Abstract Potential lethal diarrhoea caused by enterotoxigenic Escherichia coli strains is one of the most common diseases in young pigs. It can be cured by single-chain antibody fragments (scFv), which can be produced in recombinant microorganisms. Pichia pastoris, a methylotrophic yeast, is generally considered an interesting production system candidate, as it can secrete properly folded proteins. These proteins accumulate in high concentrations during fermentation, reducing the cost for product recovery. Strong inducible AOX1 promoter, widely used in P. pastoris for fast, inexpensive production, is typically induced by methanol. The high oxygen demand of methanol metabolism makes oxygen supply a major parameter in cultivations requiring special process design strategies. In standard fed-batch cultivation, dissolved oxygen concentration inside a bioreactor is kept at a certain level by pumping air and pure oxygen into the reactor. There are safety concerns over the handling of oxygen, especially at a large scale. Therefore, there is a need to develop a production process under oxygen-limited conditions. This dissertation studies the development of a cost-efficient production process of scFv in P. pastoris. Both methanol and oxygen parameters influence the production process and the objective was to find a robust production process. Fed-batch cultivations were performed in a 10 L scale bioreactor. The effects of lower oxygen level, methanol concentration, glycerol feeding duration and specific substrate-uptake rates on product formation were studied. A P. pastoris GS115 his4 strain under an AOX1 promoter system expressing scFv was used in this study. The fed-batch fermentations were carried out in a bioreactor with basal salt media. In this doctoral dissertation, a process was developed for a single-chain antibody fragment (scFv) production in P. pastoris. The product levels of 3.5 g L-1 scFv in culture supernatant were achieved and a production process was designed without additional need of pure oxygen, thus relieving safety requirements and lowering the amount of methanol. The process developed during this research may potentially be utilised by both academia and industry having interests in expressing proteins in P. pastoris. The methanol-uptake control strategy is beneficial for those products that suffer from degradation or modification during limited feeding of methanol. / Tiivistelmä Enterotoksigeenisten E.coli kantojen aiheuttama ripuli on porsaiden tavallisimpia tauteja, joka voi johtaa jopa kuolemaan. Tautia voidaan hoitaa yhdistelmä-DNA-tekniikalla tuotetuilla vasta-ainefragmenteilla (scFv). Metylotrofista Pichia pastoris hiivaa pidetään kiinnostavana vasta-ainefragmenttien tuottoisäntänä, koska se pystyy erittämään oikealla tavalla laskostuneita proteiineja. Näitä proteiineja kertyy fermentointiprosessissa solujen ulkopuolelle korkeina pitoisuuksina, mikä vähentää tuotteiden talteenottokustannuksia. Vahva metanolilla indusoituva AOX1-promoottori on laajassa käytössä P. pastoris tuottosysteemissä tuoton nopeuden ja alhaisten kustannusten ansiosta. Metanolin aineenvaihdunta vaatii paljon happea, joten riittävän tehokas hapen liuottaminen on tärkeimpiä fermentointiparametreja ja vaatii erityisiä prosessin toteutusstrategioita. Perinteisessä fed-batch-fermentoinnissa liuenneen hapen pitoisuus bioreaktorissa pidetään halutulla tasolla lisäämällä ilmaa ja puhdasta happea reaktoriin. Koska hapen käsittelyyn liittyy turvallisuusriskejä erityisesti teollisuusmittakaavassa, happirajoitteisissa olosuhteissa toimiva tuotantoprosessi olisi hyödyllinen. Tässä väitöstutkimuksessa kehitettiin kustannustehokasta prosessia scFv-:n tuottoon P. pastoris hiivalla. Metanoliin ja happeen liittyvät parametrit ovat olennaisia prosessiin vaikuttavia tekijöitä. Tavoite oli kehittää yksinkertainen ja käytännöllinen prosessi. Työssä tutkittiin alhaisen happitason, metanolin pitoisuuden, glyserolisyötön keston ja substraattien spesifisten kulutusnopeuksien vaikutuksia tuotteen muodostumiseen 10 litran bioreaktorissa. Isäntäkantana oli P. pastoris GS115 his4, jossa scFv-ekspressiota säädeltiin AOX1 promoottorilla. Fed-batch fermentointien kasvatusalustana käytettiin Basal Salt Medium alustaa (BSM). Väitöstyössä kehitettiin tavoitteiden mukainen vasta-ainefragmenttien tuottoprosessi P.pastoris hiivalle. Menetelmällä saavutettiin tuotepitoisuus 3,5 g L-1 kasvatusliemen supernatantissa ilman puhtaan hapen lisäystarvetta, ja siten metanolin kulutus väheni ja prosessiturvallisuus parani verrattuna perinteisiin prosesseihin. Kehitetty prosessi soveltuu käytettäväksi sekä akateemisessa tutkimuksessa että teollisuudessa tuotettaessa erilaisia proteiineja P. pastoris hiivalla. Metanolin kulutuksen säätöstrategia on erityisen hyödyllinen tuotteille, joilla ongelmana on proteolyysi tai muokkautuminen metanolirajoitteisessa fermentoinnissa.

Les Streptomyces sont des bactéries filamenteuses à Gram positif que l’on retrouve dans les couches superficielles du sol. La souche modèle S. coelicolor M145 est capable de produire des antibiotiques et accumuler de faibles quantités de triacylglycérol lorsqu’elle est mise en culture avec le glucose ou le glycérol comme source de carbone. La souche produit de l’actinorhodine seulement en culture avec le glycérol comme source de carbone, et nous assistons à une accumulation d’acides gras, notamment sous forme d’esters d’acide oléique avec l’une ou l’autre source de carbone. Ces accumulations d’acides gras ne durent que pendant une certaine période de temps, à l’issu de laquelle l’accumulation s’arrête, et on assiste à la production de métabolites secondaires qui peut s’accompagner de la consommation des acides gras précédemment accumulés. Ces observations suggèrent d’une part que la source de carbone est un effecteur différentiel dans la production de métabolites secondaire, et que d’autre part le métabolisme des lipides est lié au métabolisme secondaire. Ce travail de thèse va tenter d’établir les liens entre le métabolisme primaire et le métabolisme secondaire chez S.coelicolor via le métabolisme des lipides. Pour se faire, nous avons dans un premier temps défini un milieu synthètique, ainsi que des stratégies d’alimentation de type fed-batch en bioréacteur, pour permettre d’obtenir d’une part une phase de croissance exponentielle, et d’autre part une phase de croissance non exponentielle et imposée par une limitation en azote. Cette limitation nutritionnelle est classiquement utilisée à la fois lors des études de l’accumulation de lipides chez les microorganismes oléagineux tels que Yarrowia lipilytica ou Rhodotorula glutinis, et aussi dans le cas d’études de la production d’antibiotiques par les Streptomyces. L’étude du métabolome couplée à l’étude du protéome nous a permis d’établir la direction générale des flux globaux de carbone, d’énergie et de pouvoir réducteur lors des différentes phases de croissances. Ces travaux démontrent une hétérogénéité métabolique dans la population bactérienne totale, avec la présence de 2 à 3 sous-populations différentes pouvant coexister. Les évolutions temporelles du protéome et notamment des régulateurs transcritptomiques montrent des réactions tardives, très certainement dues à la présence d’effecteurs intracellulaires accumulés, ainsi qu’au dynamisme de la culture. / Streptomyces are filamentous Gram positive bacteria found in the soil upper layers. The model strain S. coelicolor M145 can produce antiobiotics and accumulate low levels of triacylglycerol when cultivated with glucose or glycerol as carbon source. This strain produces actinorhodin only when glycerol is used as carbone source, but the fatty acids accumulation as esterified oleic acid occurs with both carbon sources. However, the fatty acids accumulation only last for a short period of time, and afterward, a production of secondary metabolites is observed and the previously accumulated fatty acids are consummed. Those results suggest that the carbon source act as an effector for the production of secondary metabolites and the lipids metabolism is some ways linked to the secondary metabolism. With this work, we will try to establish the links between the primary and secondary metabolism via the lipids metabolism with S. coelicolor. First, in order to obtain cultures with an exponential growth phase and a non-exponential growth phase under a nitrogen limitation, a synthetic media and fed-batch feeding strategies have been designed. The nitrogen limitation is usually used to study lipids accumulation with oleaginous microorganisms such as Yarrowia lipilytica or Rhodotorula glutinis, and also during studies of antibiotics production with Streptomyces. The metabolomics study paired with the proteomics study made possible to establish the global directions of carbon, energy and reduced power fluxes during all the obtained growth phases. This work also showed that the microbial population is heterogeneous and 2 to 3 subpopulations can coexist with both carbon sources. The proteomics temporal changes and in particular of transciptional regulators show some late reactions to the nitrogen limitation especially when glycerol is used, probably because of the intracellular accumulation of effector compounds over time, and because of growth kinetics.

L’objectif de notre travail était d’étudier les bascules métaboliques chez Yarrowia li-polytica d’un métabolisme purement oxydatif vers l’accumulation de lipides puis à l’excretion d’acide citrique.Le développement d’un procédé D-stat et d’un mode de conduite fed-batch nous a permis, dans un premier temps, de quantifier les ratios N/C caractéristiques pour chacune des bascules étudiées. Nos résultats montrent que les ratios rN/rC critiques aux bascules métaboliques sont de 0,085 molN.Cmol-1 et de 0,018-0,022 molN.Cmol-1 pour l’accumulation de lipides et production de citrate, respectivement.L’analyse systémique des cultures réalisées nous a permis de mettre en évidence des mécanismes de co-régulation de certaines enzymes du métabolisme lipidique ainsi qu’une prépondérance de mécanismes post-transcriptionnels dans l’établissement des bascules étudiées.Enfin, l’utilisation de souches génétiquement modifiées au niveau de l’ATP citrate lyase, la malate déshydrogénase et de la glycérol-3-phosphate déshydogénase a permis d’évaluer l’impact de ces enzymes sur le métabolisme lipidique / This thesis aimed at studying the metabolic shifts in Yarrowia lipolytica from the pure oxidative metabolism to lipid accumulation and citric acid excretion.The development of a D-stat culture and of a monitoring fed-batch strategy allowed us to determine the N/C ratio characteristic for each of metabolic shifts. rN/rC ratio were determined equal to 0,085 molN.Cmol-1 and 0,018-0,022 molN.Cmol-1 for the lipid accumu-lation and the citric acid production, respectively.Systemic analysis of the cultivations showed coregulation phenomena among some enzymes of the lipidic metabolism and post-transcriptional modifications in the onset of the metabolic shifts.Finally, the impact of enzymes (ATP citrate lyase, malate dehydrogenase and gly-cérol-3-phosphate dehydrogenase) on the lipidic metabolism was evaluated through systemic analysis of 3 genetically modified strains

<p>The goal of this work was to contribute to the fed-batch process optimisation task by deriving parameters that have considerable impact on productivity as well as product quality The chosen parameters were I) the design of the glucose feed profile, II) the choice of induction strategy, with respect to the method of addition, and III) the time of the induction, with respect to the specific glucose consumption rate. </p><p>The present fed-batch experiments using the lacUV5-promoter, for production of b-galactosidase, have shown that a high glucose feed rate gives a specific production rate, q_p, that is twice as high, after induction, compared to a feed rate that is 2.5 times lower. The constant accumulation of lacZ-mRNA indicates that the translational capacity is initially limiting the synthesis machinery, but after four hours of maximum specific production and a corresponding drop in lacZ-mRNA production, the cultivation is likely to be transcription limited. The high feed-rate system resulted in high accumulation of β-galactosidase, corresponding to 40% of total cellular proteins.</p><p>By design of feed profiles in a fed-batch process the detrimental effects of overflow metabolism, giving acetic acid formation, can be avoided. However, the results show that a one-dose addition of isopropyl-β-D-galactopyranoside (IPTG), provokes a non-growth associated production of acetic acid. This response can be alleviated by; lowering the inducer concentration (in this case to below 165 μM), by further reducing the feed rate of glucose or by using alternative induction methods. The use of a stepwise addition or a feed of IPTG thus delayed and reduced the level of acetic acid accumulation. It was also shown that a small change in the time-point of induction lead to large variability, regarding both productivity and acetic acid accumulation, in a fed-batch cultivation, </p><p>In order to further investigate the protein quality two additional proteins were studied in fed-batch cultivations using high and low glucose feed. The aim was to prove the hypothesis that the feed related change in the rate of synthesis of the nascent polypeptide controls the product quality. For the two proteins: Zb-MalE (wt) and Zb-MalE31 (mutant), the transcription rate, in terms of amount of IPTG, and translation rate, in terms of changes in feed rate, influences the percentage of inclusion body formation and degradation of nascent polypeptide. The data show a higher rate of inclusion body formation for the model protein Zb-MalE31 during high feed rate cultivations, as well as at high levels of inducer. Furthermore, the rate of proteolysis was significantly higher for a high feed rate. The high feed rate thus results in a higher rate of synthesis but a lower corresponding quality, for the model proteins studied.</p><p>In the present investigation of fed-batch cultivations using several different expression vectors, it was found that the central alarmone guanosine tetraphosphate (ppGpp) was formed at both high and low feed rates upon induction. It could be shown, however, that by secretion of Zb-MalE to the periplasm, the stringent response could be avoided. This might be due to the decreased burden on the host where the secretion of product further seems to make the cell able to redirect the carbon flux from overflow metabolism, since no acetic acid was produced. The secretion also demonstrates that the growth arrest could be aborted, which is otherwise gained in the P_malKproduction system.</p><p>A novel fed-batch process based on the promoters for the universal stress proteins A and B (P_uspA, P_uspB) was designed to make use of these powerful promoters in an industrial production context. It was concluded that the process had to start from a high specific growth rate and induction was performed once a limiting feed started. This was done to purposely induce the stringent response and/or acetic acid accumulation since this was required for induction. In the suggested system, induction has to be performed and maintained at continuous substrate feeding, whilst avoiding exceeding the cellular capacity, since the stationary phase starvation alone did not lead to production. In conclusion, a new stress induction based production system was achieved resulting in high accumulations of product protein without any detected metabolic side effects.</p>

A cianobactéria \' Spirulina platensis \' possui alto teor de proteína e vem sendo cultivada fotoautotroficamente para a produção de biomassa microbiana. Embora as fontes convencionais de nitrogênio utilizadas para a produção de Spirulina spp. sejam os nitratos, há a possibilidade do emprego de uréia, utilizando o processo descontínuo alimentado, com diminuição do custo de produção. O emprego do processo descontínuo alimentado repetitivo para o cultivo desta microalga utilizando a uréia como fonte de nitrogênio poderia facilitar os cultivos em escala de produção, pois não haveria a necessidade de preparo de inóculo para cada ciclo de produção, além de possibilitar o aumento da produtividade do sistema. Este trabalho tem o objetivo de verificar o comportamento do cultivo microbiano com este processo, visto que não foram encontrados na literatura tais estudos para o cultivo de \'S. platensis\'. Foi verificada a influência dos parâmetros fração de corte, tempo de alimentação de uréia e ciclos de cultivo neste tipo de processo, tendo como variáveis dependentes a concentração celular máxima (Xm), a produtividade em células (Px) e o fator de conversão de nitrogênio em células (Yx/n), bem como a composição protéica e lipídica da biomassa obtida. Os resultados obtidos permitem concluir que, de uma forma geral, uma fração de corte de 80% associada a um tempo de alimentação de 6 dias leva a melhores condições de cultivo com bons resultados nos três parâmetros cinéticos (em média, 2101 mg.L?¹, 219 mg.L?¹.d?¹ e 10,3 mg.mg?¹ para Xm, Px e Yx/n, respectivamente), que foram reprodutíveis ao longo de 3 ciclos. / \' Spirulina platensis \', with high protein content, can be cultivated photoautotrophically for the microbial biomass production. Although nitrates are the conventional source of nitrogen for the Spirulina spp. production, there is a possibility of the use of urea, in a fed-batch process, leading to a cost reduction. The application of repeated fed-batch process, using urea as nitrogen source, could propitiate the cultivation of this microorganism in a production scale, since it would not need to prepare the inoculum for each production cycle. Moreover, there is a possibility to improve the productivity of this process. The aim of this work was to verify the behavior of the microbial cultivation employing this process, since this kind of study for \' Spirulina platensis \' production was not found in literature. The influence of withdrawn rate, urea feeding time and cultivation cycles were studied, considering maximum cell concentration (Xm), cell productivity (Px), nitrogen-to-cell conversion (Yx/n), and protein and total fat contents as dependent variables. The results show that in general a 80% of withdrawn rate associated with 6 days of feeding time leads to the best cultivation conditions with satisfactory results of Xm, Px and Yx/n (2101 mg.L?¹, 219 mg.L?¹.d?¹ and 10.3 mg.mg?¹, in average, respectively), that could be reproduced through the three cycles.

As microalgas são organismos unicelulares, eucariotos, simples em estrutura, fotossintetizantes, que requerem principalmente luz, água e nutrientes inorgânicos para seu crescimento. Estes micro-organismos podem converter eficientemente dióxido de carbono em biomassa e certos compostos bioativos com aplicações nas indústrias alimentícia, farmacêutica e cosmética. Botryococcus braunii é uma espécie de microalga capaz de sintetizar e armazenar lipídios, incluindo ácidos graxos, epóxidos, alquilfenol, éter e hidrocarbonetos de cadeia longa. O conteúdo lipídico pode alcançar de 15% a 75% da biomassa seca, sendo que os ácidos graxos livres representam mais da metade deste extrato. Os parâmetros como carbono inorgânico, nitrogênio, vitaminas e sais são vitais para o crescimento de microalgas, exercem influência tanto no crescimento como na composição da biomassa. Assim, este trabalho tem a finalidade de verificar a influência das quantidades de nitrato de sódio e de fosfato de potássio dibásico no crescimento e composição da microalga Botryococcus braunii, onde serão avaliados parâmetros cinéticos e de crescimento, bem como a composição da biomassa. Os diferentes meios de cultivo avaliados para o crescimento da microalga mostraram a importância da influência da salinidade no crescimento e composição da biomassa de B. braunii, sendo o meio Chu encontrado como o melhor meio para o crescimento, no qual para uma otimização do crescimento e produção de biomassa foram utilizadas diferentes concentrações de fontes de nitrogênio e fósforo. Com uso de meio Chu, foi possível a obtenção de concentrações celulares de 4962,9mg.L-1 em fotobiorreator tubular, com correção da concentração das fontes de nitrogênio e fósforo de acordo com o crescimento celular. Nas várias concentrações de nitrogênio e fósforo avaliadas, foi encontrada uma concentração de lipídios numa faixa de 32,56 a 36,93%, onde foram encontrados os seguintes ácidos graxos C11:0; C14:1; C16:0; C16:1; C17:1; C18:0; C18:1n9; C18:2n6; C20:0; C18:3n6; C20:1 e C18:3n3. / Microalgae are unicellular photosynthetic organisms, eukaryotes, simple in structure, requiring mainly light, water and inorganic nutrients for growth. These micro-organisms can efficiently convert carbon dioxide into biomass and certain bioactive compounds with applications in food, pharmaceutical and cosmetic industries. Botryococcus braunii is a microalga capable of synthesizing and storaging lipids, including fatty acids, epoxides, ether, and hydrocarbons. Lipid content can reach 15% to 75% of dry matter, and free fatty acids represent more than half of this extract. Parameters such as inorganic carbon, nitrogen, vitamins and salts are important parameters regulating algal growth and the composition of biomass. This work aims to study the influence of the amounts of sodium nitrate and potassium phosphate dibasic in the growth and composition of microalgae Botryococcus braunii, by evaluating kinetic and growth parameters, as well as biomass composition. The culture media evaluated for the growth of microalgae, showed the importance of the influence of salinity on growth and biomass composition of Botryococcus braunii. Chu medium was found as the best medium for growth, for which an optimization of growth and biomass concentrations different sources of nitrogen and phosphorus was used. Using the Chu medium, it was possible to obtain cell concentrations of 4962.9mg.L-1 in tubular phobioreactor, with correction of concentrations of both nitrogen and phosphorus sources according to cell growth. Taking into account the various nitrogen and phosphorus evaluated, A concentration of lipids was found in a range from 32.56 to 36.93%, where the fatty acids are: C11:0; C14:1; C16:0; C16:1; C17:1; C18:0; C18:1n9; C18:2n6; C20:0; C18:3n6; C20:1 and C18:3n3.

A cianobactéria \' Spirulina platensis \' possui alto teor de proteína e vem sendo cultivada fotoautotroficamente para a produção de biomassa microbiana. Embora as fontes convencionais de nitrogênio utilizadas para a produção de Spirulina spp. sejam os nitratos, há a possibilidade do emprego de uréia, utilizando o processo descontínuo alimentado, com diminuição do custo de produção. O emprego do processo descontínuo alimentado repetitivo para o cultivo desta microalga utilizando a uréia como fonte de nitrogênio poderia facilitar os cultivos em escala de produção, pois não haveria a necessidade de preparo de inóculo para cada ciclo de produção, além de possibilitar o aumento da produtividade do sistema. Este trabalho tem o objetivo de verificar o comportamento do cultivo microbiano com este processo, visto que não foram encontrados na literatura tais estudos para o cultivo de \'S. platensis\'. Foi verificada a influência dos parâmetros fração de corte, tempo de alimentação de uréia e ciclos de cultivo neste tipo de processo, tendo como variáveis dependentes a concentração celular máxima (Xm), a produtividade em células (Px) e o fator de conversão de nitrogênio em células (Yx/n), bem como a composição protéica e lipídica da biomassa obtida. Os resultados obtidos permitem concluir que, de uma forma geral, uma fração de corte de 80% associada a um tempo de alimentação de 6 dias leva a melhores condições de cultivo com bons resultados nos três parâmetros cinéticos (em média, 2101 mg.L?¹, 219 mg.L?¹.d?¹ e 10,3 mg.mg?¹ para Xm, Px e Yx/n, respectivamente), que foram reprodutíveis ao longo de 3 ciclos. / \' Spirulina platensis \', with high protein content, can be cultivated photoautotrophically for the microbial biomass production. Although nitrates are the conventional source of nitrogen for the Spirulina spp. production, there is a possibility of the use of urea, in a fed-batch process, leading to a cost reduction. The application of repeated fed-batch process, using urea as nitrogen source, could propitiate the cultivation of this microorganism in a production scale, since it would not need to prepare the inoculum for each production cycle. Moreover, there is a possibility to improve the productivity of this process. The aim of this work was to verify the behavior of the microbial cultivation employing this process, since this kind of study for \' Spirulina platensis \' production was not found in literature. The influence of withdrawn rate, urea feeding time and cultivation cycles were studied, considering maximum cell concentration (Xm), cell productivity (Px), nitrogen-to-cell conversion (Yx/n), and protein and total fat contents as dependent variables. The results show that in general a 80% of withdrawn rate associated with 6 days of feeding time leads to the best cultivation conditions with satisfactory results of Xm, Px and Yx/n (2101 mg.L?¹, 219 mg.L?¹.d?¹ and 10.3 mg.mg?¹, in average, respectively), that could be reproduced through the three cycles.

<p>The present Master thesis describes the development and optimization of a fed-batch process for production of recombinant proteins in Escherichia coli BL21(DE3) in a multi-fermentor system. The system consists of six 1-liter fermentors, capable of producing 500-1500 μg/mL with present protocol.</p><p>Response surface methodology (RSM) was used for multivariable optimization regarding cultivation time, pH, temperature and feed rate. Optimal protein expression conditions were found out to be 17.8 h cultivation time, 36.7 ºC, pH 6.8 and a feed rate corresponding to specific growth of 0.23 h-1, on glucose substrate. The aggregation of expressed proteins to inclusion bodies, could not be affected by the various growth conditions employed during cultivations.</p><p>A study was conducted regarding growth conditions effect on phosphogluconoylation of expressed proteins. In ten fed-batch cultivations on glucose, LC/MS analysis showed a gluconoylated fraction with additional 178 Da mass, but no correlation between growth conditions and gluconoylation could be found. In two fed-batch cultivations on glycerol-feed, a lower feed rate resulted in no gluconoylation, while a higher did. An explanation would be that the lower amount of available intra-cellular carbon limits formation of gluconoylation precursors.</p>

With the increasing demand for biodiesel, the accumulation of byproduct, crude glycerol has become a problem which needs to be solved. Lactic acid is one of the value-added chemical which can be produced from glycerol that has wide uses in food and chemical industry. Although glycerol can be converted to lactic acid with an alkali as the catalyst at high glycerol conversion (100 mol%) and lactic acid yield (around 90 mol%), the high alkalinity would cause severe corrosiveness to a stainless steel reactor. In this study two tasks were performed to convert glycerol to lactic acid with satisfactory conversion and selectivity, and to reduce the corrosiveness of reaction medium. First, CaO was used as solid base catalyst. The highest lactic acid yield achieved was 40.8 mol% with a 97.8 mol% glycerol conversion, when operating at 290°C after 150 min reaction with molar ratio of CaO: glycerol=0.3. Also CaO has advantages such as high lactic acid productivity (3.35 g/(min·L)) and reusability. Meanwhile, CaO can be used as the catalyst for both biodiesel production and the following crude glycerol conversion to lactic acid. Second, for glycerol conversion with NaOH as catalyst, a fed-batch reactor was applied to continuously supply NaOH during reaction process, compensating the OH- neutralized by newly formed lactic acid. The optimal lactic acid yield of 80.5 mol%, with 92.8% glycerol conversion was obtained at 300 °C for 220min, with 1.1 M glycerol initial concentration. A first-order kinetic model for glycerol concentration versus time was developed and verified experimentally under conditions with different initial glycerol concentration and reaction temperature. Although crude glycerol samples contained large amount of impurities, both methods, conversion with solid base catalyst and with fed-batch reactor, were applied successfully to three crude glycerol samples provided by biodiesel manufacturers, and the lactic acid yield reached 52.3 mol% and 72.7 mol% respectively. Finally, the corrosion issue of different methods was compared based on the Fe3+ concentration (analyzed with atomic-absorption spectroscopy) in the products. Both methods of glycerol conversions, with solid base catalyst and fed-batch reactor, can reduce the corrosiveness of glycerol conversion with an alkali as the catalyst.

No description available.