Interest in the aliphatic carbon compound 1,3-propanediol (1,3-PDO) has risen over the past 15 years. In part, this interest is due to the ability of 1,3-PDO to generate a variety of industrially relevant products such as the biodegradable polymer, polytrimethylene terephtalate. Our research group previously reported the identification of a novel Lactobacillus panis PM1 isolate capable of converting glycerol to 1,3-PDO. In this body of work, the effects of various process parameters and the ability of the novel L. panis isolate to produce 1,3-PDO in static and fed-batch cultures were examined. Data collected indicated that the concentrations of glycerol, and glucose, and pH, play a vital role in the optimized production of 1,3-PDO. Optimal conditions for the production of 1,3-PDO were determined to include: i) carbon-limited culture, defined as below 50 mM glucose and ii) growth at 37°C without agitation in the presence of glycerol (150 – 250 mM) at an elevated pH of 9 – 10. Factors such as inoculum size and temperature (OD600 in the range of 0.5 – 2 and a temperature range from 15° - 37°C) in a two-step fermentation showed insignificant variance in the production of 1,3-PDO. Initial fed-batch trials reflected the importance of pH on culture viability. A pH of 8 was determined to be necessary within culture parameters for the fed-batch production of 1,3-PDO. Further, the molar concentrations of 1,3-PDO produced were found to vary only slightly between fed batch culture and a static culture. The variance of 1,3-PDO production between the static and fed-batch trials was found to be 9.1 ± 4.9 mM for an average culture producing 85.3 ± 12.0 mM of 1,3-PDO. However, the mol concentrations of 1,3-PDO produced were found to be significantly higher with 22.3 ± 1.6 versus 5.3 ± 0.7 mmol 1,3-PDO produced for the fed batch versus the static cultures, respectively. The duration of 1,3-PDO production was found to be extended in the fed-batch model of production with increased levels of 1,3-PDO being produced over 120 hours. The cloning and characterization of the recombinant 1,3-PDO NAD+-dependent oxidoreductase also were explored to gain further insight into the native production of 1,3-PDO. Initial kinetic studies determined a Km value of 1.28 ± 0.57 mM for NAD+ versus 23.8 ± 1.1 mM for 1,3-PDO. The Km values demonstrated that the availability of NAD+/NADH may be a determining factor in 1,3-PDO concentration. These findings support the literature and the conclusion that the bottleneck in 1,3-PDO production lies in maintaining an available pool of NAD+/NADH while mitigating negative effects associated with the accumulation of toxic byproducts.

Ce travail se proposait de développer, à l’échelle du laboratoire, un procédé de production microbienne d’acide hyaluronique (AH), biopolymère d’intérêt pour la cosmétique et la santé, chez une bactérie lactique naturellement productrice, Streptococcus zooepidemicus. Une étude bibliographique a permis d’identifier les points potentiellement critiques pour ce travail qui sont la composition du milieu de culture (identification de nutriments essentiels à la synthèse d’acide hyaluronique), les conditions opératoires et plus particulièrement d’oxygénation (transfert d’oxygène mais aussi maintien du potentiel d’oxydo-réduction), en relation avec les performances de production et le métabolisme des souches considérées. Une étape préliminaire a consisté en un développement et une amélioration de techniques analytiques pour disposer d’outils pertinents de suivi du métabolisme de Streptococcus zooepidemicus. La quantification de la biomasse hors acide hyaluronique ainsi que la détection de l’activité hyaluronidasique ont ainsi été développées tandis que d’autres méthodes chromatographiques et enzymatiques ont été simplement appliquées et validées aux substrats et métabolites considérés. La mise en œuvre de souches « environnementales » prélevées sur des sites infectieux chez le cheval, ainsi que de souches de collection, a permis dans un premier temps de formuler un milieu de culture et de définir des conditions de mise en œuvre exploitables à plus grande échelle pour la production d’AH. Des résultats très encourageants ont pu être obtenus avec des productions d’AH supérieures aux essais de la littérature, tout en mettant en avant des facteurs influents cruciaux tels que la concentration initiale en glucose ou encore l’oxygénation des cultures. L’influence de ces facteurs a été étudiée par la suite par le biais de cultures en bioréacteur de laboratoire en modes discontinu et discontinu-alimenté avec pour résultat l’obtention d’un mode de mise en œuvre et de conditions physico-chimiques de production améliorées. En parallèle, une étape importante de ce travail a consisté en une approche d’amélioration de souches « environnementales » par mutagénèse aléatoire, les performances de ces souches s’avérant initialement décevantes. Des mutants surproducteurs ont ainsi été générés, caractérisés au niveau de leurs performances métaboliques et conservés. En dernier lieu, un des mutants les plus performant a été mis en œuvre dans les conditions et le mode de culture sélectionnés précédemment. Tant le niveau de production d’AH, la productivité associée, que les tailles obtenues ont permis de valider ce travail de développement d’un procédé microbien de production d’AH, tout en identifiant de nouvelles voies d’amélioration / This work intended to develop a laboratory scaleproduction process ofhyaluronic acid (HA), a biopolymer of health and cosmetic interest, using a naturally AH producing lactic acid bacterium, Streptococcus zooepidemicus. A literature review allowed to identify the following critical points: firstly, the composition of the culture medium (identification of essential nutrients for microbial growth and synthesis of HA), secondly, the oxygenation level (oxygen transfer and associated redox modifications), and finally, in relation with, the production and metabolism abilities of the considered strains. A preliminary step was dedicated to the developmentorthe improvementof analytical techniques in order todispose of appropriate tools for the monitoring of Streptococcus zooepidemicus metabolism. The quantification of the biomass without considering capsular HA fraction as well as detection of hyaluronidase activity have been developed while other chromatographic and enzymatic methods have been more basically applied to and validated with the substrates and metabolites considered. The laboratory scale cultures of collection (ATCC) as well as “environmental” strains were initially used to formulate a workable cultivation broth and to define suitable culture conditionsfor a use at a larger scale to produce HA. Very positive results were obtained with higher production of HA in comparison with literature assays, while critical influencing factors such as the initial glucose concentration or the oxygenation levelin cultures were highlighted. The influence of these factors was thoroughly studied with bioreactor cultures in both batch and fed-batch modes leading to improved cultivation conditions and culture mode. In parallel, another important step consisted in the highly performance improvement of initially low HA producing "environmental" strains via random mutagenesis. Very promising overproducing mutants have therefore been generated, characterized in their kinetic and metabolic capabilities and long-term stored. At last, one of the best and most reliable mutant has been cultivated with the best previously selected medium composition and operating conditions. Both the HA production level, productivity and size observed validated the findings of this process development work, while helping to identify new improvement domains and strategies

Made available in DSpace on 2015-03-26T13:55:30Z (GMT). No. of bitstreams: 1 texto completo.pdf: 481163 bytes, checksum: dff710e12faa7426a857bca7ddcd71a4 (MD5) Previous issue date: 2007-03-20 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Diets formulated with protein sources presenting different resistance to ruminal degradation were compared by evaluating intake, total and partial digestibility, nitrogen balance, fluxes of nutrients to omasum, production and microbial efficiency and ruminal parameters in goats. Eight rumen cannulated non-lactating, non- pregnant goats were distributed in a 4 x 4 Latin square design with two repeats. Treatments consisted of four diets where different source of plant protein account for the major protein source named soybean meal (SM), roasted soybean (RS), corn gluten meal (CGM), and cottonseed cake (CC). No difference was found as both intake and apparent digestibility of dry matter and nutrients were compared among diets. Reduction of ruminal digestibility for dry matter, crude protein and non fiber carbohydrates by using ration with RS, CGM and CC. Biological values and true digestibility for diets were similar. Amount of rumen protein were similar among rations, however fluxes of dry matter, protein and non fiber carbohydrate to omasum were higher for rations using roasted soybean, corn gluten meal and cottonseed cake. Ammonia nitrogen concentration was higher for ration with soybean meal as major protein source. Values of pH higher values were obtained for rations with RS and CC. Regarding kinetic of transit similar values were found among rations. Rations containing RS, CGM and CC as major protein source presented degradability smaller than diet using soybean meal as the major source of protein, but no difference among rations were found as availability and use by animals were compared. Diets with protein sources presenting reduced ruminal degradation do not compromised the bacterial growth, increasing flux of nutrients to omasum. Although pH and rumen ammonia are altered no compromise is observed in both production and microbial efficiency as those sources are used for dairy goats, which grant their use with similar efficiency to rations using source of more degradable protein. / Objetivou-se com este estudo, avaliar o efeito do uso de fontes protéicas apresentando diferentes graus de resistência à degradação ruminal sobre o consumo, digestibilidade total e parcial, balanço de compostos nitrogenados, fluxo de nutrientes para o omaso, produção e eficiência microbiana e características ruminais. Foram utilizadas oito cabras fistuladas no rúmen, não-gestantes e não lactantes, distribuídas em um delineamento em quadrado latino 4 x 4, duplicado. Foram utilizadas quatro dietas com diferentes fontes de proteína: farelo de soja (FS), soja grão tostada (SGT), farelo de glúten de milho (FGM) e torta de algodão (TA). As rações contendo FS, SGT, FGM e TA como fontes principais de proteína não influenciaram o consumo, a digestibilidade aparente total da matéria seca e dos nutrientes. Houve redução da digestibilidade ruminal da matéria seca, da proteína bruta e dos carboidratos não-fibrosos com o uso das rações contendo SGT, FGM e TA. Os valores biológicos e a digestibilidade total das dietas contendo diferentes fontes de proteína foram semelhantes, independente do perfil da degradação ruminal. As rações com diferentes fontes protéicas não apresentaram diferença na quantidade de proteína presente no rúmen, porém as rações contendo SGT, FGM e TA promoveram um maior fluxo de matéria seca, de proteína e de carboidratos não-fibrosos para o omaso. Maiores valores para a concentração de nitrogênio amoniacal foram obtidos com a ração contendo farelo de soja como principal fonte principal de proteína. Na avaliação de pH foram obtidos maiores valores para as rações contendo SGT e TA. Não houve diferença entre as rações com diferentes fontes de protína quanto à cinética de trânsito. As rações contendo SGT, FGM e TA como principais fontes protéicas apresentam menor degradabilidade ruminal comparativamente ao FS, no entanto as mesmas não diferem entre si, quanto sua disponibilidade e aproveitamento pelos animais. O uso destas fontes de proteína de menor degradabilidade ruminal promovem aumento no fluxo de nutrientes para o omaso e alteram parâmetros digestivos (como nitrogênio amoniacal e pH), sem comprometer a produção e eficiência microbiana, podendo ser utilizadas na alimentação de cabras leiteiras, o que garante o seu uso com eficiência similar à rações utilizando fontes protéicas com maior degradabilidade.