failing flaps and for validation of other methods.

The determinants of offshore production by Multinational Corporations (MNCs) : a comparison of Japanese and US MNCs

This study employs confidential affiliate-level panel data to improve measures of foreign affiliate activities of Japanese firms in manufacturing sectors. Combining existing data on U.S. MNCs with the Japanese data, we illustrate the pattern and determinant of their foreign affiliate sales by destination market across countries and industries for the period 1989-2005. Among our results, Japanese and U.S. MNCs are similar in the substantial growth of their foreign affiliate sales and the importance of sales to local markets. However, Japanese MNCs are distinctive from U.S. MNCs in that Japanese affiliate sales in Asia were prominently higher in host markets with lower educational attainment.

Characterization Of Friction Stir Welded Joints Of Aa 2024-T351 - Native Vs. Treated By Laser Shock Processing

The paper presents a consistent set of results showing the ability of Laser Shock Processing (LSP) in modifying the overall properties of the Friction Stir Welded (FSW) joints made of AA 2024-T351. Based on laser beam intensities above 109 W/cm2 with pulse energies of several Joules and pulses durations of nanoseconds, LSP is able of inducing a compression residual stress field, improving the wear and fatigue resistance by slowing crack propagation and stress corrosion cracking, but also improving the overall behaviour of the structure. After the FSW and LSP procedures are briefly presented, the results of micro-hardness measurements and of transverse tensile tests, together with the corrosion resistance of the native joints vs. LSP treated are discussed. The ability of LSP to generate compressive residual stresses and to improve the behaviour of the FSW joints is underscored.

Mechanical properties enhancement of high reliability metallic materials by laser shock processing

Laser shock processing (LSP) is being increasingly applied as an effective technology for the improvement of metallic materials surface properties in different types of components as a means of enhancement of their corrosion and fatigue life behavior. As reported in previous contributions by the authors, a main effect resulting from the application of the LSP technique consists on the generation of relatively deep compression residual stresses field into metallic alloy pieces allowing an improved mechanical behaviour, explicitly the life improvement of the treated specimens against wear, crack growth and stress corrosion cracking. Additional results accomplished by the authors in the line of practical development of the LSP technique at an experimental level (aiming its integral assessment from an interrelated theoretical and experimental point of view) are presented in this paper. Concretely, follow-on experimental results on the residual stress profiles and associated surface properties modification successfully reached in typical materials (especially Al and Ti alloys) under different LSP irradiation conditions are presented along with a practical correlated analysis on the protective character of the residual stress profiles obtained under different irradiation strategies and the evaluation of the corresponding induced properties as material specific volume reduction at the surface, microhardness and wear resistance. Additional remarks on the improved character of the LSP technique over the traditional “shot peening” technique in what concerns depth of induced compressive residual stresses fields are also made through the paper.

Effect of Thermal Treatments on the Mechanical Properties Enhancement of High Reliability Metallic Materials by Laser Shock Processing

Laser shock processing (LSP) is increasingly applied as an effective technology for the improvement of metallic materials mechanical properties in different types of components as a means of enhancement of their fatigue life behavior. As reported in previous contributions by the authors, a main effect resulting from the application of the LSP technique consists on the generation of relatively deep compression residual stresses fields into metallic components allowing an improved mechanical behaviour, explicitly the life improvement of the treated specimens against wear, crack growth and stress corrosion cracking. Additional results accomplished by the authors in the line of practical development of the LSP technique at an experimental level (aiming its integral assessment from an interrelated theoretical and experimental point of view) are presented in this paper. Concretely, experimental results on the residual stress profiles and associated mechanical properties modification successfully reached in typical materials under different LSP irradiation conditions are presented. In this case, the specific behavior of a widely used material in high reliability components (especially in nuclear and biomedical applications) as AISI 316L is analyzed, the effect of possible “in-service” thermal conditions on the relaxation of the LSP effects being specifically characterized. I.

Induction of engineered residual stresses fields and enhancement of fatigue life of high reliability metallic components by laser shock processing

Laser shock processing (LSP) is being increasingly applied as an effective technology for the improvement of metallic materials mechanical and surface properties in different types of components as a means of enhancement of their corrosion and fatigue life behavior. As reported in previous contributions by the authors, a main effect resulting from the application of the LSP technique consists on the generation of relatively deep compression residual stresses field into metallic alloy pieces allowing an improved mechanical behaviour, explicitly the life improvement of the treated specimens against wear, crack growth and stress corrosion cracking. Additional results accomplished by the authors in the line of practical development of the LSP technique at an experimental level (aiming its integral assessment from an interrelated theoretical and experimental point of view) are presented in this paper. Concretely, follow-on experimental results on the residual stress profiles and associated surface properties modification successfully reached in typical materials (especially Al and Ti alloys characteristic of high reliability components in the aerospace, nuclear and biomedical sectors) under different LSP irradiation conditions are presented along with a practical correlated analysis on the protective character of the residual stress profiles obtained under different irradiation strategies. Additional remarks on the improved character of the LSP technique over the traditional “shot peening” technique in what concerns depth of induced compressive residual stresses fields are also made through the paper

Residual stress distribution in ferritic-austenitic steel joints made by laser welding

The present investigation addresse the influence of laser welding process-ing parameters used for joining dis-similar metals (ferritic to austenitic steel), on the induced residual stress field. Welding was performed on a Nd:YAG laser DY033 (3300 W) in a continuous wave (CW), keyhole mode. The base metals (BM) employed in this study are AISI 1010 carbon steel (CS) and AISI 304L austenitic stainless steel (SS). Pairs of dissimilar plates of 200 mm x 45 mm x 3 mm were butt joined by laser welding. Different sets of parameters were used to engineer the base metals apportionment at joint formation, namely distinct dilution rates. Residual strain scanning, carried out by neutron diffraction was used to assess the joints. Through-thickness residual stress maps were determined for the laser welded samples of dis-similar steels using high spatial reso-lution. As a result, an appropriate set of processing parameters, able to mi-nimize the local tensile residual stress associated to the welding process, was found.