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The methylotrophic yeast Pichia pastoris has become an efficient expression system for heterologous protein production. Different methods have been studied to enhance cell growth as well as the production of products of interest. Two of the major strategies for improving the product or biomass yields are optimizing bioprocess controls and cultivation conditions. In this work, the characteristics of this yeast system and of its different promoters are discussed, and the effect of operational strategies on cell growth and recombinant protein expression is also studied. The effect of different feeding strategies were studied and optimized for pGAP (glyceraldehyde-3-phosphate dehydrogenase)-regulated phytase production in P. pastoris. Alternative carbon sources were screened and the feasibility of using citric acid as a carbon source for recombinant protein production was also investigated. The effects of parameters such as the carbon source concentration and culture pH were studied using shake-flasks, and the effect of different feeding profiles on bioreactor performance was also investigated. Three feeding strategies, Stepwise feeding, Exponential feeding and DO-stat feeding were tested and DO-stat was found to be more efficient and led to a high phytase activity. A modified DO-stat method was investigated to overcome the oxygen limited condition in the standard DO-stat method. For the carbon source, citric acid showed promise in improving phytase expression. Further experiments in bioreactors performed with the presence of certain amount of citric acid showed that less glycerol could be used to achieve the same level of phytase activity.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18842号 / 理博第4100号 / 新制||理||1589(附属図書館) / 31793 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 幸島 司郎, 教授 伊谷 原一, 教授 村山 美穂 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Este trabalho apresenta a avaliação da estabilidade e da eficiência de dois reatores anaeróbios operados em batelada seqüencial no tratamento de água residuária que simula o efluente de indústria de produtos de higiene pessoal, sendo um com biomassa imobilizada em espuma de poliuretano (AnSBBR) e recirculação da fase líquida, e outro com biomassa granular autoimobilizada (AnSBR) e agitação mecânica. O AnSBBR foi operado em batelada e em batelada alimentada, seguida de batelada, com tempo de ciclo (tc) de 8 h e carga orgânica volumétrica aplicada (COVA) de 0,9 e 1,5 gDQO/L.d, tratando 2,5 L de meio líquido por ciclo. Os valores de eficiência de remoção de matéria orgânica de amostras filtradas do efluente ficaram abaixo de 71% nas condições operadas em batelada, e acima de 74% nas condições operadas em batelada alimentada, seguida de batelada. Por outro lado, o AnSBR foi operado em batelada, com tc de 8, 12 e 24 h e COVA de 0,75, 0,50 e 0,25 gDQO/L.d, tratando 2,0 L de meio líquido por ciclo. O aumento de tc resultou em ganho de eficiência nas condições de mesma COVA, chegando a 77% para tc de 24 h, contra 69% para tc de 8 h. Porém, houve queda de eficiência nas condições em que a COVA foi reduzida em função do aumento de tc. / This study presents the evaluation of stability and efficiency of two anaerobic sequencing batch reactors for wastewater treatment simulating the effluent from industry of personal care products, one containing immobilized biomass on polyurethane foam (AnSBBR) and external circulation of the liquid phase, and the other with self-immobilized granulated biomass (AnSBR) and mechanical agitation. The AnSBBR was operated in batch and fed batch/batch modes, with cycle time (tc) of 8 h and applied organic loading rates (COVA) of 0.9 and 1.5 gDQO/L.d, treating 2.5 L of liquid medium per cycle. The values of organic matter removal efficiency for filtered samples of effluent were below 71% in batch operations, and over 74% in fed-batch/batch operations. On the other hand, the AnSBR was operated in batch, with tc of 8, 12 and 24 h and COVA of 0.75, 0.50 and 0.25 gDQO/L.d, treating 2.0 L of liquid medium per cycle. The increase of tc resulted in higher values of efficiency for conditions with the same COVA, reaching 77% for a 24-h cycle against 69% for an 8-h cycle. However, there was an efficiency drop at the conditions in which COVA was reduced due to an increase of tc.

In this study, it was aimed to investigate the effects of different co-carbon sources on therapeutically important glycoprotein, recombinant human erythropoietin (rHuEPO) production by Pichia pastoris by designing feeding strategies which were applied in the production phase of the bioprocess. During the experiments, the cell growth, sorbitol, mannitol, and methanol consumptions, recombinant human EPO production, alcohol oxidase activity, total protease concentrations and the by-products organic acid concentrations were analyzed. In this context, firstly, laboratory scale air filtered shake bioreactor experiments were performed by P. pastoris Mut+ strain to investigate the effects of mannitol and sorbitol. 50 gL-1 initial concentration of co-substrates was found more affordable and appropriate for cell concentration and recombinant protein production. Thereafter, six pilot scale bioreactor operations were designed and performed. In the first designed strategy (named as SSM strategy), batch-wise 50 g L-1 sorbitol was fed at t=0 h of the production phase and then sorbitol concentration was kept constant at 50 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Srb0=0.025 h-1 within t=0-15 h of the production phase together with fed-batch methanol feeding with a pre-determined specific growth rate of &mu / M0=0.03 h-1. In the following bioreactor experiments co-substrate mannitol was fed to the system with different feeding strategies together with fed-batch methanol feeding with a pre-determined specific growth rate of &mu / M0=0.03 h-1. In the second strategy (MM), only 40 g L-1 mannitol was added to the system at t=0 h of the production phase. In the third strategy (MMM), after adding 50 g L-1 mannitol at t=0 h, mannitol concentration was kept constant at 50 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Man0=0.11 h-1 within t=0-9 h of the production phase when the same cell concentration was attained in SSM strategy. In the fourth one (MLM), limiting amount of mannitol, 3 g L-1, was added at t=0 h and then mannitol concentration was kept constant at 3 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Man0=0.005 h-1 within t=0-10 h of the production phase. After these strategies, several pulses, batch-wise, mannitol feeding strategies were performed. In the fifth strategy (MPM), besides 50 g L-1 initial mannitol feeding at t=0 h, adding second batch-wise mannitol at t=6 h, and third one at t=12 h were applied. In the last strategy (MPMG), four 50 g L-1 pulse feeding of mannitol were performed at t=0 h, 7 h, 14 h, and 24 h, containing glycerol, with an initial concentration in the fermentation medium being 8 g L-1. The highest extracellular rHuEPO production was achieved in the fifth strategy MPM as CrHuEPO=645 mg L-1 at t=9 h while the highest cell concentration was achieved in the first strategy SSM as Cx=109 gL-1 at t=48 h. The overall cell and product yields on total substrate were calculated as YX/St=0.22 g g-1 and YP/St=2.23 mg g-1 in the highest rHuEPO production case.

In this study, the aim was to achieve high concentration of recombinant human growth hormone (rhGH) production by recombinant Pichia pastoris by investigating the effects of various operation parameters and to determine the suitable peptide ligand sequence that shows affinity and specificity to hGH. In this context, firstly the effect of temperature and Tween-20/80 addition on production and cell growth were investigated. While at T=30 and 32&deg / C, there was no difference, at 27 and 25&deg / C cell growth slowed down and production decreased significantly. The addition of Tween-20/80 in existence of co-substrate sorbitol did not affect the bioprocess while in absence of sorbitol Tween alone did not show the same positive effect on product formation and cell growth. Thereafter at T=30&deg / C, without addition of Tween, three sets of pilot scale bioreactor experiments were performed. In the first set, the effect of methanol feeding rate on bioprocess characteristics were investigated at the specific growth rates of &mu / =0.02, 0.03 and 0.04 h-1. While the highest cell concentration was achieved at &mu / =0.04 h-1, the highest rhGH concentration was achieved at &mu / =0.03 h-1. Secondly, conducting methanol feeding at &mu / =0.03 h-1, pH=5.5 experiment was conducted. The highest cell concentration, 45 g L-1 and maximum rhGH concentration 0.25 g L-1 were achieved at t=18 h of the process. Finally, the effect of batch sorbitol feeding on bioprocess was observed by the addition of 50 g L-1 sorbitol at t=0, 14 and 31 h of the production phase. It was shown that sorbitol addition to the medium increased process duration / hence cells enter stationary phase after a longer production phase. However, the protease concentration continued increasing with respect to time and at the end of the process reached twice the concentration it was obtained with single sorbitol addition case decreasing the rhGH concentration. In selection of the peptide sequence that shows affinity towards hGH, phage display method was conducted. Additionally the sequences from literature and computational design were used as alternatives. The interaction between these peptides and hGH was investigated by isothermal titration calorimetry and surface plasmon resonance.

In this study, recombinant human growth hormone (rhGH) production by Pichia pastoris-Mut+ strain was improved by designing feeding strategies which were applied in the production phase of the bioreactor operations. During the bio-reactor experiments the cell growth, sorbitol and methanol consumptions, recom-binant hGH production, alcohol oxidase (AOX) activity, the by-products protease and organic acid concentrations were followed and analyzed. In this context, in the first part of the study, three bioreactor operations were designed and per-formed. In general, the designed strategies are fundamentally based on simulta-neous feeding of the two substrates starting at t=0 h of the production phase, i.e., batch-wise 50 gL-1sorbitol feeding, together with fed-batch methanol feeding with a specific growth rate of &mu / 0=0.03 h-1 or &mu / 0=0.04 h-1, and fed-batch sorbitol feeding with a specific growth rate of &mu / 0=0.025h-1 which was calculated based on the specific consumption rate qS=0.152 g g-1h-1 of sorbitol. Consequently, sorbitol concentration was kept constant at 50 gL-1 within t=0-15h of the production phase / where, sorbitol feeding was terminated at t=15h. Amongst, in the first strategy (SSM1), methanol was fed to the system with the specific growth rate of &mu / 0=0.03 h-1, and the H+ concentration (pH) in the bioreactor was kept constant at pH=5.0. In the second strategy (SSM2), pH was kept constant at 5.5 until t=24h of the induction phase (production phase), thereafter, was reduced to pH= 5.0 / where methanol was fed to the bioreactor with the specific growth rate of &mu / 0=0.03 h-1. In the third strategy (SSM3), methanol was fed with the specific growth rate of &mu / 0=0.04 h-1, and the pH in the bioreactor was kept constant at pH 5.0. The highest rhGH production and cell concentration were achieved in the first strategy SSM1 as CrhGH=640 mg L-1 and CX=105.3 g L-1, and the overall cell and product yields on total substrate were calculated as YX/S =0.21 g g-1 and YCrhGH/S =1.83 mg g-1. In the second part of this study the two-substrates sorbitol and methanol were fed simultaneously in a solution compose of 1.37 mol sorbitol and 6.21 mol methanol in 13.88 mol water, which is named as SM. In this strategy (SM), the two-substrate solution was fed to the medium with the specific growth rate of &mu / 0=0.03 h-1 on sorbitol until t=30h / thereafter, only methanol was fed to the bio-reactor with the specific growth rate of &mu / 0=0.03 h-1. The highest cell and rhGH concentrations obtained in SM were, respectively, Cx=104.7 g L-1 and CrhGH=124 mg L-1 / and the overall cell and product yields on the total substrate were calcu-lated as YX/S=0.21 g g-1 and YCrhGH/S=0.39 mg g-1. Although the highest cell con-centration obtained at SM is close to that of the SSM1, the rhGH concentration obtained at SM is 5.2-fold lower than that of the strategy SSM1.

In this study, the effects of pH and different feeding modes on beta-lactamase production and cell metabolism were investigated with Bacillus licheniformis (ATCC 25972). For this purpose, first, the effects of pH on beta-lactamase activity, cell formation, substrate consumption, as well as intracellular sodium, potassium, ammonium ion, amino acid and organic acid concentrations were investigated in V= 3.0 dm3 batch bioreactors consisting of temperature, pH, foam, stirring rate and dissolved oxygen controls. Among the investigated uncontrolled pH operation with pH0=7.5 and controlled pH operations, pHc=6.75 yielded the highest cell concentration and beta-lactamase activity as Cx=0.60 kg/m3 and A= 54 U/cm3, respectively. Next, the production medium was redesigned in terms of initial glucose and phosphate ion concentrations in order to increase the enzyme activity and cell growth rate, and to determine the feeding strategy in laboratory scale batch-bioreactors using shake bioreactors having V=33 ml working volumes. The medium containing (kg/m3), glucose 2.5 (7.0) / Na2HPO4, 1.0 / K2HPO4 1.0 / (NH4)2HPO4, 7.1 and salt solution at pHc=6.75 was accepted as optimized medium for fed-batch (batch) processes. Using this optimized medium the feeding strategy was investigated for linear and exponential feeding profiles and compared with batch operation. Throughout the fermentation, cell, substrate and intracellular and extracellular by-product, sodium, potassium, ammonium ion concentrations, beta-lactamase activity, yield coefficients, specific rates, oxygen uptake rates and liquid phase mass transfer coefficient values were determined. The highest beta-lactamase activity was obtained at fed-batch operation with exponential feeding (FB1) condition as A= 108 U/cm3, which is ca. 1.7-fold higher than that of the batch operation with optimized medium. Finally, to invesitigate the physiological state of the culture media, viability of the cells was monitored throughout the cultivation time for repeated FB1, pHc=6,75, and pHuc=7.5 experiments. About 9% of the cells were found to be dead through the end of FB1 and pHuc=7.5 operations.

Este trabalho apresenta a avaliação da estabilidade e da eficiência de dois reatores anaeróbios operados em batelada seqüencial no tratamento de água residuária que simula o efluente de indústria de produtos de higiene pessoal, sendo um com biomassa imobilizada em espuma de poliuretano (AnSBBR) e recirculação da fase líquida, e outro com biomassa granular autoimobilizada (AnSBR) e agitação mecânica. O AnSBBR foi operado em batelada e em batelada alimentada, seguida de batelada, com tempo de ciclo (tc) de 8 h e carga orgânica volumétrica aplicada (COVA) de 0,9 e 1,5 gDQO/L.d, tratando 2,5 L de meio líquido por ciclo. Os valores de eficiência de remoção de matéria orgânica de amostras filtradas do efluente ficaram abaixo de 71% nas condições operadas em batelada, e acima de 74% nas condições operadas em batelada alimentada, seguida de batelada. Por outro lado, o AnSBR foi operado em batelada, com tc de 8, 12 e 24 h e COVA de 0,75, 0,50 e 0,25 gDQO/L.d, tratando 2,0 L de meio líquido por ciclo. O aumento de tc resultou em ganho de eficiência nas condições de mesma COVA, chegando a 77% para tc de 24 h, contra 69% para tc de 8 h. Porém, houve queda de eficiência nas condições em que a COVA foi reduzida em função do aumento de tc. / This study presents the evaluation of stability and efficiency of two anaerobic sequencing batch reactors for wastewater treatment simulating the effluent from industry of personal care products, one containing immobilized biomass on polyurethane foam (AnSBBR) and external circulation of the liquid phase, and the other with self-immobilized granulated biomass (AnSBR) and mechanical agitation. The AnSBBR was operated in batch and fed batch/batch modes, with cycle time (tc) of 8 h and applied organic loading rates (COVA) of 0.9 and 1.5 gDQO/L.d, treating 2.5 L of liquid medium per cycle. The values of organic matter removal efficiency for filtered samples of effluent were below 71% in batch operations, and over 74% in fed-batch/batch operations. On the other hand, the AnSBR was operated in batch, with tc of 8, 12 and 24 h and COVA of 0.75, 0.50 and 0.25 gDQO/L.d, treating 2.0 L of liquid medium per cycle. The increase of tc resulted in higher values of efficiency for conditions with the same COVA, reaching 77% for a 24-h cycle against 69% for an 8-h cycle. However, there was an efficiency drop at the conditions in which COVA was reduced due to an increase of tc.

La forte prévalence des problèmes digestifs chez les lapins en croissance autour du sevrage, et le taux élevé de mortalité chez les lapines associé à une forte prévalence de femelles ayant un état corporel dégradé pourrait être le témoin d’une discordance entre les apports nutritionnels des animaux et leurs besoins. Nous avons visé, dans le cadre d’une alimentation séparée entre les lapines et leurs lapereaux avant le sevrage, de répondre à deux objectifs complémentaires : i) de mieux comprendre les relations entre les apports nutritionnels, la mise en place de l’ingestion d’aliment, la maturation de l’écosystème caecal, la santé et la croissance des jeunes lapereaux, et ii) d’explorer le lien entre la nature des nutriments apportés au cours du cycle de reproduction et les performances chez les lapines reproductrices. Nous avons observé que dans le cadre d’une stratégie de restriction alimentaire après le sevrage, la distribution, de 18 à 70 jours d’âge, d’un aliment plus riche en énergie (+7% ED), mais contenant un niveau de fibres suffisant, permet d’optimiser la croissance des lapereaux avant et après sevrage,sans pénaliser la santé des lapereaux(1,49% de mortalité). Nous avons aussi montré, que lors de l’application d’une restriction alimentaire après le sevrage, la distribution d’un aliment riche soit en amidon (166 vs 72 g/kg),soit en fibres (173 vs 220 g/kg d’ADF),avant le sevrage conditionne le poids des lapereaux non seulement au moment du sevrage (+7,7%) mais également jusqu’au moment de la vente (2522 vs 2584 g). Enfin, nous avons constaté que le niveau d’ingestion de l’aliment chez les lapereaux au moment de la mise en place de l’ingestion d’aliment solide influence la maturation de l’écosystème caecal. Chez les lapines, notre étude a apporté des résultats mitigés. Nous avons observé peu d’effet sur les performances de reproduction chez les lapines, mais nous avons observé des conséquences importantes sur le bilan énergétique des femelles (-4,94 vs -2,05 MJ), la quantité (3911 vs 3415 g)et la qualité du lait produit. Ce dernier élément a des effets sur la croissance des lapereaux et leur ingestion précoce d’aliment solide (7 vs 9 g/j entre 18 à 25 jours d’âge), ce qui en conséquence affecte leur santé après le sevrage (5,8 vs 1,7 % de mortalité).Cela a été le cas avec l’utilisation d’un aliment riche en lipides (49 g/kg de lipides brute) au début de la lactation.Toutefois, nous avons aussi montré que les coefficients d’utilisation digestive des nutriments sont très différents chez les lapereaux et chez les femelles à différents stades physiologiques. Par conséquent, en l’absence de mesures réelles de la valeur nutritionnelle des aliments sur les femelles elles-mêmes et au stade physiologique considéré, nos estimations des apports nutritionnels et donc l’estimation des bilans peuvent être erronés. Au final, nos résultats suggèrent que la stratégie qui consisterait à valoriser le système d’alimentation séparée pour n’utiliser qu’un seul aliment pour les lapines pendant toute leur vie reproductive pourrait être le meilleur compromis entre simplicité, efficacité et rentabilité. Celui-ci devrait être riche en énergie sous forme d’amidon pour favoriser le maintien de la fertilité et des réserves corporelles sur le long terme. Ce système d’alimentation séparée permettrait aussi chez les lapereaux de limiter les conséquences néfastes d’un changement d’aliment et d’optimiser le compromis entre performances de croissance et de santé par l’introduction d’un aliment plus riches en énergie et en protéine mais avec un niveau de fibres suffisant dans le cadre d’une restriction alimentaire après le sevrage. Mieux comprendre l’adaptation digestive et la gestion des priorités métaboliques au cours du cycle de reproduction chez la femelle ainsi que la relation entre ingestion précoce et maturation de l’écosystème caecal chez le jeune lapereau sont des perspectives de recherche intéressante à nos travaux. / The high prevalence of digestive problems in growing rabbits around weaning and the high rate of mortality in rabbit does associated with a high prevalence of does having a degraded body condition can contest to a mismatch between the nutritional intake of animals and their needs. This thesis aimed, through the use of a separate feeding system between rabbits does and their kits before weaning, to respond to two complementary objectives: i) to better understand the relationship between nutrient intake, the establishment of solid feed intake, the maturation of the cecal ecosystem, and the health and growth of young rabbits, and ii) to explore the relationship between the nature of nutrients provided throughout the reproduction cycle and performance in breeding does. We observed that in the context of a feed restriction strategy after weaning, the distribution, from 18 to 70 days of age, of a more energy-rich feed (+7% DE), but containing sufficient level of fiber, optimizes young rabbit growth before and after weaning, without compromising their health (1.49% mortality). We also showed that when a feed restriction plan is implemented after weaning, the nutritional strategy used before weaning, the distribution of a feed either rich in starch (166 vs 72 g/kg) or in fiber (173 vs 220 g/kg ADF), conditions the weight of growing rabbits not only at weaning (+7.7%) but also at the time of sale (2522 vs 2584 g). Finally, we found that the solid feed intake level of rabbit kits at the establishment of solid feed intake influences the maturation of the cecal ecosystem. In reproductive does, our study has produced mixed results. We observed little effect on reproductive performances in does, but observed a significant impact on their energy balance (-4.94 vs -2.05 MJ), and the quantity (3911 vs 3415 g) and quality of the milk produced. The latter had an effect on the growth of young rabbits and early ingestion of solid food (7 vs 9 g/day from 18 to 25 days of age), which consequently affects their health after weaning (5.8 vs 1.7% mortality).This was the case with the introduction of a feed rich in fat (49 g/kg crude fat) during early lactation. However, our latest work revealed that, in the absence of actual measurements of the nutritional value of feed using the females themselves and taking into consideration their physiological state, our estimation of nutrient intake, and therefore the estimation of the energy balance may be wrong. Finally, our results suggest that the strategy which introduces the separate feeding system in order to distribute only one type of feed for the rabbit does throughout their reproductive life might be the best compromise between simplicity, efficiency and profitability. The feed used should be rich in energy, in the form of starch, to help maintain fertility and body reserves of the does in the long term. This separate feeding system would also allow the rabbits to limit the negative consequences of a diet change and optimize the trade off between growth performance and health by introducing a richer food energy and protein but with a sufficient level of fiber as part of a feed restriction strategy after weaning. A better understanding of the digestive adaptation and management of metabolic priorities during the reproductive cycle in the female and the relationship between early maturation and ingestion of cecal ecosystem in young rabbit are interesting research perspectives to our work.