Orientador: Rubens Maciel Filho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-06T23:09:13Z (GMT). No. of bitstreams: 1 Gouveia_VeraLuciaReisde_D.pdf: 3830222 bytes, checksum: 41e2cf7f0901948710226a6037c7ed76 (MD5) Previous issue date: 2006 / Resumo: Os processos biotecnológicos ou Bioprocessos necessitam de maiores cuidados do que a maioria dos processos químicos comuns devido, principalmente, à sensibilidade a variações nos estados do sistema como, por exemplo, temperatura e concentração. O crescente aumento da importância dos Bioprocessos e a maior dificuldade em seu controle e simulação resultaram na procura e utilização de novas ferramentas como a Inteligência Artificial (IA). Inteligência Artificial é uma parte da ciência da computação relacionada ao projeto de sistemas computacionais inteligentes. Nos últimos anos houve um aumento substancial do uso de ferramentas de IA tais como Redes Neurais Artificiais (R.N.A), Sistemas Especialistas e Lógica Fuzzy no controle e simulação de Bioprocessos. Este trabalho teve como objetivo o desenvolvimento de um software que atue como um assistente, auxiliando na simulação de Bioprocessos. O Bio-PSA (BioProcess Simulation Assistant) possui quatro módulos: Módulo 1: Simulação de Bioprocessos, incluindo funcionalidades para simulação de produção de etanol e dextrana, ambos com modelagem normal e híbrida. Módulo 2: Simulação de processos para purificação de proteínas, incluindo a modelagem de processos baseados no modelo CARE (Continuous Affinity Recycle Extraction). ?Módulo 3: Sistema Especialista e uma base de conhecimento que auxilia o usuário no design de processos biotecnológicos. Módulo 4: Software para treinamento de R.N.A / Abstract: The biotechnical processes or Bioprocesses require larger cares than common chemical processes due, mainly, the states of the system variations sensibility, for example, temperature and concentrations. The increasing raise of the Bioprocess importance and the largest difficulty in its control and simulation resulted in the search and use of Artificial Intelligence (AI) tools. Artificial Intelligence is a part of the computer science related to the project of intelligent computational systems. In the last years there was a substantial increase in the use of AI tools such as Artificial Neural Networks (A.N.N), Expert Systems and Fuzzy Logic in the Bioprocess control and simulation. This work had the objective of the development of software that acts as an assistant, aiding in the Bioprocess simulation. The Bio-PSA (BioProcess Simulation Assistant) has four modules: Module 1: Bioprocess simulation, including applications for simulation of ethanol and dextran production, both with normal and hybrid modeling. Module 2: Simulation of proteins purification processes, including processes modeling based in the CARE (Continuous Affinity Recycle Extraction) model. Module 3: Expert System and a knowledge base that aids the user in the Bioprocesses design.?Module 4: Software for A.N.N training / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutor em Engenharia Química

Monoclonal antibodies (MAbs) have an expanding market for use in diagnostic and therapeutic applications. Industrial production of these biopharmaceuticals is usually achieved based on fed-batch cultures of mammalian cells in bioreactors (Chinese hamster ovary (CHO) and Hybridoma cells), which can express different kinds of recombinant proteins. In order to reach high cell densities in these bioreactors, it is necessary to carry out an optimization of their production processes. Hence, macroscopic model equations must be developed to describe cell growth, nutrient consumption and product generation. These models will be very useful for designing the bioprocess, for developing robust controllers and for optimizing its productivity.<p>This thesis presents a new kinetic model of hybridoma cell metabolism in fed batch culture and typical illustration of a systematic methodology for mathematical modelling, parameter estimation and model-based optimization and state estimation of bioprocesses. <p>In the first part, a macroscopic model that takes into account phenomena of overflow metabolism within glycolysis and glutaminolysis is proposed to simulate hybridoma HB-58 cell cultures. The model of central carbon metabolism is reduced to a set of macroscopic reactions. The macroscopic model describes three metabolism states: respiratory metabolism, overflow metabolism and critical metabolism. The model parameters and confidence intervals are obtained via a nonlinear least squares identification. It is validated with experimental data of fed-batch hybridoma cultures and successfully predicts the dynamics of cell growth and death, substrate consumption (glutamine and glucose) and metabolites production (lactate and ammonia). Based on a sensitivity analysis of the model outputs with respect to the parameters, a model reduction is proposed. <p>In the next step, the effort is directed to the maximization of biomass productivity in fed-batch cultures of hybridoma cells based on the overflow metabolism model. Optimal feeding rate, on the one hand, for a single feed stream containing both glucose and glutamine and, on the other hand, for two separate feed streams of glucose and glutamine are determined using a Nelder-Mead simplex optimization algorithm. Two different objective functions (performance criteria) are considered for optimization; the first criterion to be maximized is the biomass productivity obtained at the end of the fed-batch culture, the second criterion to be minimized is the difference between global substrate consumption and the maximum respiratory capacity.<p>The optimal multi exponential feed rate trajectory improves the biomass productivity by 10% as compared to the optimal single exponential feed rate. Moreover, this result is validated by the one obtained with the analytical approach in which glucose and glutamine are fed to the culture so as to control the hybridoma cells at the critical metabolism state, which allows maximizing the biomass productivity. The robustness analysis of optimal feeding profiles obtained with different optimization strategies is considered, first, with respect to parameter uncertainties and, finally, with respect to model structure errors.<p>Finally, the overflow metabolism model is used to develop an extended Kalman filter for online estimation of glucose and glutamine in hybridoma cell fed-batch cultures based on the considered available measurements (biomasses (on-line), lactate and ammonia (on-line or off-line)). The observability conditions are examined, and the performances are analysed with simulations of hybridoma cell fed-batch cultures. Glutamine estimation sensitivity is enforced by minimizing a cost function combining a usual least-squares criterion with a state estimation sensitivity criterion. <p> / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished

Acute liver failure (ALF) is a rare but devastating clinical syndrome with multiple causes and a variable course. The mortality rate is high. Orthotopic liver transplantation is the only therapy of proven survival benefit but the limited supply of donor organs, the rapidity of progression and the variable course of ALF limit its use. A need therefore exists for a method to ‘bridge’ patients, that is, provide temporary support, to either the spontaneous regeneration of the innate liver or transplantation. One possibility includes bio-artificial liver support systems (BALSS). This technology is composed of an extracorporeal circulation system incorporating a bioreactor that contains parenchymal liver cells (hepatocytes) to perform the detoxifying, transforming and synthetic properties of a liver. However, the development of a BALSS holds particular challenges. Despite approximately four decades of research, bio-artificial liver (BAL) technology globally remains in a pre-commercial stage. The University of Pretoria (UP) and the Council for Scientific and Industrial Research (CSIR) have developed a BALSS with novel characteristics. These include a computationally optimized radial-flow primary porcine hepatocyte bioreactor perfused with blood plasma, and a perfluorocarbon oxygen carrier which replaces hemoglobin. There are also novel design properties in the circulation system itself. Demonstrating the metabolic and clinical efficacy of a BAL device requires implementing, in vitro (cell biology), in vivo (animal) and mathematical modeling studies. These studies are a formal necessity but are inherently ‘models’ of the in vivo human clinical circumstance. That is, they are limited by their experimentally controlled configuration/s. In investigating these, this thesis firstly provides a foundation by reviewing the clinical and biological context of ALF and BAL technology, then presents and evaluates particular studies/models that have been implemented over several years in the course of the UP-CSIR BAL project. For each section, thoughts and recommendations regarding future work that will facilitate the development of BAL technology are discussed in detail. The thesis is concluded with an evaluation of success and the consensus-agreed requirement of continued research and innovation in the field. / Thesis (PhD)--University of Pretoria, 2010. / Chemical Engineering / unrestricted

Uma abordagem promissora para a obtenção de coquetéis enzimáticos eficientes para a etapa de hidrólise na produção de etanol de 2ª geração é o enriquecimento em termos de atividades que lhes faltam, mas podem ser obtidas através de sistemas heterólogos. O trabalho teve como objetivo avaliar o sistema toxina -antitoxina (TA) para estabilização plasmidial em E. coli na produção de expansina e endoglucanase recombinantes. Os resultados indicaram que o sistema de expressão com estabilização TA é tão eficaz quanto o dependente de antibiótico para estabilização plasmidial. Além disso, mostrou-se mais eficiente pelo fato de não permitir a sobrevivência de células sem o plasmídeo. Estudos indicaram a quantidade mínima de 0,05mM de IPTG para expressão das proteínas nesta linhagem, cerca de 20 vezes menos que a concentração usualmente aplicada no momento da indução. Sendo assim, o sistema de estabilização plasmidial TA mostrou-se uma ótima ferramenta para o desenvolvimento de uma plataforma alternativa para a produção de proteínas recombinantes. / A promising approach to obtain efficient enzyme cocktails for the hydrolysis step in the production of 2nd generation ethanol is the enrichment in terms of activities that are lacking, but can be obtained by heterologous systems. The study aimed to evaluate the toxina-antitoxin (TA) system for plasmid stabilization in E. coli in the production of recombinant expansin and endoglucanase. The results indicated that the expression system with TA stabilization is as effective as the antibiotic dependent for plasmid stabilization. Moreover, it proved to be more efficient by not allo wing the survival of cells without the plasmid. Studies have indicated the minimum amount of 0.05 mM IPTG for expression of proteins in this strain, about 20 times less than the concentration usually applied for induction. Thus, the plasmid stabilization TA system proved to be a great tool for the development of an alternative platform for producing recombinant proteins.

One of the main interests in commercial methane production is to maximize the gas yield, and it is thus appealing to use material with relative high methane potential. However, such material often results in process instability whereas ammonia inhibition is common. Removal of ammonia through adsorption is a fairly unexplored method in the field of biogas production, and could prove to be cost-effective.The adsorption capacity of pulp fibres from the paper making industry were investigated through batch adsorption experiments. Additionally, the fibres effect on small scale batch digesters in terms of methane production and cellulase activity was explored. Overall, the adsorption capacity of the pulp fibres was low, whereas Kraft hardwood had the highest adsorption capacity in both an aqueous ammonium solution and digester fluid at 11±3 and 60±20 mg g-1, respectively. The initial total ammonium nitrogen concentration had the highest effect on the adsorption capacity with a positive correlation. The pulp fibres seemingly had no effect on the ammonia inhibited anaerobic digestion systems. However, the cellulase activity was higher after day 5 in the anaerobic digestion systems with a high ammonia concentration.In essence, the overall results showed that the adsorption of the fibres was relatively low and most likely not suitable as a material to prevent ammonia inhibition in an AD.

Food waste and fashion pollution are two of the significant global environmental issues throughout the recent past. In this research, it was investigated the feasibility of making a leather-like material from bread waste using biotechnology as the bridging mechanism. The waste bread collected from the supermarkets were used as the substrate to grow filamentous fungi species Rhizopus Delemar and Fusarium Venenatum. Tanning of fungal protein fibres was successfully performed using vegetable tanning, confirmed using FTIR and SEM images. Furthermore, glycerol and a biobased binder treatment was performed for the wet-laid fungal microfibre sheets produced. Overall, three potential materials were able to produce with tensile strengths ranging from 7.74 ± 0.55 MPa to 6.92 ± 0.51 MPa and the elongation% from 16.81 ± 1.61 to 4.82 ± 0.36. The binder treatment enhanced the hydrophobicity even after the glycerol treatment, an added functional advantage for retaining flexibility even after contact with moisture. The fungal functional material produced with bread waste can be tailored successfully into leather substitutes using an environmentally benign procedure.

>Magister Scientiae - MSc / Saccharomyces cerevisiae is an important host in industrial biotechnology. This yeast is the host of choice for the first and second-generation biofuels for ethanol production. Genome modification in S. cerevisiae has been extremely successful largely due to this yeast’s highly efficient homology-directed DNA repair machinery. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing technology has made multi-gene editing in yeast more accessible. In this study, we aimed at targeting the Cas9 to multiple genomic positions for integrating multiple genes at different sites. We have developed two CRISPR-Cas9 systems, based on published one- and two-plasmid systems, for application in S. cerevisiae strains. In this study, these CRISPR-Cas9 systems were used to transform fungal heterologous genes into yeast using the electroporation transformation method. We first utilized the CRISPR systems for targeting the T.r.eg2 gene to single locus chromosomal sites for single copy integration. Subsequently, we then targeted the same gene to repeated sequences in the genome, namely the delta sites, for multi-copy integration. The procedure was repeated with a different gene, T.e.cbh1, integrated into the same sites to ascertain reporter gene specific effects. High integration efficiency was achieved, since all the strains successfully integrated the genes. However, we discovered significant differences in enzyme activities between the two genes when targeted to different loci, as well as varying copy numbers as determined by qPCR. The T.e.cbh1 gene was highly expressed by yeast transformants targeted at the repeated delta sequences used for multi-copy integration, reaching maximum levels of 248 mU/gDCW. The T.r.eg2 gene was highly expressed in yeast transformants targeted to the single locus site on chromosome 12, reaching a maximum of 160U/gDCW, though it was shown that off-target integration likely occurred. We then used the information from these observations to construct a CBP yeast strain containing three cellulase genes: T.r.eg2, T.e.cbh1, and S.f.BGL1. Significant differences in enzyme activities were observed between the three genes, and it was shown that the S.f.BGL1 gene was poorly expressed by the CBP yeast strain, whereas the T.r.eg2 gene was highly expressed. Notably, due to the fact that marker containing plasmids could be cured from these strains, many additional genetic changes can still be made. Overall, our two CRISPR-Cas9 systems were efficient at engineering strains that produce recombinant proteins and can be used in future studies for a variety of applications, including metabolic engineering in S. cerevisiae

Syftet med projektet är att undersöka effekten av att leda över vattenverksslam från Norrvattens vattenverk, Görvälnverket, till Käppalaverkets reningsverk som ett steg i en mer hållbar slamhantering. Detta slamhanterings alternativ jämförs med en framtida lokal slamhantering vid Görvälnverket. Studien undersöker möjligheten att leda vattenverksslam över till Käppalaverket utifrån fyra huvudaspekter, vattenrening, drift, kostnad och miljöpåverkan. En litteraturstudie genomfördes med syfte att utvärdera hur vattenverksslammet kan påverka reningsprocesserna vid Käppalaverket. En Livscykelanalys genomfördes med syfte att utvärdera miljöpåverkan av att leda över vattenverksslam till Käppalaverket. Genomförbarheten utvärderades med hjälp av en multikriterieanalys, där tekniska, miljömässiga och ekonomiska aspekter utvärderades.  Resultat från studien visade att den framtida lokala slamhanteringen är mer fördelaktig från ett tekniskt och ekonomiskt perspektiv, än överledning av vattenverksslammet till Käppalaverket. En nackdel med överledning av vattenverksslam till Käppalaverket är att vattenverksslammet sannolikt kommer påverka avvattningen av avloppsslammet, vilket resulterar i en högre polymerförbrukning och en ökad hydraulisk belastning på centrifuger och rötkammare. Överledningen av vattenverksslam antas däremot inte ha en negativ påverka på kvaliteten av reningen vid Käppalaverket. Att leda vattenverksslam över till Käppalaverket ger en lägre miljöpåverkan med avseende på kemikalieutsläpp till vattenmiljön men en högre miljöpåverkan med avseende på transporter och energiförbrukning.  Överledning av vattenverksslam till Käppalaverket bedöms som genomförbart, men denna studie visar att den framtida lokala slamhanteringen på Görvälnverket är ett mer fördelaktigt alternativ. Om överledning av vattenverksslam till Käppalaverket fortsatt är ett aktuellt alternativ rekommenderas det att en mer djupgående studie utförs, där vattenverksslammet tillsätts till Käppalaverket för att utvärdera dess påverkan. / The aim with this project is to investigate the impact of leading the produced waterworks sludge (WWS) from Norrvattens drinking water treatment plant (DWTP), Görvälnverket, over to Käppalaverkets wastewater treatment plant (WWTP) as a step in a more sustainable sludge management. This alternative is compared to a future sludge management at Görvälnverket. The study, investigating the feasibility of leading the WWS over to Käppalaverket, is based on four main aspects, water treatment, operation, cost, and environmental impact. A literature study was performed to evaluate the effect of WWS on Käppalaverket. A life cycle assessment (LCA) analysis was performed to evaluate the environmental impact of leading the WWS over to Käppalaverket. The feasibility was evaluated using a multi-criteria decision analysis (MCDA), where technical, environmental, and economic aspects were considered. The study shows that the future sludge management is more favourable than leading the WWS over to Käppalaverket from a technical and economic aspect. The main drawback with leading the WWS over to Käppalaverket is that the WWS will likely impact the dewatering of the sewage sludge, resulting in a higher polymer consumption and an increased hydraulic load on centrifuges and digesters. However, the addition of WWS at Käppalaverket is not assumed to negatively impact the quality of the treatment at Käppalaverket. Leading the WWS over to Käppalaverket results in a lower environmental impact regarding chemical emissions but results in a higher environmental impact regarding transportation and energy consumption. Leading the WWS over to Käppalaverket was found to be feasible, although the future sludge management at Görvälnverket was found to be more favourably in this study. A more in depth study on the feasibility of leading the WWS over to Käppalaverkets is required to fully assess this aspect. A trial where the WWS is added to Käppalaverket is recommended to further evaluate the impact of the WWS.

Trä är den bästa förnyelsebara källan för att producera många produkter på grund av dess biokompatibla och biologiskt nedbrytbara natur. Träets biomassa har en motstridig natur mot enzymatisk uppgradering. Det beror på olika orsaker som ligninhalt, acetylhalt i hemicellulosa och cellulosakristallinitet som blockerar enzymets bindningsställe. Denna studie kommer under BioUPGRADE, som är en samarbetsplattform för att skapa högvärdiga och mångsidiga material på ett hållbart sätt med hjälp av biokatalys. Det allmänna syftet med denna studie är att producera holocellulosa och hemicellulosasubstrat från olika träslag och producera, rena och validera galaktosoxidas som en potentiell biokatalysator för träfibermodifiering. Studien undersöker effekten av kemisk perättiksyradelignifiering på två träslag, lövträ av eukalyptus (HW), och barrträ av gran (SW), undersökta vid olika tidsintervall, där PAA framställdes exsitu med ett volymetriskt förhållande på 1:3. Med resultaten från PAA-behandlingen avlägsnades 38,53 % lignin i eukalyptus och 31,80 % i barrved. Hemicellulosautbytet ökade med 47,40 % för eukalyptus och 19,05 % för gran med en ökning av tiden för PAA-behandling. Acetylhalten i hemicellulosan minskade från 2 % till 0,6 % i lövträ och 1,96 % till 0,6 % i barrträ. Cellulosautvinningen efter delignifieringen var nästan 100 %. Galaktosoxidaset producerades i en skakkolv med användning av Pichia pastoris KM71H-stammen. Pichia pastoris KM71H-celler odlades med användning av det buffrade komplexa glycerolmediet (BMGY) och galaktosoxidas uttrycktes med användning av Pichia pastoris KM71H-stammen i buffrat komplex metanolmedium (BMMY). Det uttryckta GaOx-proteinet renades därefter med användning av AKTA-kromatografi med användning av en 5 ml Histrap FF-kolonn. För att bestämma proteinkoncentrationen utfördes bicinchoninsyra (BCA) analys och GaOx som producerades i skakkolvsodling var 286,25 mg/L. Den specifika aktiviteten hos skakkolven som produceras GaOx är 164,24 U/mg. Det kan observeras att PAA-behandling visar sig vara en effektiv metod för delignifiering eftersom cellulosautvinningen är nära 100 % och förlusten av hemicellulosa är relativt låg med det använda volymetriska förhållandet. GaOx som produceras i skakkolvsproduktion visar ett lovande utbyte med en betydande specifik aktivitet mot galaktosen som substrat och kan användas i framtiden för enzymatisk uppgradering av träets biomassa. / Wood is the best renewable source for producing many products due to its biocompatible and biodegradable nature. The wood biomass has a recalcitrance nature towards enzymatic upgrading. It is due to various reasons such as lignin content, acetyl content in hemicellulose, and cellulose crystallinity which blocks the enzyme binding site. This study comes under BioUPGRADE, which is a collaborative platform to create high-value and multipurpose materials sustainably using biocatalysis. The general aim of this study is to produce holocellulose and hemicellulose substrates from different wood species and produce, purify, and validate galactose oxidase as a potential biocatalyst for wood fiber modification. The study investigates the effect of chemical peracetic acid delignification on two wood species, eucalyptus hardwood (HW), and spruce softwood (SW) investigated at different time intervals, where the PAA was prepared ex-situ with a volumetric ratio of 1:3. With the results from the PAA treatment, 38.53% of lignin was removed in eucalyptus and 31.80% in softwood. The hemicellulose yield increased by 47.40% for eucalyptus and 19.05% for spruce with an increase in the time of PAA treatment. The acetyl content of the hemicellulose was reduced from 2% to 0.6% in hardwood and 1.96% to 0.6% in softwood. The cellulose recovery after the delignification was nearly 100%. The galactose oxidase was produced in a shake flask using the Pichia pastoris KM71H strain. Pichia pastoris KM71H cells were cultivated using the buffered complex glycerol media (BMGY) and galactose oxidase was expressed using the Pichia pastoris KM71H strain in Buffered complex methanol media (BMMY). The expressed GaOx protein was subsequently purified using AKTA chromatography using a 5ml Histrap FF column. To determine the protein concentration Bicinchoninic acid (BCA) analysis was performed and the GaOx produced in shake flask cultivation was 286.25 mg/L. The specific activity of the shake flask produced GaOx is 164.24 U/mg. It can be observed that PAA treatment proves to be an efficient method for delignification as the cellulose recovery is near 100% and the loss of hemicellulose is relatively low with the volumetric ratio used. The GaOx produced in shake flask production shows a promising yield with a significant specific activity towards the galactose as substrate and could be used in the future for the enzymatic upgrading of the wood biomass.

I det här projektet extraherades värdefulla komponenter från äpple-pomace som är en restprodukt från cidertillverkning. Restprodukten var tillhandahållen av en svensk cidertillverkare. Extraktionen fokuserades mot pektin och fenolföreningar som uppvisar antioxiderande aktivitet. Extraktionsprocessen designades ur ett miljövänligt perspektiv, inga skadliga kemikalier användes. Fenolära substanser extraherades initialt från pomacen med en vattenlösning innehållande 50% etanol. Därefter studerades kompositionen och den antioxiderande aktiviteten i dessa extrakt. Fokuseringen i projektet låg främst på pektin. Pektin extraherades med subkritiskt vatten vid tre olika pH (3,5 och 7) samt vid två skilda temperaturer (120°C och 140°C) i antingen 5, 10 och 15 minuters sekvenser. Pektinet karakteriserades med avseende på extraktionsutvinningsgrad, sockerkomposition, molekylvikt och antioxiderande aktivitet. Resultaten jämfördes för att undersöka effekten av pH, temperatur och extraktionstid. Gelningsegenskapen hos olika pektin studerades som ett bevis på koncept i ett empiriskt experiment där hög-viskösa vätskor bestående av 5% pektin och 60% sackaros framställdes. Extraherade fenolföreningar har potential att fungera som naturligt utvunna antioxidanter i kosmetiska produkter och pektin kan tänkas användas som reologimodifierare i vattenbaserade formuleringar med låga pH-värden utan behov av kemiska modifieringar. / In this project, apple pomace from a Swedish cider making factory as a by-product was used as a raw material to extract valuable compounds. The extraction was focused on pectin and phenolic compounds with antioxidant activity. For the extraction procedures environmentally friendly processes were chosen without using any harsh chemicals. Phenolic compounds wereinitially extracted from the pomace using 50% aqueous ethanol and then the composition, total phenolic content and antioxidant activity were studied in these extracts. The main focus was on pectins, which were extracted by subcritical water at three different pH conditions (pH 3, 5 and 7) and two different temperatures (120°C and 140°C) in 5-, 10- and 15 minute sequences. Then the pectins were characterized in terms of extraction yield, sugar composition, molecular weight and antioxidant activity and the results were compared in terms of the effect of pH, temperatur eand extraction time. The gelling properties of the different pectins were also studied as a proof of concept in an empirical experiment, where highly viscous liquids were obtained at 5% pectinand 60% sucrose content. In summary, the extracted phenolic compounds have potential to function as naturally derived antioxidants in cosmetics and the pectin may be used as a rheology modifier in water-based formulations of low pH without any additional chemical modifications.