<p>Proteomics is the analysis of the total complement of proteins expressed by a cell or organism grown under a specified condition. The obtained protein profile would provide a better understanding of phenotypic characteristics of a cell grown under pre-determined conditions. Mass spectrometric-based protein analysis is currently the standard method in proteomic studies; however, there are many limitations associated with its application. The major objectives of this study included the development of a strategy to analyze the confidence of identified proteins and the development of an algorithm to interpret the experimentally obtained mass spectral data. </p> <p>A two-step strategy was developed to analyze the confidence of identified proteins. In the first step, the proteins identified by a single protein identification tool were classified into two groups: high confidence proteins that were identified by unique peptides, and low confidence proteins that were identified by non-unique peptides. In the second step, the proteins identified by different tools (e.g., SEQUEST and Mascot in our work) were cross-compared. After integrating the two-step analysis, the identified proteins were classified into four levels of confidence. The proteins that were identified by the presence of unique peptides and that were commonly identified by different tools were grouped into the highest confidence level - Level 4. Even though the number of proteins in Level 4 was reduced significantly, the conclusions drawn from the proteins were more reliable.</p> <p>According to the operation of tandem mass spectrometry and the characteristics of the peptides generated by site-specific protease digestion, a two-pass approach for identifying the species-specific proteins was developed. The approach can find all possible peptides corresponding to a precursor ion and gives detailed matching information of each peptide candidate to the experimental product ion series. According to the total number of matched product ions, the total number of matched b- and y- ions, and the contiguity characteristic of identified product ions, the peptide candidates were ranked decreasingly from the most probable to the least. Combined with the concept of unique peptide, the obtained most probable peptide can then be used to predict proteins existing in the original sample.</p> <p>The developed two-pass approach and two-step strategy were then used to study the protein profiling of <i>Saccharomyces cerevisiae</i> cultivated in various gravity conditions (10 and 300 g glucose/l) in order to investigate the changes in central metabolic pathways of <i>S. cerevisiae</i>. Our fermentation data indicated that the higher glucose contents would result in lower cell growth and higher ethanol production (e.g., high ethanol concentration in fermentation broth). However, the relative ethanol yield as related to the glucose consumption was lower under higher glucose concentrations. The protein profile showed that a higher flux of nutrient was channelled into the pentose phosphate pathway when <i>S. cerevisiae</i> was grown under a high glucose concentration. The reason for this phenomenon might be that the cell needs more reducing power (e.g., NADPH) for the synthesis of macromolecules such as proteins, nucleic acids, and lipids. These materials are essential to the cell in order to modify its structure (e.g., cell wall), to survive osmotic stress and to replicate.</p>

The objective of this study is to evaluate and compare, both technically and economically, various glucose feeding concentrations and different redox potential settings on ethanol production under very-high-gravity (VHG) conditions. Laboratory data were collected for process modeling and two process models were created by two individual process simulators. The first one is a simplified model created and evaluated by Superpro Designer. The second one is an accurate model created by Aspen Plus and evaluated by Aspen Icarus Process Evaluator (Aspen IPE). The simulation results of the two models were also compared. Results showed that glucose feeding concentration at 250±3.95 g/L to the fermentor resulted in the lowest unit production cost (1.479 $/kg ethanol in the Superpro model, 0.764 $/kg ethanol in the Aspen Plus model), with redox potential control effects accounted. Controlling redox potential at -150 mV increased the ethanol yield under VHG fermentation conditions while no significant influences were observed when glucose feeding concentration was less than 250 g/L. Results of product sales analysis indicated that for an ethanol plant with a production rate of 85~130 million kg ethanol/year, only maintaining the glucose feeding concentration to the fermentor at around 250 g/L resulted in the shortest payout period of 5.33 years in average,, with or without redox potential control. If 300±6.42 g/L glucose feeding concentration to the fermentor is applied, it is essential to have the redox potential only controlled at -150 mV in the fermentor to limit the process payout period within 6 years. In addition, fermentation processes with glucose feeding concentration at around 200 g/L to the fermentor were estimated to be unprofitable under all studied conditions. For environmental concerns, two disposal alternatives were presented for CO2 produced during fermentation process rather than emission into atmosphere. One is to sell CO2 as byproduct, which brought 1.52 million $/year income for an ethanol plant with a capacity of 100 million kg ethanol/year. Another option is to capture and transport CO2 to deep injection sites for geological underground storage, which is already a safe and mature technology in North America, and also applicable to many other sites around the world. This would roughly add 4.78 million dollars processing cost annually in the studied scenario. Deep injection of captured CO2 from ethanol plants prevents emission of CO2 into the atmosphere, thus makes it environmental friendly.

The objective of this study is to evaluate and compare, both technically and economically, various glucose feeding concentrations and different redox potential settings on ethanol production under very-high-gravity (VHG) conditions. Laboratory data were collected for process modeling and two process models were created by two individual process simulators. The first one is a simplified model created and evaluated by Superpro Designer. The second one is an accurate model created by Aspen Plus and evaluated by Aspen Icarus Process Evaluator (Aspen IPE). The simulation results of the two models were also compared. Results showed that glucose feeding concentration at 250±3.95 g/L to the fermentor resulted in the lowest unit production cost (1.479 $/kg ethanol in the Superpro model, 0.764 $/kg ethanol in the Aspen Plus model), with redox potential control effects accounted. Controlling redox potential at -150 mV increased the ethanol yield under VHG fermentation conditions while no significant influences were observed when glucose feeding concentration was less than 250 g/L. Results of product sales analysis indicated that for an ethanol plant with a production rate of 85~130 million kg ethanol/year, only maintaining the glucose feeding concentration to the fermentor at around 250 g/L resulted in the shortest payout period of 5.33 years in average,, with or without redox potential control. If 300±6.42 g/L glucose feeding concentration to the fermentor is applied, it is essential to have the redox potential only controlled at -150 mV in the fermentor to limit the process payout period within 6 years. In addition, fermentation processes with glucose feeding concentration at around 200 g/L to the fermentor were estimated to be unprofitable under all studied conditions. For environmental concerns, two disposal alternatives were presented for CO2 produced during fermentation process rather than emission into atmosphere. One is to sell CO2 as byproduct, which brought 1.52 million $/year income for an ethanol plant with a capacity of 100 million kg ethanol/year. Another option is to capture and transport CO2 to deep injection sites for geological underground storage, which is already a safe and mature technology in North America, and also applicable to many other sites around the world. This would roughly add 4.78 million dollars processing cost annually in the studied scenario. Deep injection of captured CO2 from ethanol plants prevents emission of CO2 into the atmosphere, thus makes it environmental friendly.

<p>Proteomics is the analysis of the total complement of proteins expressed by a cell or organism grown under a specified condition. The obtained protein profile would provide a better understanding of phenotypic characteristics of a cell grown under pre-determined conditions. Mass spectrometric-based protein analysis is currently the standard method in proteomic studies; however, there are many limitations associated with its application. The major objectives of this study included the development of a strategy to analyze the confidence of identified proteins and the development of an algorithm to interpret the experimentally obtained mass spectral data. </p> <p>A two-step strategy was developed to analyze the confidence of identified proteins. In the first step, the proteins identified by a single protein identification tool were classified into two groups: high confidence proteins that were identified by unique peptides, and low confidence proteins that were identified by non-unique peptides. In the second step, the proteins identified by different tools (e.g., SEQUEST and Mascot in our work) were cross-compared. After integrating the two-step analysis, the identified proteins were classified into four levels of confidence. The proteins that were identified by the presence of unique peptides and that were commonly identified by different tools were grouped into the highest confidence level - Level 4. Even though the number of proteins in Level 4 was reduced significantly, the conclusions drawn from the proteins were more reliable.</p> <p>According to the operation of tandem mass spectrometry and the characteristics of the peptides generated by site-specific protease digestion, a two-pass approach for identifying the species-specific proteins was developed. The approach can find all possible peptides corresponding to a precursor ion and gives detailed matching information of each peptide candidate to the experimental product ion series. According to the total number of matched product ions, the total number of matched b- and y- ions, and the contiguity characteristic of identified product ions, the peptide candidates were ranked decreasingly from the most probable to the least. Combined with the concept of unique peptide, the obtained most probable peptide can then be used to predict proteins existing in the original sample.</p> <p>The developed two-pass approach and two-step strategy were then used to study the protein profiling of <i>Saccharomyces cerevisiae</i> cultivated in various gravity conditions (10 and 300 g glucose/l) in order to investigate the changes in central metabolic pathways of <i>S. cerevisiae</i>. Our fermentation data indicated that the higher glucose contents would result in lower cell growth and higher ethanol production (e.g., high ethanol concentration in fermentation broth). However, the relative ethanol yield as related to the glucose consumption was lower under higher glucose concentrations. The protein profile showed that a higher flux of nutrient was channelled into the pentose phosphate pathway when <i>S. cerevisiae</i> was grown under a high glucose concentration. The reason for this phenomenon might be that the cell needs more reducing power (e.g., NADPH) for the synthesis of macromolecules such as proteins, nucleic acids, and lipids. These materials are essential to the cell in order to modify its structure (e.g., cell wall), to survive osmotic stress and to replicate.</p>

L'éthanol est un composé à usages très variés allant de la chimie à l'agroalimentaire. Cependant, la croissance actuelle du marché se fait essentiellement autour de l'utilisation de l'éthanol en tant que carburant. L'objectif de ce projet est d'intensifier la production d'éthanol à partir du sirop basse pureté, produit de la seconde cristallisation des jus d'extraction de betterave, afin de diminuer les consommations en énergie et en eau pour la production d'éthanol. Pour ce faire, en partenariat avec l'UNGDA et l'ADEME, nous avons mené des travaux de recherche sur les fermentations à haute densité afin d'obtenir des vins à teneur plus élevée en éthanol. A l'issu d'un état de l'art et de quatre visites dans des ateliers de production, une stratégie de recherche en trois points a été établie. Le premier point a consisté en la recherche d'une composition de milieu de fermentation permettant d'augmenter la concentration finale en éthanol. Le second point a eu pour but de déterminer si les besoins en nutriments se limitaient uniquement à la phase de croissance ou au contraire si l'apport de ces nutriments était bénéfique tout au long de la fermentation. Le dernier point a quant à lui utilisé l'ensemble des résultats obtenus pour définir une conduite de procédé, permettant d'obtenir la concentration finale en éthanol la plus élevée possible. Ces résultats montrent qu'il est possible de réaliser des fermentations haute densité à partir de sirop basse pureté et d'obtenir 15,2 % (v/v) d'éthanol en fin de fermentation. L'application de ces travaux dans les ateliers de production permettrait d'économiser par litre d'éthanol pur, entre 20 et 30 % d'énergie pour la distillation, entre 35 et 49 % d'eau pour la réalisation des milieux de fermentation à partir de SBP et de diminuer de 23 à 38 % le volume de déchet produit après distillation. / Ethanol is a compound with a wide usage range from chemistry to food. However, the current market growth mainly concerns the use of ethanol as fuel. The objective of this project was to intensify ethanol production from low purity syrup 2, which is a substrate from sugar beet, in order to reduce the consumptions of energy and water for its production. To do this, in partnership with UNGDA and ADEME, we have conducted research on high-gravity fermentations in order to increase the ethanol concentration at the end of the fermentation. With the coming of a state of the art and four visits in production facilities, a three points research strategy has been established. The first point consisted of fermentation medium composition finding in order to increase the final ethanol concentration at the end of the fermentation. The second point was to determine if the nutrients requirements were limited only during the growth phase or, on the contrary, if nutriments were beneficial throughout the fermentation. The latter point was to use the overall results to define a fermentation process, to obtain a final ethanol concentration as high as possible. These results show that it is possible to achieve high gravity fermentation from low purity syrup and reach a final ethanol concentration of 15.2 % (v/v). The application of this work in production facilities could save per liter of pure ethanol between 20 and 30% energy for distillation, between 35 and 49 % water for the production of fermentation media from SBP and decrease from 23 to 38 % of the volume of waste produced after distillation.

Department of Food Science and Technology / MSCPNT