Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering

Background: The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production. Results: An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate. Conclusions: An industrial yeast strain for bioethanol production with lignocellulose hydrolysates has been developed in the genetic background of a strain widely used for commercial bioethanol production. The strain uses glucose and D-xylose with high consumption rates and partial cofermentation in various lignocellulose hydrolysates with very high ethanol yield. The GS1.11-26 strain shows highly promising potential for further development of an all-round robust yeast strain for efficient fermentation of various lignocellulose hydrolysates

GOGMA: Globally-Optimal Gaussian Mixture Alignment

Gaussian mixture alignment is a family of approaches that are frequently used for robustly solving the point-set registration problem. However, since they use local optimisation, they are susceptible to local minima and can only guarantee local optimality. Consequently, their accuracy is strongly dependent on the quality of the initialisation. This paper presents the first globally-optimal solution to the 3D rigid Gaussian mixture alignment problem under the L2 distance between mixtures. The algorithm, named GOGMA, employs a branch-and-bound approach to search the space of 3D rigid motions SE(3), guaranteeing global optimality regardless of the initialisation. The geometry of SE(3) was used to find novel upper and lower bounds for the objective function and local optimisation was integrated into the scheme to accelerate convergence without voiding the optimality guarantee. The evaluation empirically supported the optimality proof and showed that the method performed much more robustly on two challenging datasets than an existing globally-optimal registration solution.Comment: Manuscript in press 2016 IEEE Conference on Computer Vision and Pattern Recognitio

Tracking the best level set in a level-crossing analog-to-digital converter

Cataloged from PDF version of article.In this paper, we investigate level-crossing (LC) analog-to-digital converters (ADC)s in a competitive algorithm framework. In particular, we study how the level sets of an LC ADC should be selected in order to track the dynamical changes in the analog signal for effective sampling. We introduce a sequential LC sampling algorithm asymptotically achieving the performance of the best LC sampling method which can choose both its LC sampling levels (from a large class of possible level sets) and the intervals (from the continuum of all possible intervals) that these levels are used based on observing the whole analog signal in hindsight. The results we introduce are guaranteed to hold in an individual signal manner without any stochastic assumptions on the underlying signal. © 2012 Published by Elsevier Inc

Engineering a Vascularized 3D Hybrid System to Model Tumor-Stroma Interactions in Breast Cancer

The stromal microenvironment of breast tumors, namely the vasculature, has a key role in tumor development and metastatic spread. Tumor angiogenesis is a coordinated process, requiring the cooperation of cancer cells, stromal cells, such as fibroblasts and endothelial cells, secreted factors and the extracellular matrix (ECM). In vitro models capable of capturing such complex environment are still scarce, but are pivotal to improve success rates in drug development and screening. To address this challenge, we developed a hybrid alginate-based 3D system, combining hydrogel-embedded mammary epithelial cells (parenchymal compartment) with a porous scaffold co-seeded with fibroblasts and endothelial cells (vascularized stromal compartment). For the stromal compartment, we used porous alginate scaffolds produced by freeze-drying with particle leaching, a simple, low-cost and non-toxic approach that provided storable ready-to-use scaffolds fitting the wells of standard 96-well plates. Co-seeded endothelial cells and fibroblasts were able to adhere to the surface, spread and organize into tubular-like structures. For the parenchymal compartment, a designed alginate gel precursor solution load with mammary epithelial cells was added to the pores of pre-vascularized scaffolds, forming a hydrogel in situ by ionic crosslinking. The 3D hybrid system supports epithelial morphogenesis in organoids/tumoroids and endothelial tubulogenesis, allowing heterotypic cell-cell and cell-ECM interactions, while presenting excellent experimental tractability for whole-mount confocal microscopy, histology and mild cell recovery for down-stream analysis. It thus provides a unique 3D in vitro platform to dissect epithelial-stromal interactions and tumor angiogenesis, which may assist in the development of selective and more effective anticancer therapies.We would like to acknowledge FEDER – Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operational Programme Competitiveness and Internationalization (POCI), Portugal 2020, and Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of Project ANGIONICHE (POCI-01-0145-FEDER-028744 and PTDC/BTMMAT/28744/2017). The authors thank FCT for research contract DL 57/2016/CP1360/CT0006 (SB), junior research contract in the framework of the project Angioniche (AT), and IF research position IF/00296/2015 (CB)

Phosphate turnover during anaerobic digestion of chicken, pig and dairy manure

Phosphate (P) is used extensively in agriculture. This has led to a reliance on P imports. Meanwhile, the framework for fertilization with digestate and manure in the European Union has become more stringent in recent years. Therefore, nutrients should be recovered as fertilizer to reduce dependencies, redistribute nutrient and amplify the product portfolio of biogas plants. Current nutrient recovery processes have in common that they are post digestion treatments of digestate, which neglect the phosphate behavior during digestion. It is necessary to closely evaluate P behavior during AD to optimize post digestion treatments of digestate by using digestion as a pretreatment for digestate. Therefore, it was the overall objective of this work to evaluate the turnover of P during anaerobic digestion in laboratory scale batch and continuous digestion systems. In laboratory experiments with batch reactor systems three different manures, namely pig, dairy and chicken manure were digested. Activated sludge served as inoculum. A set of 120 mL batch digesters were filled and individual bottles were opened after defined times and discarded afterwards until the last reactors were opened on day 30. The results showed that H2O-P and NaHCO3-P decreased over the digestion period by up to 40.1 %. Meanwhile, NaOH-P increased. Overall, it could be concluded that anaerobic digestion leads to a mineralization of P. The mineralization was especially profound during the first few days after the substrate was mixed with the inoculum, concluding that the ions in the inoculum played a significant role in this mineralization. In effect, AD reduces immediate plant availability but increases slow-release fertilization effects. During the batch experiments it was found that for a defined measurement wavelength for digestate the absorbance spectrum of digestate extracts needed to be analyzed and a drying temperature needed to be determined for sample treatment. For the evaluation of these two aspects samples were dried at 50°C and at 105°C and freeze dried. These samples and undried digestate were extracted by Hedley fractionation. The coloring agent was added to the extracts and the spectra between 600 nm and 1100 nm were measured. The spectral lines showed two peaks (709 nm and 889 nm). The lower wavelength proved to be more stable at low absorbance, making this the better wavelength for analysis. The analysis of the Hedley extracts showed that drying increases the H2O-P and NaHCO3-P fraction by up to 70 %. The samples were rinsed with preceding solvent to increase accuracy. Overall, the adapted method achieved higher accuracy for H2O-P, NaHCO3-P than the former method. The adapted fractionation was used for the analysis of samples during experiments in continuously stirred tank reactors. Chicken and dairy manure were each co-digested with straw and the parameters OLR and temperature were varied. The results showed that OLR had a negative correlation with H2O-P, which decreased by up to 50.49 %. Meanwhile, HCl-P increased significantly in chicken manure digestate, showing a positive correlation with OLR. It was proven that temperature has a minor effect on P transformation with a slightly higher mineralization of P under thermophilic conditions. Especially the high calcium concentration in chicken manure dominated the P turnover during the digestion, which can also be seen in the positive correlation of OLR with HCl-P as well as a high Pearson correlation coefficient above 0.85 for calcium and phosphate in chicken manure digestion. The results of this work have proven that P changes its chemical composition significantly during anaerobic digestion. The parameters of the digestion process had a decisive effect on the final composition with OLR and substrate composition being the major drivers. The results further showed that gas production and high P solubility are in conflict because for increased H2O-P OLR needs to be reduced. Future work should focus specifically on the combination of this anaerobic digestion and post-digestion treatments for cost effective recovery. This can play a key role for future profitability of biogas projects.Phosphat (P) wird in großem Umfang in der Landwirtschaft verwendet. Das hat zu Anhängigkeiten von P importen geführt. Gleichzeitig wurde der rechtliche Rahmen zur Düngung mit Gärrest und Gülle in der Europäischen Union in den letzten Jahres strenger. Daher sollten Nährstoffe aus Gärresten zurückgewonnen werden um die Abhängigkeiten zu reduzieren, Nährstoffe neu zu verteilen und das Produktportfolie von Biogasanlagen zu erweitern. Bei derzeitigen Verfahren zur Nährstoffrückgewinnung handelt es sich um Nachbehandlungen von Gärresten, die das Verhalten von Phosphat während der Vergärung vernachlässigen. Es ist erforderlich, das P-Verhalten während der anaeroben Vergärung genau zu untersuchen, um die Nachbehandlung von Gärresten zu optimieren, indem die Vergärung als Vorbehandlung genutzt wird. Daher war die Gesamtzielsetzung dieser Arbeit die P Umwandlung während der anaeroben Vergärung in Batch- und kontinuierlichen Vergärungssystemen im Labormaßstab zu analysieren. In Laborexperimenten mit Batch-Reaktorsystemen wurden drei verschiedene tierische Exkremente, Schweine-, Rinder- und Hühnermist, vergoren. Belebtschlamm diente als Inokulum. Mehrere 120 mL Batch-Fermenter wurde gefüllt und einzelne Reaktoren wurden nach bestimmten Zeiten geöffnet und anschließend verworfen, bis die letzten Reaktoren am dreißigsten Tag geöffnet wurden. Die Ergebnisse zeigten, dass H2O-P und NaHCO3-P während der Vergärungszeit um bis zu 40,1 % abnahmen. Gleichzeitig stieg NaOH-P an. Insgesamt ließ sich feststellen, dass die anaerobe Vergärung zu einer Mineralisierung von P führte. Die Mineralisierung war in den ersten Tagen nach der Vermischung des Substrats mit dem Inokulum besonders ausgeprägt, was darauf schließen lässt, dass die Ionen im Inokulum eine wichtige Rolle bei dieser Mineralisierung spielten. Daraus resultiert, dass AD die unmittelbare Pflanzenverfügbarkeit verringert, aber die Langzeitdüngewirkung erhöht. Bei den Batch-Versuchen wurde festgestellt, dass für eine definierte Messwellenlänge für Gärreste das Absorptionsspektrum von Gärrestextrakten analysiert und eine Trocknungstemperatur für die Probenbehandlung bestimmt werden muss. Zur Beurteilung dieser beiden Aspekte wurden Proben bei 50C und 105°C getrocknet und gefriergetrocknet. Anschließend wurden diese Proben und ungetrocknete Gärreste durch Hedley-Fraktionierung extrahiert. Die Indikatorlösung wurde den Extrakten zugesetzt und die Spektren zwischen 600 nm und 1100 nm gemessen. Die Spektrallinien zeigten zwei Maxima (709 nm und 889 nm). Die niedrigere Wellenlänge war bei geringen Konzentrationen stabiler, weshalb diese die bessere Wellenlänge zur Analyse war. Die Analyse der Hedley-Extrakte zeigte, dass die Trocknung den Anteil an H2O-P und NaHCO3-P um bis zu 70 % erhöht. Hinzu kommt, dass die Proben mit dem vorherigen Lösungsmittel gespült wurden, um die Genauigkeit zu erhöhen. Insgesamt erzielte die angepasste Methode eine höhere Genauigkeit für H2O-P, NaHCO3-P als die frühere Methode. Die angepasste Fraktionierung wurde für die Analyse von Proben bei Versuchen in kontinuierlich gerührten Tankreaktoren verwendet. Hühnerkot und Rindergülle wurden jeweils mit Stroh co-fermentiert und die Parameter Faulraumbelastung und Temperatur wurden variiert. Die Ergebnisse zeigten, dass die Faulraumbelastung eine negative Korrelation mit H2O-P hatte, das um bis zu 50,49 % abnahm. Gleichzeitig stieg der HCl-P-Gehalt in Gärresten aus Hühnerkot signifikant an und zeigte eine positive Korrelation mit der Faulraumbelastung. Es wurde nachgewiesen, dass die Temperatur einen geringen Einfluss auf die P-Umwandlung hat, wobei die P-Mineralisierung unter thermophilen Bedingungen etwas höher war. Insbesondere die hohe Kalziumkonzentration in Hühnerkot dominierte den P-Umsatz während der Vergärung, was sich auch in der positiven Korrelation der Faulraumbelastung mit HCl-P, sowie einem hohen Pearson-Korrelationskoeffizienten von über 0,85 für Kalzium und Phosphat in Hühnerkot zeigte. Die Ergebnisse dieser Arbeit haben gezeigt, dass P seine chemische Zusammensetzung während der anaeroben Vergärung erheblich verändert. Die Parameter des Vergärungsprozesses haben einen entscheidenden Einfluss auf die endgültige Zusammensetzung, wobei die Faulraumbelastung und die Substratzusammensetzung die wichtigsten Faktoren waren. Die Ergebnisse zeigten außerdem, dass Gasproduktion und hohe P-Löslichkeit in Konflikt stehen, da für eine erhöhte H2O-P Konzentration die Faulraumbelastung reduziert werden muss. Zukünftige Arbeiten sollten sich speziell auf die Kombination dieser anaeroben Vergärung und Nachbehandlungen für eine kosteneffiziente Rückgewinnung konzentrieren. Dies kann eine Schlüsselrolle für die künftige Rentabilität von Biogasprojekten spielen