Saccharomyces cerevisiae tem sido empregada como modelo experimental em testes de mutagenicidade, devido à presença de um sistema citocromo P-450 capaz de metabolizar substâncias promutagênicas a sua forma ativa. O grande número de linhagens e as diferenças na capacidade de produção de citocromo P-450 dificultam a definição das condições ideais de cultivo para obtenção de células com alto conteúdo desta enzima, sendo que poucos são os trabalhos publicados a este respeito. O objetivo deste trabalho é avaliar as melhores condições de cultivo para produção de citocromo P-450 em S. cerevisiae. Para tanto, quatro diferentes linhagens foram cultivadas sob diferentes condições, a fim de avaliar a influência de fatores como fonte de carbono, tempo de incubação e adição de etanol ao meio de cultura. Foi verificado que células cultivadas na presença de fontes de carbono altamente fermentáveis e coletadas no final da fase de crescimento exponencial apresentavam o maior conteúdo da enzima. Observou-se também que o nível de citocromo P-450 variou entre as linhagens estudadas, bem como o tempo de incubação para atingir a concentração máxima, sendo que o conteúdo mais alto foi encontrado na linhagem ATCC 44953. A adição de 2 % de etanol (v/v) ao meio de cultura contendo 2 % de glicose (p/v) resultou em aumento do nível de citocromo P-450, indicando o efeito indutor do etanol. Os resultados deste trabalho mostram a importância de estabelecer condições de cultivo para obtenção de células com altas concentrações da enzima, uma vez que fatores como a composição do meio de cultura, o tempo de incubação e a linhagem empregada podem influenciar a produção de citocromo P-450 por S. cerevisiae. / Saccharomyces cerevisiae has been employed as experimental model in mutagenicity tests due to the presence of a cytochrome P-450 system capable of metabolizing promutagens to its active form. The large number of S.cerevisiae strains and their different capacity of producing cytochrome P-450 make the definition of the ideal cultivation conditions to obtain cells with high enzyme content difficult. Moreover, there are only a few reports related to this subject. The aim of this work is evaluate the ideal cultivation conditions to produce cytochrome P-450 by S. cerevisiae. For this purpose, four different S. cerevisiae strains were cultured under different cultivation conditions in order to evaluate the influence of factors such as carbon source, incubation time and addition of ethanol to the culture media. It was found that the cytochrome P-450 content was higher in cells growth on strongly fermentable carbon source and harvested at the end of the exponential growth phase. Our results also showed that the cytochrome P-450 concentration varied among the strains studied, as well as the incubation time to reach the maximum level. The highest content was found in the ATCC 44953 strain and the addition of 2 % ethanol (w/w) to the culture media containing 2 % glucose (w/v) increased the cytochrome P-450 amount, indicating the induction of cytochrome P-450 production by ethanol action. These results show the importance of establishment of the cultivation conditions to obtain cells with high cytochrome P-450 concentrations, considering that factors such as the culture media, incubation time and strain employed can influence the cytochrome P-450 production by S.cerevisiae.

Saccharomyces cerevisiae tem sido empregada como modelo experimental em testes de mutagenicidade, devido à presença de um sistema citocromo P-450 capaz de metabolizar substâncias promutagênicas a sua forma ativa. O grande número de linhagens e as diferenças na capacidade de produção de citocromo P-450 dificultam a definição das condições ideais de cultivo para obtenção de células com alto conteúdo desta enzima, sendo que poucos são os trabalhos publicados a este respeito. O objetivo deste trabalho é avaliar as melhores condições de cultivo para produção de citocromo P-450 em S. cerevisiae. Para tanto, quatro diferentes linhagens foram cultivadas sob diferentes condições, a fim de avaliar a influência de fatores como fonte de carbono, tempo de incubação e adição de etanol ao meio de cultura. Foi verificado que células cultivadas na presença de fontes de carbono altamente fermentáveis e coletadas no final da fase de crescimento exponencial apresentavam o maior conteúdo da enzima. Observou-se também que o nível de citocromo P-450 variou entre as linhagens estudadas, bem como o tempo de incubação para atingir a concentração máxima, sendo que o conteúdo mais alto foi encontrado na linhagem ATCC 44953. A adição de 2 % de etanol (v/v) ao meio de cultura contendo 2 % de glicose (p/v) resultou em aumento do nível de citocromo P-450, indicando o efeito indutor do etanol. Os resultados deste trabalho mostram a importância de estabelecer condições de cultivo para obtenção de células com altas concentrações da enzima, uma vez que fatores como a composição do meio de cultura, o tempo de incubação e a linhagem empregada podem influenciar a produção de citocromo P-450 por S. cerevisiae. / Saccharomyces cerevisiae has been employed as experimental model in mutagenicity tests due to the presence of a cytochrome P-450 system capable of metabolizing promutagens to its active form. The large number of S.cerevisiae strains and their different capacity of producing cytochrome P-450 make the definition of the ideal cultivation conditions to obtain cells with high enzyme content difficult. Moreover, there are only a few reports related to this subject. The aim of this work is evaluate the ideal cultivation conditions to produce cytochrome P-450 by S. cerevisiae. For this purpose, four different S. cerevisiae strains were cultured under different cultivation conditions in order to evaluate the influence of factors such as carbon source, incubation time and addition of ethanol to the culture media. It was found that the cytochrome P-450 content was higher in cells growth on strongly fermentable carbon source and harvested at the end of the exponential growth phase. Our results also showed that the cytochrome P-450 concentration varied among the strains studied, as well as the incubation time to reach the maximum level. The highest content was found in the ATCC 44953 strain and the addition of 2 % ethanol (w/w) to the culture media containing 2 % glucose (w/v) increased the cytochrome P-450 amount, indicating the induction of cytochrome P-450 production by ethanol action. These results show the importance of establishment of the cultivation conditions to obtain cells with high cytochrome P-450 concentrations, considering that factors such as the culture media, incubation time and strain employed can influence the cytochrome P-450 production by S.cerevisiae.

Abstract Small-scale cultivation methods are a necessity for the development of new biotechnological processes. The most common method for submerged microbial cultivation is a shake flask used with a batch operation protocol. Well plate cultivation formats have also increased their importance, due to the need to utilize high-throughput cultivations for efficient product development. However, batch cultivation is often not the optimal method for obtaining high cell densities and good product quality, due to unlimited microbial growth. The aim of this dissertation was to improve small-scale microbial cultivations for microbial growth and product formation. Hydrolytic enzymes were utilized to relieve nutrient limitation by hydrolysis of proteins in lactic acid bacteria cultures to improve lactic acid production from dairy side products. Hydrolytic enzymes were also utilized in the enzymatic release of glucose from starch to create a fed-batch-like cultivation system applicable on small scale. The wireless sensor system developed was applied in shake flask cultivations to monitor oxygen and pH levels. Enzymatic polymer processing was applicable for small-scale cultivations. Lactic acid production by Lactobacillus salivarius ssp. salicinius was enhanced four-fold when the proteins were hydrolyzed either by proteases or by proteolytic microbes. The fed-batch-mimicking controlled glucose feeding and growth control were obtained by means of the simultaneous enzymatic hydrolysis of starch-polymer during cultivation. Controlled growth, higher cell densities, decreased side product formation and increased amount of soluble protein product were obtained in Escherichia coli cultivations. When this method was applied to the cultivation and recombinant protein production of the methylotrophic yeast Pichia pastoris, higher cell densities and higher amounts of active protein were obtained. The glucose concentration remained low enough to avoid the substrate repression of the alcohol oxidase promoter. The fed-batch method is suitable for high-throughput cultivations since the method can be utilized in well plate formats without external feeding devices. The method can be utilized in the development of new biotechnological products, especially when the production system is sensitive to growth conditions, and growth control is preferred. / Tiivistelmä Pienen mittakaavan mikrobikasvatusmenetelmiä tarvitaan kehitettäessä uusia bioteknologisia prosesseja. Tavallisin menetelmä mikrobien liuoksessa tapahtuvaan kasvatukseen on panostyyppisesti tehtävä sekoituspullokasvatus. Kuoppalevykasvatukset ovat myös tulleet entistä tärkeämmiksi, koska tuotekehityksen tehostamiseksi on tarvetta käyttää high-throughput-menetelmiä. Tavoiteltaessa korkeita mikrobisolutiheyksiä ja tuotteen hyvää laatua, panostyyppinen kasvatus ei ole usein paras vaihtoehto, johtuen mikrobien rajoittamattomasta kasvusta. Tämän työn tarkoituksena oli parantaa mikrobien kasvua ja tuotteen muodostusta pienen mittakaavan kasvatuksissa. Meijeriteollisuuden sivutuotteiden proteiineja pilkottiin entsyymien avulla, jotta maitohappobakteerit pystyivät hyödyntämään proteiinit tehokkaammin ja tuottamaan enemmän maitohappoa. Hydrolyyttisiä entsyymejä hyödynnettiin myös glukoosin vapauttamiseen tärkkelyksestä, jolloin saatiin luotua pieneen mittakaavaan sopiva panossyöttötyyppinen kasvatusmenetelmä. Työn aikana kehitettyä langatonta mittausjärjestelmää hyödynnettiin sekoituspullokasvatuksissa happipitoisuuden ja pH:n seurantaan. Entsymaattinen polymeerien käsittely oli soveltuva menetelmä pienen mittakaavan kasvatuksiin. Maitohapon tuotto Lactobacillus salivarius ssp. salicinius -mikrobilla nelinkertaistui, kun ravinneproteiinit pilkottiin joko proteaasien tai proteolyyttisten mikrobien avulla. Panossyöttömenetelmää muistuttava hallittu glukoosin syöttö ja mikrobin kasvun hallinta saavutettiin pilkkomalla tärkkelystä glukoosiksi kasvatuksen aikana. Escherichia coli kasvatuksissa saavutettiin hallittu solumäärän kasvu, korkeammat solutiheydet, vähentynyt sivutuotteiden muodostus ja suurempi liukoisen tuoteproteiinin määrä. Tätä menetelmää sovellettiin myös vierasproteiinin tuottoon metylotrofisella Pichia pastoris -hiivalla, jolloin saavutettiin korkeammat solutiheydet ja suurempi aktiivisen tuoteproteiinin määrä. Glukoosin määrä kasvatusliuoksessa pysyi riittävän alhaisena, jotta se ei repressoinut hiivan alkoholioksidaasi-promoottoria. Panossyöttömenetelmä on sopiva high-throughput-mikrobikasvatuksiin, koska sitä voidaan käyttää kuoppalevyillä ilman syöttölaitteita. Menetelmää voidaan hyödyntää uusien bioteknisten tuotteiden kehittämisessä erityisesti silloin, kun tuottoisäntä on herkkä kasvuolosuhteiden suhteen ja mikrobin kasvua halutaan hallita.

Control of cultivation conditions in the~live cell imaging extends the possibilities of biological experiments and makes the experimental results more reliable. In order to change the~cultivation conditions in a controlled manner and increase the reproducibility of the experiments, it is necessary to reduce the amount of manual operations and replace them with automated procedures. Therefore, the concept of a new automated culture device (bioreactor) was created. This device controls the exchange of medium in the observation chamber, ensures the circulation and exchange of the atmosphere and controls its composition. The bioreactor is intended for use in the Laboratory of Experimental Biophotonics. This laboratory is equipped with coherence-controlled holographic microscope (CCHM), which uses quantitative phase imaging (QPI) method. Thus, the bioreactor is adapted to the current requirements of this laboratory and optical elements of the bioreactor meet the requirements of the QPI method. This text specifies the cultivation conditions of the living cells and summarizes, how the conditions could be controlled in the live cell microscopy. Next some commercially available culture devices are described and assessed, whether they are convenient for the~use in Laboratory of Experimental Biophotonics. The crucial part of the thesis is the~design, construction and testing of the new bioreactor.