Estudou-se a influência da concentração inicial de substrato no cultivo em fermentador para produção de biomassa de Candida utilis IZ-1840 em meio sintético tamponado contendo glicerol (1,2,3-propanotriol) como fonte exclusiva de carbono nas seguintes concentrações: 15, 25, 35, 45, 55, 65 e 75 g/L com ensaios em duplicata. Observou-se um comportamento cinético característico de fenômeno de inibição por substrato, tendo-se então ajustada uma equação de modelo de inibição baseada na literatura para os dados de velocidade específica máxima de crescimento celular em função da concentração inicial de substrato. A partir de tal equação determinou-se o valor da concentração inicial de substrato onde não ocorre inibição, definido como S* ( S*=17,511 g/L) e o valor teórico acima do qual a inibição é máxima definido como Sm, (Sm=175,4 g/L). O valor teórico de velocidade específica máxima de crescimento celular, definido como µ* correspondente ao valor S* como concentração de substrato inicial foi calculada em 0,225 1/h. / The influence of initial substrate concentration in the growth of Candida utilis IZ-1840 in a buffered synthetic mediurn was studied utilizing glycerol (1,2,3-propanetriol) as an exc1usive carbon source, in the following concentrations: 15, 25, 35, 45, 55, 65 and 75 of in double assays. Characteristic kinetic behavior of substrate inhibition was detected, then one model equation of inhibition based on literature was adjusted for the maximum specific growth rate in function of the initial substrate concentration. From this equation it was possible to determine the value of the initial substrate concentration that do not occur growth inhibition, defined as S* (S* = 17,511 g/L).and the theoretical value of limit concentration where above this the growth inhibition becomes maximum which was defined as Sm (Sm = 175,4 g/L). The theoretical value for maximum specific growth rate, defmed as µ* corresponding to determined S* as initial substrate concentration was calculated in 0,225 1/h.

Estudou-se a influência da concentração inicial de substrato no cultivo em fermentador para produção de biomassa de Candida utilis IZ-1840 em meio sintético tamponado contendo glicerol (1,2,3-propanotriol) como fonte exclusiva de carbono nas seguintes concentrações: 15, 25, 35, 45, 55, 65 e 75 g/L com ensaios em duplicata. Observou-se um comportamento cinético característico de fenômeno de inibição por substrato, tendo-se então ajustada uma equação de modelo de inibição baseada na literatura para os dados de velocidade específica máxima de crescimento celular em função da concentração inicial de substrato. A partir de tal equação determinou-se o valor da concentração inicial de substrato onde não ocorre inibição, definido como S* ( S*=17,511 g/L) e o valor teórico acima do qual a inibição é máxima definido como Sm, (Sm=175,4 g/L). O valor teórico de velocidade específica máxima de crescimento celular, definido como µ* correspondente ao valor S* como concentração de substrato inicial foi calculada em 0,225 1/h. / The influence of initial substrate concentration in the growth of Candida utilis IZ-1840 in a buffered synthetic mediurn was studied utilizing glycerol (1,2,3-propanetriol) as an exc1usive carbon source, in the following concentrations: 15, 25, 35, 45, 55, 65 and 75 of in double assays. Characteristic kinetic behavior of substrate inhibition was detected, then one model equation of inhibition based on literature was adjusted for the maximum specific growth rate in function of the initial substrate concentration. From this equation it was possible to determine the value of the initial substrate concentration that do not occur growth inhibition, defined as S* (S* = 17,511 g/L).and the theoretical value of limit concentration where above this the growth inhibition becomes maximum which was defined as Sm (Sm = 175,4 g/L). The theoretical value for maximum specific growth rate, defmed as µ* corresponding to determined S* as initial substrate concentration was calculated in 0,225 1/h.

The increasing cost of effluent treatment in the wool scouring industry is rapidly becoming a determining factor in the viability of existing scouring operations and new installations alike. This thesis details the development of an integrated effluent treatment process capable of treating the worst polluted effluent from a wool scour "heavy flow-down", to the point where it can either be economically discharged to local trade waste sewer, or directly discharged to river or ocean outfall with minimal environmental impact. The existing proprietary chemical flocculation process, Sirolan CF™, was improved by the addition of a bio-flocculation stage and turbidity monitoring and control, and the product from this process fed to an aerobic biological treatment system based upon the traditional activated sludge process. The biological treatment process was found to remove up to 98% of the BOD5 loading from the pre-treated liquor with a hydraulic residence time of at least 50 hours being required in the aerobic digestion vessels. A residual biorefractory COD of approximately 3,600mg/L was identified which could not be removed by biological treatment. When operating continuously, the biological process was observed to metabolically neutralise the pH 3.0 - 4.5 feed from the chemical flocculation system to pH &gt 7.0 without the need for supplemental addition of neutralising agents such as sodium hydroxide. This in itself provides a significant economic incentive for implementation of the process. Kinetic analysis of the biological process carried out under controlled laboratory conditions using a Bioflo 3000 continuous fermentor showed that the bio-chemical process followed substrate inhibition kinetics. An appropriate kinetic model was identified to represent the behaviour of the substrate degradation system, and modified by inclusion of a pseudo toxic concentration to account for the effect of pH inhibition upon the biological growth rate. The process was verified both at pilot plant scale and at demonstration plant scale at an operational wool scour. The demonstration plant was of sufficient size to handle the full heavy effluent flow-down from a small wool scour. At the time of publishing three full-scale effluent treatment systems based on this research had been sold to both domestic and international clients of ADM Group Ltd. who funded the research.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18342号 / 農博第2067号 / 新制||農||1024(附属図書館) / 学位論文||H26||N4849(農学部図書室) / 31200 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 三上 文三, 教授 平竹 潤 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Serine racemase (SR) is a pyridoxal-5'-phosphate-dependent enzyme responsible for biosynthesis of D-serine, a recognized neurotransmitter acting as a co-activator of N-methyl- D-aspartate (NMDA) type of glutamate receptors in the mammalian central nervous system. The hyperfunction of the mentioned receptors have been shown to be implicated in many neuropathological conditions including Alzheimer's disease, amyotrophic lateral sclerosis and epilepsy. To alleviate the symptoms of these diseases, several artificial blockers of NMDA receptors have been introduced into the clinical practice. However, many of these compounds cause undesirable side effects and it is thus necessary to search for either less harmful blockers or regulators of other targets of pharmaceutical intervention that are involved in NMDA receptor activation. In this context, specific inhibition of serine racemase seems to be a promising strategy for regulation of NMDA receptor overstimulation. Mouse serine racemase shares 89% identity with its human ortholog and it was also shown that both enzymes possess similar kinetic parameters and inhibitor specificity. Therefore, the mouse models can be used to search for a potent human serine racemase inhibitor. Although many different compounds for their inhibitory potency towards serine...

La levure Saccharomyces cerevisiae est capable d’utiliser une grande variété de sucres comme source de carbone et d’énergie. La plupart des enzymes impliquées dans l’utilisation de ces sucres sont codées par des gènes issus de familles multigéniques. C’est le cas de la famille IMA identifiée comme impliquée dans l’utilisation de l’isomaltose. Cette famille comprend cinq gènes qui codent pour quatre isomaltases partageant une forte identité de séquence (de 65% à 100 %). Dans ce travail , la diversitéfonctionnelle de la famille IMA a été étudiée, en caractérisant de façon exhaustive in vitro leurs propriétés biochimiques et enzymologiques. Ima1p et Ima2p possèdent des propriétés biochimiques identiques (pH, température, et thermostabilité) mais Ima3p se distingue par rapport à ces deux protéines bien que n’ayant que trois acides de différence avec Ima2p (thermostabilité plus faible). Ima5p quant à elle, est la protéine la plus dissemblable (température optimale plus faible et demi-vie basse dès 37°C). Les quatre isomaltases sont cependant très sensibles au Tris et aux ions Fe3+. Les quatre isoenzymes présentent une préférence pour les disaccharides liés en α-1,6 (isomaltose et palatinose), avec une cinétique de type Michaëlis-Menten et une inhibition par le substrat à une concentration élevée. Les isomaltases Imap sont cependant aussi capables d'hydrolyser les disaccharides α-1,2, α-1,3 et α-1,5 ainsi que les trisaccharides portant une liaison α-1,6, ce qui met en évidence leur ambiguïté de substrat .Nos résultats ont toutefois montré de nombreuses singularités dans cette famille de protéines. Alors que Ima1p et Ima2p présentent des propriétés très semblables, l’activité catalytique de Ima3p est globalement très faible malgré sa forte ressemblance avec Ima2p. Le variant Ima3p_R279Q retrouve des niveaux d'activité proches de ceux d’Ima2p, tandis que la substitution d’une leucine par une proline à la position 240 a permis d’augmenter de manière significative la stabilité d’Ima3p confirmant le rôle des prolines dans la thermostabilité des protéines. L’hydrolyse de l’isomaltose par Ima5p réfute lesconclusions précédemment publiées sur l'exigence d'acides aminés spécifiques pour déterminer la spécificité de α-1,6 puisque le variant IMA5-MQH ne permet pas de restaurer une activité semblable à Ima1p malgré la présence des trois résidus MQH. Nous avons également trouvé qu’Ima5p est inhibé par le maltose suivant une inhibition mixte tandis qu’Ima1p est inhibée de façon compétitive à faible concentration et de manière incompétitive à forte concentration en isomaltose / Most enzymatic systems for sugar uptake and assimilation rely on multigene families in theyeast Saccharomyces cerevisiae. The IMA / MAL family has been used as a model system to study themolecular mechanisms that govern evolution of duplicated genes. The five IMA multigene familymembers encode four isomaltases sharing high sequence identity from 65% to 99%, of which IMA3and IMA4 are 100% identical to encode the same isomaltase. In this work, the functional diversity ofIMA family was further explored, with exhaustive in-vitro characterization of their biochemical andenzymological properties.Ima1p and Ima2p were similar to biochemical properties; Ima3p showed some differences fromthe two proteins; amongst them, Ima5p was the most distant protein. The four isomaltases were highlysensitive to Tris and Fe3+, but were unaffected by the addition or the removal of Ca2+ despiteconservation of the calcium binding site. Besides, four isoenzymes exhibited a preference for the α-(1,6)disaccharides isomaltose and palatinose, with Michaelis-Menten kinetics and inhibition at highsubstrates concentration. They were also able to hydrolyse trisaccharides bearing an α-(1,6) linkage,but also α-(1,2), α-(1,3) and α-(1,5) disaccharides including sucrose, highlighting their substrateambiguity. While Ima1p and Ima2p presented almost identical characteristics, the results neverthelessshowed many singularities within this protein family. In particular, Ima3p presented lower activitiesthan Ima2p despite only 3 different amino acids between these two isoforms. The Ima3p_R279Qvariant recovered activity levels of Ima2p, while the Leu-to-Pro substitution at position 240significantly increased the stability of Ima3p and supported the role of prolines inthermostability.Ima5p presented the lower optimal temperature and was also extremely sensitive to temperature. Isomaltose hydrolysis by Ima5p challenged previous conclusions about the requirement of specificamino acids for determining the specificity for α-(1,6) substrates. We finally found a mixed inhibitionby maltose for Ima5p while, contrary to a previous work, Ima1p inhibition by maltose was competitiveat very low isomaltose concentrations and uncompetitive as the substrate concentration increased.The presented Ph.D’s work provided preliminary insights into determining structural factorswithin this family, exemplifying for example the role of proline residues for thermosability. Moreover,it was illustrated that a gene family encoding proteins with strong sequence similarities can lead toenzyme with notable differences in biochemical and enzymological properties.

A digestão anaeróbia é uma potencial solução para a destinação dos grandes volumes de vinhaça gerada em biorrefinarias de cana-de-açúcar. A alta concentração de matéria orgânica presente na vinhaça é uma questão importante a ser considerada, uma vez que as populações microbianas anaeróbias, principalmente metanogênicas, são sensíveis a sobrecargas orgânicas. Uma série de configurações de reatores, bem como diferentes estratégias de partida e operação tem sido aplicadas ao tratamento de vinhaça, como a recirculação do efluente tratado, a qual é usualmente empregada para prevenir eventos de sobrecarga orgânica. O objetivo deste estudo foi determinar a influência da recirculação de efluentes em reatores anaeróbios compartimentados (RAC) tratando águas residuárias concentradas, uma vez que a literatura não é conclusiva sobre os efeitos da recirculação neste tipo de reator. Para atingir esse objetivo, foi utilizada uma abordagem dupla: experimental e modelagem matemática. Um RAC com 4 compartimentos em série em escala de bancada foi alimentado com vinhaça de cana-de-açúcar (DQO = 18,0 g/L) e monitorado durante 273 dias. Os parâmetros cinéticos e de transferência de massa do reator foram obtidos e utilizados para criar um modelo matemático para a conversão da matéria orgânica no software Matlab®. Tanto os dados operacionais como os parâmetros cinéticos mostraram que a elevada concentração de DQO fez com que o RAC não se comportasse como um sistema de duas fases, bem como a recirculação não resultasse em melhorias relevantes de desempenho. O modelo matemático concordou com os dados operacionais e mostrou que, em termos de capacidade de conversão, o uso de elevadas taxas de recirculação não é necessário para um adequado desempenho do RAC, uma vez que a cinética bioquímica observada não foi expressivamente limitada pela concentração de substrato. / Anaerobic digestion is a potential approach to handle the large volume of vinasse generated in sugarcane biorefineries. The high concentration of organic matter present in vinasse is an important issue to be considered, since anaerobic microbial populations, mainly methanogens, are sensitive to organic overloads. A serie of reactors configurations, as well as different startup and operating strategies, have been applied to the treatment of vinasse, so that the recirculation of the treated effluent is usually used to prevent organic overloading events. The aim of this study was to determine the influence of effluent recirculation in anaerobic baffled reactors (ABR) treating high concentrated wastewater, since literature is not conclusive about its effects. To achieve this aim an experimental and mathematical modeling dual approach was employed. A bench-scale ABR with four serial baffleds was fed with sugarcane vinasse (COD = 18,0 g/L) and monitored for 273 days. Kinetic and mass transfer parameters were obtained and used to create a mathematical model for the organic matter conversion in Matlab®. Both the operating data and the kinetic parameters showed that at high COD concentrations the ABR does not behave as a two-stage system, as well as recirculation did not result in relevant performance improvements. The mathematical model agreed with the operational data and showed that, in terms of conversion capacity, the use of high recirculation ratios is not necessary for suitable ABR performance, since the observed biochemical kinetics were not high limited by the concentration of substrate.

A digestão anaeróbia é uma potencial solução para a destinação dos grandes volumes de vinhaça gerada em biorrefinarias de cana-de-açúcar. A alta concentração de matéria orgânica presente na vinhaça é uma questão importante a ser considerada, uma vez que as populações microbianas anaeróbias, principalmente metanogênicas, são sensíveis a sobrecargas orgânicas. Uma série de configurações de reatores, bem como diferentes estratégias de partida e operação tem sido aplicadas ao tratamento de vinhaça, como a recirculação do efluente tratado, a qual é usualmente empregada para prevenir eventos de sobrecarga orgânica. O objetivo deste estudo foi determinar a influência da recirculação de efluentes em reatores anaeróbios compartimentados (RAC) tratando águas residuárias concentradas, uma vez que a literatura não é conclusiva sobre os efeitos da recirculação neste tipo de reator. Para atingir esse objetivo, foi utilizada uma abordagem dupla: experimental e modelagem matemática. Um RAC com 4 compartimentos em série em escala de bancada foi alimentado com vinhaça de cana-de-açúcar (DQO = 18,0 g/L) e monitorado durante 273 dias. Os parâmetros cinéticos e de transferência de massa do reator foram obtidos e utilizados para criar um modelo matemático para a conversão da matéria orgânica no software Matlab®. Tanto os dados operacionais como os parâmetros cinéticos mostraram que a elevada concentração de DQO fez com que o RAC não se comportasse como um sistema de duas fases, bem como a recirculação não resultasse em melhorias relevantes de desempenho. O modelo matemático concordou com os dados operacionais e mostrou que, em termos de capacidade de conversão, o uso de elevadas taxas de recirculação não é necessário para um adequado desempenho do RAC, uma vez que a cinética bioquímica observada não foi expressivamente limitada pela concentração de substrato. / Anaerobic digestion is a potential approach to handle the large volume of vinasse generated in sugarcane biorefineries. The high concentration of organic matter present in vinasse is an important issue to be considered, since anaerobic microbial populations, mainly methanogens, are sensitive to organic overloads. A serie of reactors configurations, as well as different startup and operating strategies, have been applied to the treatment of vinasse, so that the recirculation of the treated effluent is usually used to prevent organic overloading events. The aim of this study was to determine the influence of effluent recirculation in anaerobic baffled reactors (ABR) treating high concentrated wastewater, since literature is not conclusive about its effects. To achieve this aim an experimental and mathematical modeling dual approach was employed. A bench-scale ABR with four serial baffleds was fed with sugarcane vinasse (COD = 18,0 g/L) and monitored for 273 days. Kinetic and mass transfer parameters were obtained and used to create a mathematical model for the organic matter conversion in Matlab®. Both the operating data and the kinetic parameters showed that at high COD concentrations the ABR does not behave as a two-stage system, as well as recirculation did not result in relevant performance improvements. The mathematical model agreed with the operational data and showed that, in terms of conversion capacity, the use of high recirculation ratios is not necessary for suitable ABR performance, since the observed biochemical kinetics were not high limited by the concentration of substrate.