Obtenção e caracterização de linhagem de Escherichia coli adaptada ao glicerol bruto proveniente da síntese de biodiesel por engenharia evolutiva
Autor(a) principal: | |
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Data de Publicação: | 2016 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/8628 |
Resumo: | Biodiesel is a renewable fuel and its production generate raw glycerol (RG) as main byproduct. The use of RG as carbon source in microorganism cultivations poses as an alternative to add value and reduce the environmental impact of this residue. However, RG impurities (salts, esters, alcohol and soap) can inhibit cell growth. Techniques that aims adapting microorganisms to environments containing contaminants by adaptive evolution have been employed to overcome inhibition problems. Adaptation strategies allows imposing a certain selective pressure upon the population, favoring the appearance of mutants and selection of most beneficial mutations, which will make the cell more suited to develop itself in a hostile environment. This work employed Adaptive Evolution methodology to obtain an E. coli K12 strain adapted to RG concentrated by rotary evaporation (RGRota). Cultivations were carried out in plates (E. coli – USP strain) incubated at 37 ºC, as well as shaken flasks (E. coli – UMinho strain), kept at 37 ºC and 300 rpm, involving transfers to defined media gradually enriched with RGRota. Obtained evolved strain as well as the wild-type strain E. coli – UMinho were characterized in cultivations using 2 L, bench-scale bioreactor, equipped with monitoring and control system. During shaken flask experiments, growth was followed by optical density (OD) readings. In bioreactor cultures, samples were withdrawal to analyze cell concentration of the suspension (OD and dry cell weight), concentrations of glycerol, ethanol and organic acids (liquid chromatography), concentration of viable cells (colony forming units counting) and morphology. Cultures characterization were carried out with E. coli – USP in shaken flasks, the values of maximum specific growth rate (μmax) remained between 0.40 e 0.45 h-1 and they showed little influence of strain or media composition. These results suggest that the selected strain did not have differentiated characteristics from the wild-type strain. For E. coli – UMinho, two adaptation strategies were evaluated: successive transfer during exponential growth phase (OD = ~2.5) and during stationary growth phase (OD = ~10). In both cases cells evolved, showing increased μmax values, with more homogeneous populations being observed for adaptation conducted under the first strategy. After 26 days of adaptation, corresponding to 534 generations, an evolved strain, exhibiting μmax of 0.60 h-1 and capable of growing in medium containing 29 g/L of glycerol from RGRota was selected by the methodology of successive transfers in exponential phase. This growth rate was 27.6 % superior to that achieved by the wild-type strain (0.47 h-1). Evolved and wild-type strains were cultivated in bioreactor, containing defined medium prepared with GBRota to have 40 g/L of glycerol. The evolved one maintained μmáx of 0.61 h-1. Acetate formation was observed, with yield of 0.19 g acetate/g glycerol, which caused growth inhibition and limited biomass yield to 0.26 gbiomass/gglycerol. When the wild-type strain was cultivated in bioreactor, exponential growth started after 24 h of lag phase and it presented μmax of 0.28 h-1, biomass yield of 0,39 gbiomass/gglycerol and acetate yield of 0.19 gacetate/gglycerol. The evolved strain obtained, capable of growing in the biodiesel production residue, showed a μmax value similar to the best results reported in the literature for E. coli adaptation in pure glycerol (0.7 h-1), what demonstrates the successful application of the adaptive evolution methodology. Acetate accumulation can be reduced by Genetic Engineering techniques to manipulate metabolic pathways and this will lead to development of an industrial strain which can be employed as a platform of high value products using unrefined glycerol as substrate. |
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Miranda, Letícia PassosZangirolami, Teresa Cristinahttp://lattes.cnpq.br/4546701843297248Horta, Antonio Carlos Lupernihttp://lattes.cnpq.br/5923938048634505http://lattes.cnpq.br/61089027226813896070f3b4-6583-4b08-9523-9f5b4847e0302017-04-19T13:42:20Z2017-04-19T13:42:20Z2016-03-31MIRANDA, Letícia Passos. Obtenção e caracterização de linhagem de Escherichia coli adaptada ao glicerol bruto proveniente da síntese de biodiesel por engenharia evolutiva. 2016. Dissertação (Mestrado em Engenharia Química) – Universidade Federal de São Carlos, São Carlos, 2016. Disponível em: https://repositorio.ufscar.br/handle/ufscar/8628.https://repositorio.ufscar.br/handle/ufscar/8628Biodiesel is a renewable fuel and its production generate raw glycerol (RG) as main byproduct. The use of RG as carbon source in microorganism cultivations poses as an alternative to add value and reduce the environmental impact of this residue. However, RG impurities (salts, esters, alcohol and soap) can inhibit cell growth. Techniques that aims adapting microorganisms to environments containing contaminants by adaptive evolution have been employed to overcome inhibition problems. Adaptation strategies allows imposing a certain selective pressure upon the population, favoring the appearance of mutants and selection of most beneficial mutations, which will make the cell more suited to develop itself in a hostile environment. This work employed Adaptive Evolution methodology to obtain an E. coli K12 strain adapted to RG concentrated by rotary evaporation (RGRota). Cultivations were carried out in plates (E. coli – USP strain) incubated at 37 ºC, as well as shaken flasks (E. coli – UMinho strain), kept at 37 ºC and 300 rpm, involving transfers to defined media gradually enriched with RGRota. Obtained evolved strain as well as the wild-type strain E. coli – UMinho were characterized in cultivations using 2 L, bench-scale bioreactor, equipped with monitoring and control system. During shaken flask experiments, growth was followed by optical density (OD) readings. In bioreactor cultures, samples were withdrawal to analyze cell concentration of the suspension (OD and dry cell weight), concentrations of glycerol, ethanol and organic acids (liquid chromatography), concentration of viable cells (colony forming units counting) and morphology. Cultures characterization were carried out with E. coli – USP in shaken flasks, the values of maximum specific growth rate (μmax) remained between 0.40 e 0.45 h-1 and they showed little influence of strain or media composition. These results suggest that the selected strain did not have differentiated characteristics from the wild-type strain. For E. coli – UMinho, two adaptation strategies were evaluated: successive transfer during exponential growth phase (OD = ~2.5) and during stationary growth phase (OD = ~10). In both cases cells evolved, showing increased μmax values, with more homogeneous populations being observed for adaptation conducted under the first strategy. After 26 days of adaptation, corresponding to 534 generations, an evolved strain, exhibiting μmax of 0.60 h-1 and capable of growing in medium containing 29 g/L of glycerol from RGRota was selected by the methodology of successive transfers in exponential phase. This growth rate was 27.6 % superior to that achieved by the wild-type strain (0.47 h-1). Evolved and wild-type strains were cultivated in bioreactor, containing defined medium prepared with GBRota to have 40 g/L of glycerol. The evolved one maintained μmáx of 0.61 h-1. Acetate formation was observed, with yield of 0.19 g acetate/g glycerol, which caused growth inhibition and limited biomass yield to 0.26 gbiomass/gglycerol. When the wild-type strain was cultivated in bioreactor, exponential growth started after 24 h of lag phase and it presented μmax of 0.28 h-1, biomass yield of 0,39 gbiomass/gglycerol and acetate yield of 0.19 gacetate/gglycerol. The evolved strain obtained, capable of growing in the biodiesel production residue, showed a μmax value similar to the best results reported in the literature for E. coli adaptation in pure glycerol (0.7 h-1), what demonstrates the successful application of the adaptive evolution methodology. Acetate accumulation can be reduced by Genetic Engineering techniques to manipulate metabolic pathways and this will lead to development of an industrial strain which can be employed as a platform of high value products using unrefined glycerol as substrate.O biodiesel é um combustível renovável cuja produção gera o glicerol bruto (GB) como principal subproduto. O aproveitamento de GB como fonte de carbono em cultivos de microrganismos se apresenta como uma alternativa para agregar valor e reduzir o impacto ambiental deste resíduo. Contudo, as impurezas do GB (sais, ésteres, álcool e sabão) podem inibir o crescimento das células. Técnicas que visam adaptar os microrganismos via evolução adaptativa a ambientes contendo contaminantes vêm sendo empregadas para contornar problemas de inibição. As estratégias de adaptação permitem impor uma certa pressão seletiva sobre a população, favorecendo o aparecimento de mutantes e a seleção de mutações benéficas, que tornam a célula mais apta a se desenvolver em um ambiente hostil. O trabalho empregou a metodologia de Evolução Adaptativa para obter uma linhagem de E. coli K12 adaptada ao GB concentrado por rotaevaporação (GBRota). Os cultivos foram realizados tanto em placas (linhagem E. coli – USP) incubadas a 37ºC, como em frascos agitados (linhagem E. coli – UMinho), mantidos a 37ºC e 300 rpm, envolvendo transferências para meios definidos gradualmente enriquecidos com GBRota. A linhagem evoluída obtida assim como a linhagem selvagem E. coli – UMinho foram caracterizadas em cultivos em biorreator de bancada de 2 L, dotado de sistema de monitoramento e controle. Durante os experimentos em frascos agitados, o crescimento foi acompanhado por leitura de densidade ótica (DO). Nos cultivos em biorreator, amostras foram coletadas para análises de concentração celular da suspensão (DO e massa seca), da concentração de glicerol, etanol e ácidos orgânicos (por cromatografia líquida), da concentração de células viáveis (por contagem de unidades formadoras de colônia) e de morfologia. Para os cultivos de caracterização da E. coli – USP realizados em frascos agitados, os valores da velocidade máxima específica de crescimento (max) permaneceram entre 0,40 e 0,45 h-1, sendo pouco influenciados pela linhagem ou pela composição dos meios, sugerindo que a metodologia adotada para adaptação em placa não foi eficiente, já que a linhagem selecionada não possuía características diferenciadas em relação à linhagem selvagem. Para a E. coli – UMinho foram avaliadas duas estratégias de adaptação: transferências sucessivas na fase exponencial do cultivo (DO = ~2,5) e na fase estacionária (DO = ~10). Em ambos os casos, as células evoluíram, apresentando aumento nos valores de max., sendo que populações mais homogêneas foram observadas na adaptação realizada pela primeira estratégia. Após 26 dias de adaptação, correspondendo a 534 gerações, foi selecionada pela metodologia de transferências sucessivas na fase exponencial, uma linhagem evoluída apresentando velocidade máxima específica de 0,60 h-1, resultado superior em 27,6% ao da linhagem selvagem (0,47h-1), capaz de crescer em meio contendo ~30 g/L de glicerol proveniente do GBRota. As linhagens selvagem e evoluída foram cultivadas em biorreator contendo meio preparado com GBRota na concentração de 40 g/L de glicerol. A linhagem evoluída manteve o μmáx de 0,61 h-1. Foi observada formação de acetato, com rendimento de 0,19 gacetato/gglicerol, o que causou inibição do crescimento e limitou o rendimento em biomassa a 0,26 gbiomassa/gglicerol. Enquanto que, para a linhagem selvagem o cultivo em biorreator apresentou uma fase lag de 24 h, um max de 0,28 h-1, rendimento em biomassa de 0,39 gacetato/gglicerol e rendimento em acetato 0,19 gacetato/gglicerol. A linhagem evoluída obtida no presente trabalho, capaz de crescer no resíduo da produção de biodiesel, apresenta max semelhante aos melhores resultados relatados na literatura para adaptação de E. coli em glicerol puro (0,7 h-1), demonstrando o sucesso da aplicação da metodologia de evolução adaptativa. O acúmulo de acetato pode ser amenizado utilizando técnicas de Engenharia Genética para manipulação das vias metabólicas e permitindo o desenvolvimento de uma linhagem industrial que poderá ser empregada como plataforma para obtenção de produtos de alto valor agregado usando o glicerol não refinado como substrato.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)porUniversidade Federal de São CarlosCâmpus São CarlosPrograma de Pós-Graduação em Engenharia Química - PPGEQUFSCarEscherichia coliEvolução adaptativaGlicerol brutoAdaptive evolutionUnrefined glycerolENGENHARIAS::ENGENHARIA QUIMICAObtenção e caracterização de linhagem de Escherichia coli adaptada ao glicerol bruto proveniente da síntese de biodiesel por engenharia evolutivainfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisOnline6006004c81169f-86ab-4df0-8284-9cb6516960a4info:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINALDissLPMoc.pdfDissLPMoc.pdfapplication/pdf5018835https://repositorio.ufscar.br/bitstream/ufscar/8628/1/DissLPMoc.pdf6e118e7b00ba50c95eaca8d0df293f3aMD51LICENSElicense.txtlicense.txttext/plain; charset=utf-81957https://repositorio.ufscar.br/bitstream/ufscar/8628/2/license.txtae0398b6f8b235e40ad82cba6c50031dMD52TEXTDissLPMoc.pdf.txtDissLPMoc.pdf.txtExtracted texttext/plain202120https://repositorio.ufscar.br/bitstream/ufscar/8628/3/DissLPMoc.pdf.txte4ac90c27d546b4f2283d086e9594573MD53THUMBNAILDissLPMoc.pdf.jpgDissLPMoc.pdf.jpgIM Thumbnailimage/jpeg5866https://repositorio.ufscar.br/bitstream/ufscar/8628/4/DissLPMoc.pdf.jpg74c3a0e3f242d6e75d4c4cf6d3f8b851MD54ufscar/86282023-09-18 18:31:09.593oai:repositorio.ufscar.br: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Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:31:09Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.por.fl_str_mv |
Obtenção e caracterização de linhagem de Escherichia coli adaptada ao glicerol bruto proveniente da síntese de biodiesel por engenharia evolutiva |
title |
Obtenção e caracterização de linhagem de Escherichia coli adaptada ao glicerol bruto proveniente da síntese de biodiesel por engenharia evolutiva |
spellingShingle |
Obtenção e caracterização de linhagem de Escherichia coli adaptada ao glicerol bruto proveniente da síntese de biodiesel por engenharia evolutiva Miranda, Letícia Passos Escherichia coli Evolução adaptativa Glicerol bruto Adaptive evolution Unrefined glycerol ENGENHARIAS::ENGENHARIA QUIMICA |
title_short |
Obtenção e caracterização de linhagem de Escherichia coli adaptada ao glicerol bruto proveniente da síntese de biodiesel por engenharia evolutiva |
title_full |
Obtenção e caracterização de linhagem de Escherichia coli adaptada ao glicerol bruto proveniente da síntese de biodiesel por engenharia evolutiva |
title_fullStr |
Obtenção e caracterização de linhagem de Escherichia coli adaptada ao glicerol bruto proveniente da síntese de biodiesel por engenharia evolutiva |
title_full_unstemmed |
Obtenção e caracterização de linhagem de Escherichia coli adaptada ao glicerol bruto proveniente da síntese de biodiesel por engenharia evolutiva |
title_sort |
Obtenção e caracterização de linhagem de Escherichia coli adaptada ao glicerol bruto proveniente da síntese de biodiesel por engenharia evolutiva |
author |
Miranda, Letícia Passos |
author_facet |
Miranda, Letícia Passos |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/6108902722681389 |
dc.contributor.author.fl_str_mv |
Miranda, Letícia Passos |
dc.contributor.advisor1.fl_str_mv |
Zangirolami, Teresa Cristina |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/4546701843297248 |
dc.contributor.advisor-co1.fl_str_mv |
Horta, Antonio Carlos Luperni |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://lattes.cnpq.br/5923938048634505 |
dc.contributor.authorID.fl_str_mv |
6070f3b4-6583-4b08-9523-9f5b4847e030 |
contributor_str_mv |
Zangirolami, Teresa Cristina Horta, Antonio Carlos Luperni |
dc.subject.lat.fl_str_mv |
Escherichia coli |
topic |
Escherichia coli Evolução adaptativa Glicerol bruto Adaptive evolution Unrefined glycerol ENGENHARIAS::ENGENHARIA QUIMICA |
dc.subject.por.fl_str_mv |
Evolução adaptativa Glicerol bruto |
dc.subject.eng.fl_str_mv |
Adaptive evolution Unrefined glycerol |
dc.subject.cnpq.fl_str_mv |
ENGENHARIAS::ENGENHARIA QUIMICA |
description |
Biodiesel is a renewable fuel and its production generate raw glycerol (RG) as main byproduct. The use of RG as carbon source in microorganism cultivations poses as an alternative to add value and reduce the environmental impact of this residue. However, RG impurities (salts, esters, alcohol and soap) can inhibit cell growth. Techniques that aims adapting microorganisms to environments containing contaminants by adaptive evolution have been employed to overcome inhibition problems. Adaptation strategies allows imposing a certain selective pressure upon the population, favoring the appearance of mutants and selection of most beneficial mutations, which will make the cell more suited to develop itself in a hostile environment. This work employed Adaptive Evolution methodology to obtain an E. coli K12 strain adapted to RG concentrated by rotary evaporation (RGRota). Cultivations were carried out in plates (E. coli – USP strain) incubated at 37 ºC, as well as shaken flasks (E. coli – UMinho strain), kept at 37 ºC and 300 rpm, involving transfers to defined media gradually enriched with RGRota. Obtained evolved strain as well as the wild-type strain E. coli – UMinho were characterized in cultivations using 2 L, bench-scale bioreactor, equipped with monitoring and control system. During shaken flask experiments, growth was followed by optical density (OD) readings. In bioreactor cultures, samples were withdrawal to analyze cell concentration of the suspension (OD and dry cell weight), concentrations of glycerol, ethanol and organic acids (liquid chromatography), concentration of viable cells (colony forming units counting) and morphology. Cultures characterization were carried out with E. coli – USP in shaken flasks, the values of maximum specific growth rate (μmax) remained between 0.40 e 0.45 h-1 and they showed little influence of strain or media composition. These results suggest that the selected strain did not have differentiated characteristics from the wild-type strain. For E. coli – UMinho, two adaptation strategies were evaluated: successive transfer during exponential growth phase (OD = ~2.5) and during stationary growth phase (OD = ~10). In both cases cells evolved, showing increased μmax values, with more homogeneous populations being observed for adaptation conducted under the first strategy. After 26 days of adaptation, corresponding to 534 generations, an evolved strain, exhibiting μmax of 0.60 h-1 and capable of growing in medium containing 29 g/L of glycerol from RGRota was selected by the methodology of successive transfers in exponential phase. This growth rate was 27.6 % superior to that achieved by the wild-type strain (0.47 h-1). Evolved and wild-type strains were cultivated in bioreactor, containing defined medium prepared with GBRota to have 40 g/L of glycerol. The evolved one maintained μmáx of 0.61 h-1. Acetate formation was observed, with yield of 0.19 g acetate/g glycerol, which caused growth inhibition and limited biomass yield to 0.26 gbiomass/gglycerol. When the wild-type strain was cultivated in bioreactor, exponential growth started after 24 h of lag phase and it presented μmax of 0.28 h-1, biomass yield of 0,39 gbiomass/gglycerol and acetate yield of 0.19 gacetate/gglycerol. The evolved strain obtained, capable of growing in the biodiesel production residue, showed a μmax value similar to the best results reported in the literature for E. coli adaptation in pure glycerol (0.7 h-1), what demonstrates the successful application of the adaptive evolution methodology. Acetate accumulation can be reduced by Genetic Engineering techniques to manipulate metabolic pathways and this will lead to development of an industrial strain which can be employed as a platform of high value products using unrefined glycerol as substrate. |
publishDate |
2016 |
dc.date.issued.fl_str_mv |
2016-03-31 |
dc.date.accessioned.fl_str_mv |
2017-04-19T13:42:20Z |
dc.date.available.fl_str_mv |
2017-04-19T13:42:20Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
MIRANDA, Letícia Passos. Obtenção e caracterização de linhagem de Escherichia coli adaptada ao glicerol bruto proveniente da síntese de biodiesel por engenharia evolutiva. 2016. Dissertação (Mestrado em Engenharia Química) – Universidade Federal de São Carlos, São Carlos, 2016. Disponível em: https://repositorio.ufscar.br/handle/ufscar/8628. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/8628 |
identifier_str_mv |
MIRANDA, Letícia Passos. Obtenção e caracterização de linhagem de Escherichia coli adaptada ao glicerol bruto proveniente da síntese de biodiesel por engenharia evolutiva. 2016. Dissertação (Mestrado em Engenharia Química) – Universidade Federal de São Carlos, São Carlos, 2016. Disponível em: https://repositorio.ufscar.br/handle/ufscar/8628. |
url |
https://repositorio.ufscar.br/handle/ufscar/8628 |
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openAccess |
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Universidade Federal de São Carlos Câmpus São Carlos |
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Programa de Pós-Graduação em Engenharia Química - PPGEQ |
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UFSCar |
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Universidade Federal de São Carlos Câmpus São Carlos |
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