Benzaldehyde (BZA), a biologically derived high-value molecule used in the flavour and fragrance industry for its characteristic almond-like aroma, has also found use in nutraceutical, pharmaceutical, cosmetics, agrochemical, and dye applications. Although, nature-identical BZA is most commonly produced by chemical synthesis, biologically derived BZA, whether by plant material extraction or via microbial biocatalysts, commands much higher prices. The bioproduction of high value molecules has often been characterized by low titers as results of substrate and product inhibition. The current work examined a variety of process strategies and the implementation of a solid-liquid bioreactor partitioning system with continuous integrated pervaporation to enhance the bioproduction of BZA using Pichia pastoris. Previous work on two-phase partitioning bioreactors (TPPBs) for the biotransformation of BZA using Pichia pastoris has had limitations due to long fermentation times and unutilized substrate in the immiscible polymer phase, contributing to complications for product purification. To reduce fermentation times, a mixed methanol/glycerol feeding strategy was employed and reduced the time required for high-density fermentation by 3.5 fold over previous studies. Additionally, because BZA and not the substrate benzyl alcohol (BA) had been found to be significantly inhibitory to the biotransformation reaction, a polymer selection strategy based on the ratio of partition coefficients (PCs) for the two target molecules was implemented. Using the polymer Kraton D1102K, with a PC ratio of 14.9 (BZA:BA), generated a 3.4 fold increase in BZA produced (14.4 g vs. 4.2 g) relative to single phase operation at more than double the volumetric productivity (97 mg L-1 h-1 vs. 41 mg L-1 h-1). This work also confirmed that the solute(s) of interest were taken up by polymers via absorption, not adsorption. BZA and BA cell growth inhibition experiments showed that these compounds are toxic to cells and it was their accumulation rather than low enzyme levels or energy (ATP) depletion that caused a reduction in the biotransformation rate. For this reason, the final strategy employed to enhance the bioproduction of benzaldehyde involved in situ product removal by pervaporation using polymer (Hytrel 3078) fabricated into tubing by DuPont, Canada. This aspect was initiated by first characterizing the custom-fabricated tubing in terms BZA and BA fluxes. The tubing was then integrated into an in situ pervaporation biotransformation and was shown to be effective at continuous product separation, using 87.4% less polymer by mass in comparison to polymer beads in conventional TPPB operation, and improved overall volumetric productivity by 214% (245.9 mg L-1 h-1 vs. 115.0 mg L-1 h-1) over previous work producing BZA. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-09-28 17:41:45.458

β-Casein is a major milk protein, which is synthesized in mammary alveolar secretory epithelial cells (MECs) upon the stimulation of lactogenic hormones, mainly prolactin and glucocorticoids (HP). Previous studies revealed that the proximal promoter (-258 bp to +7 bp) of the β-casein gene is sufficient for induction of the promoter activity by HP. This proximal region contains the binding sites for the signal transducer and activator of transcription 5 (STAT5), glucocorticoid receptor (GR), and octamer transcription factors (Oct). STAT5 and GR are essential downstream mediators of prolactin and glucocorticoid signaling, respectively. This study investigated the functions of Oct-1 and Oct-2 in HP induction of β-casein gene expression. By transiently transfection experiment, we showed that individual overexpression of Oct-1 and Oct-2 further enhanced HP-induced β-casein promoter activity, respectively, while Oct-1 and Oct-2 knockdown significantly inhibited the HP-induced β-casein promoter activity, respectively. HP rapidly induced the binding of both Oct-1 and Oct-2 to the β-casein promoter, and this induction was not mediated by either increasing their expression or inducing their translocation to the nucleus. In MECs, Oct-2 was found to physically interact with Oct-1 regardless of HP treatment. However, HP induced physical interactions of Oct-1 or Oct-2 with both STAT5 and GR. Although the interaction between Oct-1 and Oct-2 did not synergistically stimulate HP-induced β-casein gene promoter activity, the synergistic effect was observed for the interactions of Oct-1 or Oct-2 with STAT5 and GR. The interactions of Oct-1 with STAT5 and GR enhanced or stabilized the binding of STAT5 and GR to the promoter. Abolishing the interaction between Oct-1 and STAT5 significantly reduced the hormonal induction of β-casein gene transcription. Thus, our study indicates that HP activate β-casein gene expression by inducing the physical interactions of Oct-1 and Oct-2 with STAT5 and GR in mouse MECs. There is a high and increasing demand for insulin because of the rapid increase in diabetes incidence worldwide. However, the current manufacturing capacities can barely meet the increasing global demand for insulin, and the cost of insulin production keeps rising. The mammary glands of dairy animals have been regarded as ideal bioreactors for mass production of therapeutically important human proteins. We tested the feasibility of producing human proinsulin in the milk of transgenic mice. In this study, four lines of transgenic mice were generated to harbor the human insulin gene driven by the goat β-casein gene promoter. The recombinant human proinsulin was detected in the milk by Western blotting and enzyme-linked immunosorbent assay. The highest expression level of human proinsulin was as high as 8.1 μg/µl in milk of transgenic mice at mid-lactation. The expression of the transgene was only detected in the mammary gland during lactation. The transgene expression profile throughout lactation resembled the milk yield curve, with higher expression level at middle lactation and lower expression level at early and late lactation. The blood glucose and insulin levels and major milk compositions of transgenic mice were not changed. The mature insulin derived from the milk proinsulin retained biological activity. Thus, our study indicates that it is practical to produce high levels of human proinsulin in the milk of dairy animals, such as dairy cattle and goat.

Unlike thin tissues (e.g., skin) which has been successfully grown, growing thick tissues (e.g., bone and muscle) still exhibit certain limitations due to lack of nutrients (e.g., glucose and oxygen) feeding on cells in extracapillary space (ECS) region, or also known as scaffold in an in vitro static culture. The transport of glucose and oxygen into the cells is depended solely on diffusion process which results in a condition where the cells are deprived of adequate glucose and oxygen supply. This condition is termed as hypoxia and leads to premature cell death. Hollow fibre membrane bioreactors (HFMBs) which operate under perfusive cell culture conditions, have been attempted to reduce the diffusion limitation problem. However, direct sampling of glucose and oxygen is almost impossible; hence noninvasive methods (e.g., mathematical models) have been developed in the past. These models have defined that the glucose diffusivity in cell culture medium (CCM) is similar to the diffusivity in water; thus, they do not represent precisely the nutrient transport processes occurring inside the HFMB. In this research, we define glucose as our nutrient specie due to its limited published information with regard to its diffusivity values, especially one that corresponds to cell/tissue engineering (TE) experiments. A series of well-defined diffusion experiments are carried out with TE materials of varying pore size and shapes imbibed in water and CCM, namely, cellulose nitrate (CN) membrane, polyvinylidene fluoride (PVDF) membrane, poly(L-lactide) (PLLA) scaffold, poly(caprolactone) (PCL) scaffold and collagen scaffold. A diffusion cell is constructed to study the diffusion of glucose across these materials. The glucose diffusion across cell-free membranes and scaffolds is investigated first where pore size distribution, porosity and tortuosity are determined and correlated to the effective diffusivity. As expected, the effective diffusivity increases correspondingly with the pore size of the materials. We also observe that the effective glucose diffusivity through the pores of these materials in CCM is smaller than in water. Next, we seeded human osteoblast cells (HOSTE85) on the scaffolds for a culture period of up to 3 weeks. Similar to the first series of the diffusion experiments, we have attempted to determine the effective glucose diffusivity through the pores of the scaffolds where cells have grown at 37°C. The results show that cell growth changes the morphological structure of the scaffolds, reducing the effective pore space which leads to reduced effective diffusivity. In addition, the self-diffusion of glucose in CCM and water has also been determined using a diaphragm cell method (DCM). The results have shown that the glucose diffusivity in CCM has significantly reduced in comparison to the water diffusivity which is due to the larger dynamic viscosity of CCM. The presence of other components and difference in fluid properties of CCM may also contribute to the decrease. We finally employ our experimentally deduced effective diffusivity and self-diffusivity values into a mathematical model based on the Krogh cylinder assumption. The glucose concentration is predicted to be the lowest near the bioreactor outlet, or in the scaffold region, hence this region becomes a location of interest. The governing transport equations are non-dimensionalised and solved numerically. The results shown offer an insight into pointing out the important parameters that should be considered when one wishes to develop and optimise the HFMB design.

Si le procédé de traitement des eaux usées urbaines par bioréacteur à membranes est en plein essor depuis quelques années, il ne reste pas moins plusieurs verrous technologiques qui ralentissent l'expansion de sa commercialisation. Ce travail de thèse s'attache à apporter des éléments de réponse sur quelques uns des freins à sa progression. Le premier réside dans la détermination d'un âge de boues adapté permettant une épuration maximale de l'azote et de la matière organique, par une biomasse active où le colmatage est limité et la production de boues faible. Le choix s'est porté sur 50 jours qui rempli l'ensemble de ces critères. Une autre barrière se situe dans le manque de données permettant l'extrapolation des informations obtenues en laboratoire à celles engrangées sur site. Une des étapes clés est de déterminer les conséquences du changement d'effluent (synthétique ou réel) sur les performances et le colmatage du procédé. L'utilisation d'une méthode de fluorescence récente a permis la détermination de l'implication des protéines et substances humiques-like extraits des polymères extracellulaires solubles dans le colmatage. / For several years, membrane bioreactor for urban wastewater treatment has been booming. Nevertheless some issues still slow down its expansion. This work tries to answer some of the brakes to its progression. First one concerns the choice of the suitable sludge adge which guarantee maximal removal rates of ammonium and organic matter, by an active biomasse where fouling is limited and slude production low. 50 days is fulling all the criteria. An other concern is about the lack of data allowing extrapolation from laboratory to wastewater treatment plant. One key step is to detremine the impact of influent nature (synthetic or real) on process results and fouling. Use of recent fluorescence method allows determining the implication of proteins and humic substances-like from soluble extra polymeric substances on fouling.

Master of Science / Department of Civil Engineering / Alok Bhandari / In the upper Midwest, subsurface tile drainage water is a major contributor of nitrate (NO[subscript]3–N) coming from fertilizers and animal manure. Movement of NO[subscript]3-N through tile drainage into streams is a major concern as it can cause eutrophication and hypoxia conditions, as in the Gulf of Mexico. Denitrifying bioreactors is one of the pollution control strategies to treat contaminated tile drainage water. These bioreactors require four conditions which are: 1) organic carbon source, 2) anaerobic conditions, 3) denitrifying bacteria and 4) influent NO[subscript]3-N. This research focuses on investigating fate of veterinary antibiotics in woodchips commonly used in in-situ reactors. Tylosin (TYL) and sulfamethazine (SMZ) are two veterinary antibiotics which are most commonly used in the United States and can be found in tile water after manure is land applied. Partition coefficients of TYL and SMZ on wood were determined by sorption experiments using fresh woodchips and woodchips from an in situ reactor. It was concluded that the woodchips were an effective means to sorb the veterinary antibiotics leached into the tile water after application of animal manure. Linear partition coefficients were calculated and phase distribution relationships were established for both the chemicals. The fresh woodchips gave inconclusive data but predictions could be made by the information determined in the experiments using woodchips from a ten year old woodchip bioreactor. Desorption was also studied and the likelihood of desorption was predicted using the Apparent Hysteresis Index. Overall, it was found that the old woodchips allowed for quick sorption of both antibiotics. It was also found that SMZ had reversible sorption on old woodchips. Thus, it was concluded that the woodchip bioreactor would not be effective for removal of veterinary antibiotics from tile drainage. More research is required for the fate of TYL and to confirm the conclusion.

Si les risques liés à la présence de résidus médicamenteux dans l'environnement sont encore méconnus, les premiers cas avérés et l'introduction récente de trois médicaments (2013) sur une liste de surveillance de l'UE imposent d'ores et déjà le développement de procédés de traitement capables d'éliminer cette pollution spécifique. C'est dans ce contexte que le traitement des effluents d'un service d'oncologie par un bioréacteur à membranes a été évalué dans cette thèse. Les effluents collectés sont caractérisés par une importante variabilité de la charge polluante et des concentrations médicamenteuses très élevées, parfois supérieures à 1 mg.L-1. Ces conditions n'ont pas favorisé le développement continu de la biomasse épuratrice. Il a toutefois été démontré que la toxicité de ces effluents n'est pas proportionnelle à la charge appliquée puisqu'une charge massique supérieure à 0.20 kgDCO.kgMVS-1.j-1 permet la croissance de la biomasse. Le colmatage des membranes a permis une rétention significative des médicaments sélectionnés. L'élimination des médicaments sélectionnés a par ailleurs été systématiquement améliorée par les boues acclimatées avec notamment le développement de capacités de biotransformation sur des molécules parfois uniquement éliminées par sorption dans les stations d'épuration. / The risks concerning the presence of pharmaceutical residues into the environment are still unknown. However, the first confirmed cases and the recent introduction of three drugs (2013) on a surveillance list of EU already require the development of processes able to remove this specific pollution. It is against that background that oncological ward wastewater treatment by a membrane bioreactor was investigated in this thesis. These effluents are characterized by a very variable charge and high pharmaceutical concentrations, sometimes above 1 mg.L-1. These conditions did not favor the continuous development of the biomass. However, it could be demonstrated that the toxicity of these effluents is not related to the applied charge since a food to microorganisms ratio above 0.20 kgCOD.kgMLVSS-1.d-1 allows biomass growth. Membrane fouling played a major role in the significant retention of the investigated drugs. In comparison to unacclimated activated sludge from WWTP pharmaceutical removal was systematically enhanced by the acclimated biomass with the development of biotransformation possibilities.

The second-generation bioethanol production in which lignocellulosic material is used as feedstock faces some difficulties. Lignocellulosic materials have to be pretreated prior to fermentation. In the pretreatment stages several inhibitory compounds, which can negatively affect the metabolic and physiologic activity of the microorganism used, Saccharomyces cerevisiae, are released. Moreover, wild strains of Saccharomyces cerevisiae cannot co-utilize the hexose and pentose saccharides present in the lignocellulosic substrate. In this study, reverse membrane bioreactor (rMBR) was applied to address the difficulties faced in the secondgeneration ethanol production. Semi-synthetic medium and pretreated wheat straw slurry containing different level of glucose, xylose and inhibitor concentrations were fermented in rMBR using genetically-modified xylose-consuming S. cerevisiae. The diffusion rate of different substrates and metabolites during fermentation were measured and analyzed. The results showed that the application of rMBR facilitated simultaneous utilization of hexose and pentose sugars and enhanced the cell tolerance of the inhibitor present in the medium.

O tratamento de resíduos sólidos orgânicos, como lodos de estação de tratamento de esgotos e a fração orgânica de resíduos sólidos urbanos, são desafios atuais da engenharia sanitária e ambiental. Os processos biológicos são os mais apropriados para o tratamento desses resíduos. Acordos recentes como o Protocolo de Kyoto e os mecanismos de desenvolvimento limpo (MDL) estão contribuindo para o crescimento da digestão anaeróbia de resíduos sólidos orgânicos em todo o mundo. Nesta pesquisa foi investigado o efeito da adição de lodo anaeróbio ao lixiviado de aterro sanitário empregado como inóculo de reator anaeróbio híbrido sólido-líquido tratando a fração orgânica de resíduos sólidos urbanos. Foi verificado que a adição de lodo melhorou a digestão anaeróbia, acelerando a degradação de ácidos graxos voláteis, antecipando a geração de biogás, aumentando a composição percentual de metano e promovendo maior variabilidade e presença de microrganismos. A adição de lodo também aumentou a eficiência de conversão de sólidos totais e sólidos totais voláteis e sólidos totais fixos / Treatment of organic solid wastes like wastewater treatment plant sludges and organic fraction of municipal solid wastes are current issues in environmental engineering. Biological processes are more appropriate to treat these wastes. Recent trends like Kyoto protocol and clean development mechanisms (CDM) are improving anaerobic digestion of organic solid wastes. It was investigated the effect of adding anaerobic sludge to bioreactor landfill leachate applied like seed in hybrid anaerobic solid-liquid bioreactor treating organic fraction of municipal solid wastes. It was verified that sludge addition improved anaerobic digestion, accelerating volatile fatty acids degradation, anticipating biogas generation, increasing methane percentile composition and promoting more variability and presence of microorganisms. Sludge addition also increased total solids, total volatile solids and total fixed solids conversion

Para produção de biofármacos protéicos, como a eritropoietina recombinante humana (EPOrh), são necessárias alterações pós-traducionais adequadas que garantam a sua especificidade e atividade biológica. Essas características são obtidas apenas em biorretores baseados em células eucarióticas, como as da glândula mamária. Sistemas baseados nesse tipo celular, tanto in vivo quanto in vitro, já são utilizados para produção estratégica e viável de proteínas recombinantes biologicamente ativas. Assim, tanto o estabelecimento de novas linhagens de células mamárias que apresentem boa expressão protéica quanto o desenvolvimento de sistemas in vivo que utilizem a estrutura da glândula mamária para essa produção de proteínas recombinantes são de grande valia. O presente trabalho teve como objetivo comparar dois métodos de estabelecimento de uma cultura de células de glândula mamárias ovinas, enzimático e não enzimático, e verificar sua capacidade de expressão das proteínas do leite β-lactoglobulina, α-caseína, β-caseína e κ-caseína mediante o tratamento com SFB (soro fetal bovino) ou SOL (soro de ovelha lactante), na presença ou não de Matrigel. Para isso, foi realizado um experimento in vitro, no qual foi estabelecido o cultivo celular até a passagem 12 (P12) de duas linhagens celulares: digerida (LD) e não digerida (LND). Para a LD na P12 foi observado apenas um tipo celular, o qual era positivo para a marcação com vimentina. Essa linhagem apresentou expressão gênica de β-caseína e β-lactoglobulina apenas quando tratada com meio de cultivo acrescido de SFB, sendo a expressão inferior (P=0,001) ao grupo da LND submetido ao mesmo tratamento. Já a LND, quando tratada com meio adicionado com SFB expressou κ-caseína além da β-caseína e β-lactoglobulina. A troca do SFB do meio de cultivo por SOL aumentou a expressão gênica de β-lactoglobulina (P=0,001) para ambas linhagens. Foi realizada a curva de crescimento para LD e LND na P12 com o meio de cultivo acrescido com SFB ou SOL. Para a LND observou-se o efeito do meio na velocidade de crescimento celular, sendo que foi maior para o grupo tratado com SFB (P<0,05). Para a LD, não ocorreu o efeito do meio na velocidade de crescimento celular (P>0,05), não sendo observada diferença com a LND tratada com SOL (P>0,05). A LND apresentou marcação positiva para a presença de vimentina e citoqueratina. Este trabalho visou, ainda, estabelecer um sistema de produção da EPOrh no leite de ovelhas não transgênicas pela técnica de infusão intra-mamária in vivo de dois plasmídeos diferentes e verificar a secreção qualitativa desta proteína por Western-blotting. Assim, foi feito um experimento in vivo no qual glândulas mamárias de ovelhas foram transfectadas com dois plasmídeos diferentes: ALAC (n=2), BGL (n=2) e controle negativo (n=2). Após a infusão dos plasmídeos, foi realizada a eletroporação de cada teto (3 choques de 500 volts com a duração de 15ms cada, sendo realizada a inversão da polaridade). Os animais foram ordenhados durante 20 dias após a transfecção, porém não foi possível detectar a presença de EPOrh nas amostras de leite analisadas. O limiar de detecção do teste utilizado foi de 67,5pg de EPOrh (Eritromax®) em leite controle negativo de ovelha. Concluindo, foi possível estabelecer o cultivo in vitro das LD e LND com capacidade de expressar proteínas do leite, sendo a expressão da β-lactoglobulina aumentada pelo tratamento com SOL. Ambas as linhagens apresentaram marcação positiva para vimentina, mas apenas LND para citoqueratina. Ainda, para o experimento in vivo, não foi possível detectar a expressão de EPOrh no leite das ovelhas transfectadas com os plasmídeos ALAC e BGL. / Some post-translational modifications are necessary for the production of biopharmaceutical proteins, such as recombinant human erythropoietin (rhEPO), with a good specific action and a high biological activity. These modifications are obtained only by bioreactors based on eukaryotic cell as mammary cells. Bioreactors, in vivo or in vitro, with this kind of cell have been used for a viable and strategic production of biologically active recombinant proteins. For this reason, the establishment of a new line of mammary cells with high milk protein expression and the development of systems for production of recombinant proteins by the mammary gland in vivo are essential studies. One of the main objectives of this study was to compare two methods, enzymatic and non-enzymatic, to establish ovine mammary cells culture and verify their gene expression of milk proteins such as β-lactoglobulin, α-casein, β-casein and κ-casein with different treatments: LOS (lactating ovine serum) or FBS (fetal bovine serum) added to the culture medium, in the presence or absence of Matrigel®. In this manner, an in vitro study was performed and the culture of two lines were established, digested (DL) and non-digested (NDL), of ovine mammary cell until the passage 12 (P12). In DL was observed just one cellular type that was positive for staining with vimentin. This cell line expressed β-lactoglobulin and β-casein genes with the FBS treatment and without Matrigel. The gene expression was lower (P=0,001) when compared to the NDL under the same conditions of culture. Then, the NDL expressed β-lactoglobulin, β-casein and κ-casein genes when treated with FBS without Matrigel. The treatment with LOS in the culture medium increased the gene expression of β-lactoglobulin for both cell lines. The growth curve was determined with both cell lines in P12 with FBS or LOS treatment. For the NDL, the type of medium had effect on the cell growth speed and was highest with the FBS treatment (P<0,05). However, the medium did not have effect on growth speed of LD (P>0,05) and no difference was observed at the NDL treated with LOS (P>0,05). The NDL was positive for staining with vimentin and cytokeratin. The second main objective of this study was to establish an in vivo system for the production of rhEPO in milk of non-transgenic ewes by the intra-mammary infusion of two different plasmids and verify the qualitative milk secretion of this protein by western-blotting. In this way, in the in vivo experiment ovine mammary glands were transfected with two different plasmids: ALAC (n=2), BGL (n=2) and negative control (n=2). Each half udder was filled with plasmid solution and three 3 electric pulses of 500 volts were applied for 15ms each, followed by another three pulses with reversed polarity. The three animals were milked for 20 days after transfection, nevertheless it was not possible to identify rhEPO in any milk sample. The test threshold to identify rhEPO (Eritromax®) in milk from a negative control animal was 67,5pg. In conclusion, the in vitro culture of NDL and DL was established up to the P12 with expression of milk protein and the LOS treatment increased the expression of β-lactoglobulin. The two cell lines culture were positive for staining of vimentina but only NDL was positive for cytokeratin. In the in vivo experiment, rhEPO secretion was not detected in the milk from ewes transfected with ALAC and BGL plasmids.

As doenças cardiovasculares (DCVs) são um grupo de desordens do coração e dos vasos sanguíneos capazes de causar ataques cardíacos e acidentes vasculares cerebrais, eventos geralmente agudos oriundos principalmente de bloqueios que impedem o sangue de fluir para o coração ou o cérebro. Proteases fibrinolíticas são agentes que degradam a fibrina, principal componente de trombo sanguíneo, capazes de evitar e tratar DCVs. Fungos filamentosos têm se mostrado uma boa alternativa para a produção de enzimas fibrinolíticas, dentre esses os fungos da divisão dos zigomicetos. A cepa UCP 1262 do zigomiceto Mucor subtilissimus foi estudada com o objetivo geral de produzir protease com atividade fibrinolítica com base em trabalhos publicados em fermentação em estado sólido (FES) e cultivo submerso com a mesma cepa. Foram realizados cultivos submersos desta cepa em frascos agitados e em 2 diferentes biorreatores, com determinação da cinética de crescimento, consumo de glicose e produção de enzima. O meio utilizado foi o MS-2, com farelo de trigo como fonte de nitrogênio e glicose como fonte preferencial de carbono. A FES com a mesma fonte de nitrogênio e o cultivo submerso em diferentes condições de taxa de inóculo, pH e agitação deste estudo foram comparados, assim como duas metodologias diferentes para dosagem de concentração de biomassa, gravimetria e ergosterol. As maiores concentrações de atividade fibrinolítica e produtividade volumétrica obtidas foram de, respectivamente, 12,0 U/mL e 0,22 U/(mL.h), 92% e 89% menores, respectivamente, do que o valor reportado em FES. O valor de ?Xmax foi calculado com base no perfil de biomassa, glicose e produção de CO2 e não apresentou correlação com a produção da enzima. A maior simplicidade operacional e os dados de rendimento obtidos indicam que a FES é uma alternativa melhor para a produção dessa enzima com esta cepa. O zigomiceto Mucor subtilissimus demonstrou ser muito difícil de ser cultivado em sistemas submersos com o meio MS-2, em especial pela sua morfologia e aderência ao biorreator. / Cardiovascular diseases (CVDs) are a group of heart and blood vessels disorders capable of causing heart attacks and strokes, usually acute events caused mainly by blockages that prevent blood from flowing to the heart and brain. Fibrinolytic proteases are agents that degrade fibrin, the major component of blood thrombus. Filamentous fungi were shown to be a good alternative for the production of fibrinolytic enzymes, among them fungi of the zygomycetes division. The UCP 1262 strain of the zygomycete Mucor subtilissimus was studied with the general objective of producing protease with fibrinolytic activity based on published works on solid state fermentation (SSF) and submerged culture with the same strain. Submerged cultures of this strain were carried out in shaken flasks and in 2 different bioreactors, with determination of growth kinetics, glucose consumption and enzyme production. The medium used was MS-2, with wheat bran as the nitrogen source and glucose as the preferred carbon source. The SSF with the same nitrogen source and the submerged culture under different inoculum ratios, pH and agitation conditions of this study were compared, as well as two different methodologies for biomass, gravimetry and ergosterol dosage. The highest concentration of fibrinolytic activity and volumetric productivity obtained were, respectively, 12.0 U/mL and 0.22 U/(mL.h), 92% and 89% lower, respectively, than the value reported in SSF. The value of ?Xmax was calculated based on the biomass concentration, glucose and CO2 production profile and showed no correlation with the enzyme production. The greater operational simplicity and yield data obtained indicate that SSF is a better alternative for the production of this enzyme with this strain. The zygomycete Mucor subtilissimus showed to be very difficult to cultivate in submerged culture with the MS-2 medium, especially for its morphology and adherence to the bioreactor.