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Metabolic engineering of Zymomonas mobilis for improved production of ethanol from lignocellulosesAgrawal, Manoj 27 February 2012 (has links)
Ethanol from lignocellulosic biomass is a promising alternative to rapidly depleting oil reserves. However, natural recalcitrance of lignocelluloses to biological and chemical treatments presents major engineering challenges in designing an ethanol conversion process. Current methods for pretreatment and hydrolysis of lignocelluloses generate a mixture of pentose (C5) and hexose (C6) sugars, and several microbial growth inhibitors such as acetic acid and phenolic compounds. Hence, an efficient ethanol production process requires a fermenting microorganism not only capable of converting mixed sugars to ethanol with high yield and productivity, but also having high tolerance to inhibitors. Although recombinant bacteria and yeast strains have been developed, ethanol yield and productivity from C5 sugars in the presence of inhibitors remain low and need to be further improved for a commercial ethanol production.
The overarching objective of this work is to transform Zymomonas mobilis into an efficient whole-cell biocatalyst for ethanol production from lignocelluloses. Z. mobilis, a natural ethanologen, is ideal for this application but xylose (a C5 sugar) is not its 'natural' substrate. Back in 1995, researches at National Renewable Energy Laboratory (NREL) had managed to overcome this obstacle by metabolically engineering Z. mobilis to utilize xylose. However, even after more than a decade of research, xylose fermentation by Z. mobilis is still inefficient compared to that of glucose. For example, volumetric productivity of ethanol from xylose fermentation is 3- to 4- fold lower than that from glucose fermentation. Further reduction or complete inhibition of xylose fermentation occurs under adverse conditions. Also, high concentrations of xylose do not get metabolized completely. Thus, improvement in xylose fermentation by Z. mobilis is required.
In this work, xylose fermentation in a metabolically engineered Z. mobilis was markedly improved by applying the technique of adaptive mutation. The adapted strain was able to grow on 10% (w/v) xylose and rapidly ferment xylose to ethanol within 2 days and retained high ethanol yield. Similarly, in mixed glucose-xylose fermentation, the strain produced a total of 9% (w/v) ethanol from two doses of 5% glucose and 5% xylose (or a total of 10% glucose and 10% xylose). Investigation was done to identify the molecular basis for efficient biocatalysis. An altered xylitol metabolism with reduced xylitol formation, increased xylitol tolerance and higher xylose isomerase activity were found to contribute towards improvement in xylose fermentation. Lower xylitol production in adapted strain was due to a single mutation in ZMO0976 gene, which drastically lowered the reductase activity of ZMO0976 protein. ZMO0976 was characterized as a novel aldo-keto reductase capable of reducing xylose, xylulose, benzaldehyde, furfural, 5-hydroxymethyl furfural, and acetaldehyde, but not glucose or fructose. It exhibited nearly 150-times higher affinity with benzaldehyde than xylose. Knockout of ZMO0976 was found to facilitate the establishment of xylose fermentation in Z. mobilis ZM4.
Equipped with molecular level understanding of the biocatalytic process and insight into Z. mobilis central carbon metabolism, further genetic engineering of Z. mobilis was undertaken to improve the fermentation of sugars and lignocellulosic hydrolysates. These efforts culminated in construction of a strain capable of fermenting glucose-xylose mixture in presence of high concentration of acetic acid and another strain with a partially operational EMP pathway.
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Supply-demand analysis of anaerobic free-energy metabolism in Zymomonas mobilisCrous, Christiaan 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Fermentation in Zymomonas mobilis has been described as a catabolic highway, with
50 % of soluble protein comprising glycolytic and fermentative enzymes. In conjunction
with one of the fastest observed fermentations, the conversion of glucose to ethanol forms
one of the least efficient energy extractions found in nature. The low energy yield of
fermentation in Z. mobilis is a result of the usage of the Entner-Doudoroff glycolytic
pathway, which has half the energy yield per mol substrate compared to the well known
Embden-Meyerhof-Parnas glycolytic pathway.
The work presented in this thesis forms part of a larger project to compare glycolytic
regulation in different micro-organisms (i.e., Z. mobilis, Escherichia coli, Saccharomyces
cerevisiae and Lactococcus lactis). These organisms were chosen based on their usage of
different glycolytic mechanisms. By using supply-demand analysis for quantifying
glycolytic regulation as well as similar experimental conditions (e.g. using non-growing
cell cultures), we can compare the regulatory behaviour of mechanistically distinct freeenergy
supplies.
The aim of this thesis was to quantify the importance of anaerobic free-energy generation
for the regulation of the Entner-Doudoroff glycolytic pathway in Z. mobilis. We used
metabolic control analysis (MCA) and supply-demand analysis to realize this goal. The
central message of MCA is that when a metabolic parameter (e.g., a conserved metabolic
moiety) is deemed important for affecting a particular steady-state variable (i.e.,
fermentation flux), its effect on the steady state variable should be tested. An extension to
MCA, supply-demand analysis, provides a quantitative framework for analyzing the
regulatory importance of cellular commodities such as anaerobic free-energy. This is
done through comparing the elasticities of anaerobic free-energy supply and demand,
which yields the degree to which the respective reaction blocks control the flux through
anaerobic free-energy metabolism, as well as determine the cellular free-energy state
(ATP/ADP ratio). The regulation of anaerobic free-energy metabolism in Z. mobilis was investigated with
an experimental approach. The key features of our experimental setup were the use of
NMR spectroscopy for detecting metabolites, as well as employing non-growing
conditions for supply-demand experiments. With NMR spectroscopy metabolites could
be detected in real time without using invasive sampling techniques; the use of nongrowing
conditions further simplified the analysis by enabling us to correlate
fermentative behaviour exclusively with the anaerobic free-energy state.
Fermentation of glucose was investigated in the wild type Z. mobilis, a recombinant
containing a non-expressing plasmid, or expressing plasmids for over-expressing the
glucose facilitator (TCDB 2.A.1.1.4) or glucose-6-phosphate dehydrogenase (EC
1.1.1.49). In addition, ATP demand in the non-expressing recombinant and wild type was
perturbed by titrating with the uncoupler acetic acid. Our results show that the anaerobic
free-energy demand, the glucose facilitator and glucose-6-phospate dehydrogenase all
control the flux of ethanol production in Z. mobilis. The Entner-Doudoroff glycolytic
supply activity was found to be sensitive to changes in the ratios of ATP/ADP (elasticity
varied between –0.31 and –0.49) and NTP/NDP (elasticity varied between –0.31 and –
0.50). / AFRIKAANSE OPSOMMING: Fermentasie in Zymomonas mobilis word beskryf as ‘n kataboliese snelweg, waar
glikolitiese en fermentatiewe ensieme 50% van totale oplosbare proteïene in die sel
uitmaak. Hoewel dié fermentasie een van die vinnigstes is wat tot op hede waargeneem
is, is die omskakeling van glukose na etanol een van die mees ondoeltreffende energieekstraksies
in die natuur. Dié lae energie-opbrengs, soos waarneembaar in fermentasie in
Zymomonas mobilis, kan toegeskryf word aan die Entner-Doudoroff metaboliese pad.
Hierdie metaboliese pad lewer slegs die helfte van die energie-opbrengs per mol substraat
vergeleke met die meer bekende Embden-Meyerhof-Parnas glikolitiese pad.
Die navorsing in hierdie tesis is deel van ‘n omvattende projek wat poog om die
regulering van glikolise in verskillende mikro-organismes (Z. mobilis, Escherichia coli,
Saccharomyces cerevisiae en Lactococcus lactis) te vergelyk. Dié organismes is gekies
op grond van die uiteenlopende glikolitiese meganismes waarvan hulle gebruik maak.
Ten einde die reguleringsgedrag van meganisties verskillende vry-energie produksieweë
m.b.v. vraag-aanbod analise te vergelyk, moet glikolitiese regulering eers onder eenderse
eksperimentele kondisies (b.v. nie-groeiende selkulture) gekwantifiseer kan word.
Die hoofdoel van hierdie tesis was om die belang van anaerobiese vry-energie produksie
vir die regulering van die Entner-Doudoroff glikolitiese pad in Z. mobilis te kwantifiseer.
Hiervoor is van Metaboliese kontrole-analise (MKA) en vraag-aanbodanalise (‘n
uitbreiding van MKA) gebruik gemaak. MKA is ‘n tegniek waarmee die effek wat ‘n
metaboliese parameter (soos metaboliese deel-konservering) op ‘n spesifieke bestendige
toestand-veranderlike (soos fermentasiefluksie) het, gekwantifiseer kan word. Vraagaanbodanalise
daarenteen, bied ‘n kwantitatiewe raamwerk waardeur die regulatoriese
belang van sellulêre kommoditeite (byvoorbeeld anaerobiese vry-energie) geanaliseer kan
word. Tydens laasgenoemde proses word die elastisiteit van die anaerobiese vry-energie
aanbod en die elastisiteit van die vraag vergelyk. Op hierdie manier kan die mate van
beheer wat die onderskeie reaksieblokkie oor die fluksie deur anaerobiese vry-energie
metaboliese paaie, sowel as oor die sellulêre vry-energie toestand (ATP/ADP
verhouding), bepaal word. In hierdie werk is die regulering van anaerobiese vry-energie metabolisme in Z. mobilis
ondersoek deur van ‘n eksperimentele benadering gebruik te maak. Die
sleuteleienskappe van dié benadering was om kernmagnetiese-resonansiespektroskopie
(KMR spektroskopie) te gebruik om metabolietkonsentrasies te meet, en om van niegroeiende
kondisies gebruik te maak vir die vraag-aanbod eksperimente.
Metabolietkonsenstrasies kon aaneenlopend bepaal word sonder die gebruik van
monsternemingstegnieke wat die reaksie sou kon beïnvloed. Eksterne invloede op die
fermentasiegedrag kon ook uitgesluit word deur van nie-groeiende kondisies gebruik te
maak, sodat die waargenome fermentasiegedrag uitsluitelik aan die anaerobiese vryenergie
toestand toegeskryf kan word.
Glukose fermentasie was ondersoek in wilde tipe Z. mobilis, en in drie rekombinante wat
onderskeidelik ‘n glukose fasiliteerder ooruitdrukkingsplasmied (TCDB 2.A.1.1.4), ‘n
glukose-6-fosfaat dehidrogenase ooruitdrukkingsplasmied (EC 1.1.1.49), en ‘n nieuitdrukkingsplasmied
bevat het. Die ATP vraag in die wilde tipe en die nieuitdrukkingsrekombinant
is geperturbeer deur titrasies met asynsuur as ontkoppelaar.
Die resultate toon dan die anaerobiese vry-energievraag, sowel as die glukose
fasiliteerder en glukose-6-fosfaat dehidrogenase, die fluksie van etanolproduksie in Z.
mobilis beheer. Die Entner-Doudoroff glikolitiese produksie-aktiwiteit was sensitief vir
veranderinge in die ATP/ADP verhouding (elastisiteite was tussen -0.31 en -0.49) en die
NTP/NDP verhouding (elastisiteite was tussen -0.31 en -0.50).
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Towards a kinetic model of the Entner-Doudoroff pathway in Zymomonas mobilisVan Staden, Charles Theo 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Metabolic networks of cellular systems are complex, in that there are numerous components with
multiple non-linear interactions. To understand how these networks work they are often split into
manageable pieces and studied individually. However, an individual part is unable to account for
the complex properties of systems. In order to study these interactions the eld of systems biology
has developed. Systems biology makes use of computers to construct models as a method to
describe aspects of living systems. Using cellular pathways, kinetic models of metabolic pathways
can be constructed and used as a tool to study the biological systems and provide a quantitative
description. This thesis describes the quantitative analysis of a bacterium using a systems biology
approach.
Zymomonas mobilis is a rod shaped, Gram-negative, non-mobile facultative anaerobe and has
one of the fastest observed fermentations, yet least energy e cient extractions found in nature.
Furthermore it is the only known micro-organism to use the Entner-Doudoro (2-keto-3-deoxy-6-
phosphogluconate) pathway anaerobically. The low energy yield of fermentation in Z. mobilis is
a result of the usage of the Entner-Doudoro glycolytic pathway, which has half the energy yield
per mol substrate compared to the well known Embden-Meyerhof-Parnas glycolytic pathway.
The work presented in this thesis forms part of a larger project to compare glycolytic regulation
in di erent micro-organisms Z. mobilis, Escherichia coli, Saccharomyces cerevisiae and Lactococcus
lactis. These organisms were chosen based on their usage of di erent glycolytic mechanisms.
Kinetic models are suitable tools to draw a comparison between these organisms. The emphasis
here is on the construction of a kinetic model of the Entner-Doudoroff glycolytic pathway as it
occurs in Z. mobilis. The aim of this thesis was to characterise as many of the Entner-Doudoro pathway enzymes
as possible, under standard conditions. This was done using enzyme assays, to obtain the kinetic
parameters of each of the enzymes. Microtitre plate assays were used to characterise most of the
enzymes of the Entner-Doudoro pathway. However, not all characterisations could be done using
plate assay methods, as some intermediates were not commercially available to perform coupled
assays. Nuclear magnetic resonance (NMR) spectroscopy was used to characterise these enzymes.
These experimentally obtained parameters were then incorporated in a mathematical framework.
Time simulations on the initial model were unable to reach a steady-state, with a build up of
metabolic intermediates. A secondary model was constructed (using calculated maximal activities)
which allowed us to identify discrepancies in the initial model. This showed that the experimentally
determined maximal activities of three enzymes in lower glycolysis were unrealistically low,
which might be due to protein denaturation by sonication.
A nal model was constructed which incorporated a correction factor for these three enzymes.
The models' predicted output (steady-state concentrations and
ux) was compared to that of either literature or experimentally determined values, as a method to validate the model. The
model output compared well to literature values. The constructed and partially validated kinetic
model was then used as an analytical tool to identify points of control and regulation of glycolysis
in Z. mobilis.
The model presented in this work was also compared to published models. Our model relies
much less on literature obtained values, and uses kinetic parameters experimentally determined
under the same conditions. The parameters of the published models were obtained from the
literature and in many instances, the assay conditions for these parameters were set-up to yield
the maximum activity under non-physiological conditions. Furthermore, the number of excluded or
assumed parameters is much less in our model. However, introduction of a milder, more predictable
extraction technique for preparing cell lysates, should be considered for future work, to obtain the
parameters that was not determined during this study. The published models do include reactions
not included in our model (e.g ATP metabolism), which should be considered for inclusion, as we
strive to construct a detailed kinetic model of glycolysis in Z. mobilis in the future. / AFRIKAANSE OPSOMMING: Sellul^ere metaboliese netwerke is komplekse stelsels, omdat hulle bestaan uit talle komponente met
verskeie nie-lineêre interaksies. Om die funksionering van hierdie netwerke te verstaan, word hulle
dikwels in hanteerbare stukke verdeel en individueel bestudeer. 'n Enkele komponent is egter nie in
staat om die komplekse eienskappe van sulke stelsels te verklaar nie. Die veld van sisteembiologie
het ontwikkel met die doel om sulke stelsels te bestudeer. Sisteembiologie maak gebruik van
rekenaarmodelle as 'n metode om aspekte van lewende sisteme te beskryf. Kinetiese modelle van
metaboliese paaie word gebou en gebruik as gereedskap om die biologiese stelsels te bestudeer en
'n kwantitatiewe beskrywing te bekom. Hierdie tesis beskryf die kwantitatiewe ontleding van 'n
bakterie deur middel van 'n sisteembiologiese benadering.
Zymomonas Mobilis is 'n staafvormige, Gram-negatiewe, nie-mobiele fakultatiewe ana erobe,
en het een van die vinnigste waargenome fermentasies, maar met die minste energie-doeltre ende
ekstraksie wat in die natuur aangetref word. Verder is dit die enigste bekende mikro-organisme wat
die Entner-Doudoro (2-keto-3-dioksi-6-fosfoglukonaat) pad ana erobies gebruik. Die lae-energieopbrengs
van fermentasie in Z. mobilis is 'n gevolg van die gebruik van die Entner-Doudoro
metaboliese pad, wat die helfte van die energie-opbrengs per mol substraat lewer, in vergelyking
met die bekende Embden-Meyerhof-Parnas pad.
Die werk wat in hierdie tesis aangebied word, vorm deel van 'n groter projek om glikolitiese
regulering in verskillende mikro-organismes te vergelyk, naamlik Z. mobilis, Escherichia coli, Sac-
charomyces en Lactococcus lactis. Hierdie organismes is gekies op grond van hul gebruik van
verskillende glikolitiese meganismes. Kinetiese modellering is 'n handige metode om 'n vergelyking
tussen hierdie organismes te trek. Hierdie werk fokus op die bou van 'n kinetiese model van
die Entner-Doudoro glikolitiese metaboliese pad soos dit in Z. mobilis voorkom. Die doel van hierdie tesis was om so veel moontlik van die Entner-Doudoro ensieme onder
standaard-toestande te karakteriseer. Die kinetiese parameters van elk van die ensieme is met
behulp van ensimatiese essai's bepaal. Vir die meeste essai's is 96-put mikrotiterplate gebruik,
maar nie al die karakteriserings kon met behulp van hierdie metode gedoen word nie, omdat
sommige intermediate nie kommersieel beskikbaar was om gekoppelde essai's mee uit te voer nie.
Kernmagnetiese resonansie (KMR) spektroskopie is gebruik om hierdie ensieme te karakteriseer.
Die eksperimenteel bepaalde parameters is opgeneem in 'n wiskundige raamwerk. Tydsimulasies
op die aanvanklike model was nie in staat om 'n bestendige toestand te bereik nie, omdat
metaboliete opgebou het. 'n Sekond^ere model is gebou (met behulp van berekende maksimale
aktiwiteite) wat ons toegelaat om teenstrydighede in die aanvanklike model te identi seer. Dit het
getoon dat die eksperimenteel bepaalde maksimale aktiwiteite van drie ensieme in die laer gedeelte
van glikolise te laag was, waarskynlik as gevolg van prote en denaturering tydens die ultrasoniese
disintegrasieproses. 'n Finale model is gebou waarin 'n korreksiefaktor vir hierdie drie ensieme opgeneem is. Die
modelle se voorspelde uitset (bestendige toestand konsentrasies en
uksie) is vergelyk met waardes
uit die literatuur of wat ons self bepaal het, as 'n metode om die model te valideer. Die model uitset
was in goeie ooreenstemming met hierdie waardes. Die gedeeltelik gevalideerde kinetiese model is
voorts gebruik as 'n analitiese instrument om beheer en regulering van glikolise in Z. mobilis te
ondersoek.
Die model wat in hierdie werk ontwikkel is, is ook vergelyk met die vorige gepubliseerde modelle.
Ons model berus baie minder op waardes uit die wetenskaplike literatuur, en maak gebruik
van parameters wat eksperimenteel bepaal is, onder identiese toestande. Die parameters van die
gepubliseerde modelle is meesal verkry uit die literatuur, en in baie gevalle was die eksperimentele
kondisies vir hierdie analises opgestel om die maksimale aktiwiteit te lewer onder nie- siologiese
toestande. Verder bevat ons model minder parameters wat of uitgesluit is of wie se waardes aangeneem
moes word. In toekomstige werk sal daar egter klem gel^e moet word op 'n minder wisselvallige
ekstraksietegniek vir die verkryging van selekstrakte, om sodoende parameters te identi seer wat
nie in hierdie werk bepaal kon word nie.
Die gepubliseerde modelle sluit ook reaksies in wat nie ingesluit is in ons model nie (bv. ATP
metabolisme). Hierdie sou in ag geneem moet word vir insluiting in 'n toekomstige uitgebreide
model, om daarna te streef om 'n gedetailleerde kinetiese model van glikolise in Z. mobilis te bou.
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Produção de bioetanol de segunda geração pelo consórcio Zymomonas mobilis CCT4494 e Candida tropicallis em resíduos de uvas Isabel e Bordô /Furlan, Anelisa Doretto Freitas. January 2015 (has links)
Orientador: Crispin Humberto Garcia Cruz / Banca: Vanildo Luiz Del Bianchi / Banca: Marcos Lucca Jr. / Resumo: A produção de etanol combustível a partir de resíduos lignocelulósicos, como tecnologia emergente, conhecida como bioetanol de segunda geração, está sendo bastante pesquisada em virtude de uma preocupação com o desenvolvimento de fontes energéticas renováveis e mais limpas. Neste contexto as indústrias vinícolas têm destaque, já que o subproduto gerado por elas, bagaço e engaço, por exemplo, é de lenta decomposição. Ao mesmo tempo em que há buscas por novos substratos, há também por micro-organismos capazes de fermentar monossacarídeos presentes nestes resíduos para produzir bioetanol, e também que estes consigam fermentar em consórcio sem que haja competição entre eles para o melhor aproveitamento destas fontes de carbono. Neste trabalho foram realizadas fermentações pela bactéria Zymomonas mobilis CCT 4494 e pela levedura Candida tropicalis em hidrolisados ácidos dos resíduos de uvas das espécies Isabel e Bordô, para isto, foi utilizada a concentração de ácido sulfúrico de 1,5% com aquecimento em autoclave a 121 ºC e 1 kgf/cm2. Foram realizadas também, fermentações utilizando meios semi-sintéticos com glicose e xilose (50 g/L) como substratos para a Z. mobilis e C. tropicalis, respectivamente. Nas fermentações realizadas pela bactéria Z. mobilis, utilizando tanto a mistura (50% bagaço e 50% engaço) dos resíduos quanto apenas o engaço não houve detecção de etanol por cromatografia gasosa. Com o meio semi-sintético, em 8 horas de fermentação, foram produzidos 17,0 g/L de etanol. Durante as fermentações efetuadas pela levedura C. tropicalis, houve produção de etanol de 1,04 g/L e 5,89 g/L em 6 horas de fermentação utilizando a mistura dos resíduos e o engaço, respectivamente e, 18,56 g/L em 8 horas em meio semi-sintético. No consórcio dos micro-organismos, a produção de etanol, utilizando primeiramente a mistura e depois o engaço foi de 0,72 g/L e 1,15 g/L, ambas em 8 horas... / Abstract: The production of fuel ethanol from lignocellulosic residues, as an emerging technology, known as second-generation bioethanol is being extensive research because of a concern with the development of renewable and cleaner energy sources. In this context, the wine industry has highlighted, since the by-product generated by them, bagasse and stems, for example, is the slow decomposition. While there search for new substrates, there are also micro-organisms capable of fermenting monosaccharides present in these residues to produce bioethanol, and also that they are able to ferment in a consortium without competition between them for better use of these carbon sources . In this work were carried out fermentations by Zymomonas mobilis CCT 4494 bacteria and the yeast Candida tropicalis in hydrolyzed acid waste grape species Isabel and Bordô, for this, we used the sulfuric ... / Mestre
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Produção de bioetanol utilizando cascas de banana, maracujá e coco verde por co-fermentação de Zymomonas mobilis e Pachysolen tannophilus /Ferreira, Juliana. January 2017 (has links)
Orientador: Crispin Humberto Garcia Cruz / Banca: Vanildo Luiz Del Bianchi / Banca: Mauricio Boscolo / Banca: Sandra Regina Ceccato Antonini / Banca: Harvey Alexander Villa Vélez / Resumo: As crises no fornecimento de petróleo, a possibilidade de sua escassez, instabilidade dos preços e, principalmente, os efeitos negativos ao meio ambiente, aumentaram, nos últimos anos, o interesse pela procura de fontes alternativas para produção de energia. A possibilidade de produzir etanol a partir de resíduos lignocelulósicos é um grande atrativo para os pesquisadores, pois reduziria a necessidade em aumentar as áreas plantadas para incremento da produção, a competição direta com a produção de alimentos e o custo de matéria-prima. Diante do exposto, o objetivo deste trabalho foi estudar a produção de etanol por culturas individuais e co-culturas de Zymomonas mobilis e Pachysolen tannophilus usando cascas de banana, maracujá e coco verde hidrolisadas. O pré-tratamento ácido, seguido de hidrólise enzimática das cascas de banana, maracujá e coco, promoveram a liberação de 74,1, 87,2 e 13,4 g L-1 de açúcares totais, respectivamente. Foram avaliadas três concentrações de substrato (5, 10 e 15%) e três pH iniciais dos meios de fermentação (4,5; 5,0 e 5,5). Nas fermentações com a bactéria Z. mobilis, com os três resíduos, foi verificado que o substrato mais concentrado não contribuiu para um aumento significativo da produção de etanol. Além disso, a maior concentração do produto foi de 2,9 g L-1, obtido nas fermentações de cascas de banana hidrolisadas com concentração de 5%. Já nas fermentações com a levedura P. tannophilus, as maiores produções de etanol foram determinadas... / Abstract: Crises in oil supply, the possibility of their scarcity, price instability and, especially, the negative effects to the environment have increased the interest in alternative sources for energy production, in recent years. The possibility of producing ethanol from lignocellulosic residues is a great attractive to the researchers, because it would reduce the need for increasing the planted areas to increase production, the direct competition with food production and the high costs of raw materials. In this context, the objective of this work was to study the ethanol production by individual cultures and co-cultures of Zymomonas mobilis and Pachysolen tannophilus using banana, passion fruit and coconut peels hydrolysate. The acid pretreatment, followed by enzymatic hydrolysis of banana, passion fruit and coconut peels promoted the liberation of 74.1, 87.2 and 13.4 g L-1 of total sugars, respectively. Three substrate concentrations (5, 10 and 15%) and three initial pH of fermentation media (4.5; 5.0 and 5.5) were evaluated. In the fermentations with Z. mobilis bacteria, using the three residues, it was found that the most concentrated substrate did not contribute to a significant increase in ethanol production. In addition, the highest product concentration was 2.9 g L-1, obtained in hydrolysed banana peels fermentations at 5% concentration. In the fermentations with yeast P. tannophilus, the highest ethanol yields were determined using passion fruit peel hydrolysates at 10% solids concentration, providing the ethanol production of 10 g L-1 . And the association of Z. mobilis bacteria and P. tannophilus yeast, in the co-culture process, showed potential for the lignocellulosic hydrolysates fermentation. The inoculation system that showed the best results was the one in which the bacterium was inoculated at the beginning of the process, and after 7 h of fermentation, the yeast ... / Doutor
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Utilização de diferentes substratos para a produção de etanol, levana e sorbitol por Zymomonas mobilisErnandes, Fernanda Maria Pagane Guereschi [UNESP] 03 July 2009 (has links) (PDF)
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ernandes_fmpg_dr_sjrp.pdf: 1963066 bytes, checksum: 42dcae2ecfe9dfb358788c6490a13653 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O principal produto da fermentação de açúcares por Zymomonas mobilis é o etanol quando glicose e frutose são utilizadas como fontes de carbono. Entretanto, quando sacarose é empregada na fermentação, o rendimento do etanol diminui devido à formação de subprodutos como levana, sorbitol, acetaldeído, ácido acético, pequenas quantidades de alguns álcoois superiores e fenol. A utilização de produtos agroindustriais, como o caldo e melaço de cana-deaçúcar, é uma alternativa para reduzir o custo final dos produtos de fermentação devido à disponibilidade de aquisição e composição química desses substratos. Este trabalho teve como objetivo utilizar substratos alternativos e otimizar as condições de fermentação para a produção de etanol, levana e sorbitol por Zymomonas mobilis CCT 4494. Também foi considerado o efeito da variação do substrato e de sais minerais adicionados nos meios de produção (sintético, caldo e melaços de cana-de-açúcar). Para a obtenção dos produtos de fermentação, foi aplicada a metodologia de superfície de resposta, seguindo um planejamento fatorial do tipo 27-2, de acordo com o modelo proposto por Box e Hunter, onde as variáveis independentes estudadas foram: pH inicial do meio de cultivo, temperatura de incubação, concentração do substrato e efeito da adição de KCl, K2SO4, MgSO4, CaCl2. Durante a realização das fermentações foi observado que a bactéria Zymomonas mobilis CCT 4494 se adaptou nos meios de fermentação contendo altas concentrações de sacarose e suportou a variação do pH e da temperatura de fermentação. O aumento da concentração da fonte de carbono favoreceu a formação dos produtos levana e etanol, entretanto, não houve produção de sorbitol. O meio sintético proporcionou maior rendimento de levana e etanol, enquanto que, os meios alternativos caldo e melaços de cana-de-açúcar... / The main product from fermentation of sugars by Zymomonas mobilis is ethanol when glucose and fructose are used as carbon sources. However, when sucrose is used in the fermentation medium, ethanol yield decreases due to the formation of by-products such as levan, sorbitol, acetaldehyde, acetic acid, small amounts of some superior alcohols and phenol. The use of agro industrial by products, such as sugarcane juice and molasses, is an alternative to reduce the final cost of fermentation products due to the constant availability and to the chemical composition of these by substrates. This study had the aim of using alternative substrates and of optimizing fermentation conditions for the production of ethanol, levan and sorbitol by Zymomonas mobilis CCT 4494. The effect of variation of substrate and mineral salts added to the production media (synthetic, sugarcane juice and molasses) was also considered. To obtain the fermentation products, response surface methodology was employed, following a 27-2 factorial planning, according to the model proposed by Box and Hunter, where the independent variables studied were: initial medium pH, incubation temperature, substrate concentration and effect of the addition of KCl, K2SO4, MgSO4, CaCl2. During the fermentations, it was noted that the bacteria Zymomonas mobilis CCT 4494 well adapted in the media containing high concentrations of sucrose and tolerated pH and temperature variations. The increase of carbon source concentration favored the formation of levan and ethanol, however, there was no sorbitol production. The synthetic medium offered higher levan and ethanol yield, whereas alternative media sugarcane juice and molasses, favored cellular growth. Among the independent variables analyzed with the best medium (synthetic) for biosynthesis of the biopolymer and ethanol, the ones that significantly (p<0.05) affected were KCl, K2SO4, CaCl2... (Complete abstract click electronic access below)
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Cell Permeabilization Using Supercritical Carbon DioxideNg, Matthew January 2001 (has links)
Supercritical fluids have unique properties which may make them ideal as reaction media for biotransformation or extractive solvents. Supercritical fluids are ideal for reducing diffusivity limitations over conventional fluids. Depending on the polarity of the fluid, a supercritical fluid can be similar to conventional organic solvents, but with few of the environmental drawbacks. The use of supercritical fluids in enzymatic research has the advantage of removing mass transport limitations so that they can act as a suitable solvent. In this study, four permeabilization techniques were compared: control, toluene, supercritical carbon dioxide, and freeze/thaw cycles. The model cell systems studied were Z. mobilis and E. coli. The cells were analyzed for lipid profiles, recovery of proteins and enzymatic activity. The use of supercritical carbon dioxide may not be the most effective of the treatments based on total protein or enzyme recovery since the greatest protein and enzyme recovery was with the freeze/thaw treatment. However, it can be selective in removing cofactors from Z. mobilis enabling sorbitol production and minimizing side reactions. In this application, supercritical carbon dioxide does show an advantage over the freeze/thaw treatment. Aspects of the mechanism of permeabilization were investigated based on the lipid profiles of the cells, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The SEM and AFM show changes of the cell surface morphology which indicate that the treatments affect the cellular surface. The use of supercritical carbon dioxide as a reaction medium was investigated. Minute quantities of sorbitol were produced when Z. mobilis and sugars were placed in a supercritical carbon dioxide environment over a period of 24 hours.
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Modeling a Reversed β-oxidation Cycle Into the Genome Scale Model of Zymomonas mobilisDash, Satyakam 16 September 2013 (has links)
This study proposes simulations which present optimized methods for producing fatty acids, fatty alcohols and alkanes using Zymomonas mobilis bacterium by the energy efficient β-oxidation reversal pathway, an eco-friendly alternative to the present petroleum based processes. Zymomonas has advantages of higher carbon intake, higher ethanol tolerance and higher ethanol production efficiency than other organisms. I have improved an earlier Zymomonas genome scale model and used Constraint Based Reconstruction and Analysis (COBRA), a linear optimization based computational tool in Matlab, and to perform flux balance analysis (FBA) based simulations. FBA accounts for formation, consumption, accumulation and removal rate or flux of each metabolite. The results present solution spaces of cell growth rate and product formation rate, which trend with products and their carbon chain length. I have analyzed these solution space trends gaining insight into the Zymomonas’ metabolism, enabling efficient product formation and opening a way for future improvement.
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Cell Permeabilization Using Supercritical Carbon DioxideNg, Matthew January 2001 (has links)
Supercritical fluids have unique properties which may make them ideal as reaction media for biotransformation or extractive solvents. Supercritical fluids are ideal for reducing diffusivity limitations over conventional fluids. Depending on the polarity of the fluid, a supercritical fluid can be similar to conventional organic solvents, but with few of the environmental drawbacks. The use of supercritical fluids in enzymatic research has the advantage of removing mass transport limitations so that they can act as a suitable solvent. In this study, four permeabilization techniques were compared: control, toluene, supercritical carbon dioxide, and freeze/thaw cycles. The model cell systems studied were Z. mobilis and E. coli. The cells were analyzed for lipid profiles, recovery of proteins and enzymatic activity. The use of supercritical carbon dioxide may not be the most effective of the treatments based on total protein or enzyme recovery since the greatest protein and enzyme recovery was with the freeze/thaw treatment. However, it can be selective in removing cofactors from Z. mobilis enabling sorbitol production and minimizing side reactions. In this application, supercritical carbon dioxide does show an advantage over the freeze/thaw treatment. Aspects of the mechanism of permeabilization were investigated based on the lipid profiles of the cells, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The SEM and AFM show changes of the cell surface morphology which indicate that the treatments affect the cellular surface. The use of supercritical carbon dioxide as a reaction medium was investigated. Minute quantities of sorbitol were produced when Z. mobilis and sugars were placed in a supercritical carbon dioxide environment over a period of 24 hours.
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Microwave assisted pretreatment of sweet sorghum bagasse for bioethanol production / Busiswa Ndaba.Ndaba, Busiswa January 2013 (has links)
The growing demand for energy in the world, the implications of climate change, the increasing damages to our environment and the diminishing fossil fuel reserves have created the appropriate conditions for renewable energy development. Biofuels such as bioethanol can be produced by breaking down the lignocellulosic structure of plant materials to release fermentable sugars. Sweet sorghum bagasse has been shown to be an important lignocellulosic crop residue and is potentially a significant feedstock for bioethanol production. The aim of this study was to investigate suitable microwave assisted pretreatment conditions of sweet sorghum bagasse for bioethanol production. A chemical pretreatment process of sweet sorghum bagasse, using different concentrations (1 to 7 wt%) of sulphuric acid (H2SO4) and calcium hydroxide (Ca (OH)2) was applied to break up the lignocellulosic matrix of sweet sorghum bagasse. The pretreated broth, which contained pentose and hexose sugars, was fermented using a combination of Zymomonas mobilis ATCC31821 and Saccharomyces cerevisiae to produce bioethanol at pH 4.8 and 32oC for 24 hours. The highest reducing sugar yield of 0.82 g/g substrate was obtained with microwave irradiation at 180 W for 20 minutes in a 5 wt% sulphuric acid solution. The highest ethanol yield obtained was 0.5 g/g from 5 wt% H2SO4 pretreated bagasse at 180 W using a 10:5% v/v of Saccharomyces cerevisiae to Zymomonas mobilis ratio, whereas for 3 wt% Ca (OH)2 microwave pretreatment, a sugar yield of 0.27 g/g substrate was obtained at 300 W for 10 minutes. Thereafter, an ethanol yield of 0.13 g/g substrate was obtained after 24 hours of fermentation when using a 10:5% v/v of Saccharomyces cerevisiae to Zymomonas mobilis ratio. The effect of microwave pretreatment on the bagasse was evaluated using Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR) analysis. The reducing sugars formed were quantified using High Performance Liquid Chromatography (HPLC). The results showed that microwave pretreatment using 5 wt% H2SO4 is a very effective pretreatment that can be used to obtain sugars from sweet sorghum bagasse. The analytic results also showed physical and functional group changes after microwave pretreatment. This confirms that microwave irradiation is very effective in terms of breaking up the lignocellulose structure and improving fermentable sugar yield for fermentation. Bioethanol yields obtained from microwave pretreatment using different solvents also show that Saccharomyces cerevisiae and Zymomonas mobilis ATCC31821 is a good combination for producing ethanol from sweet sorghum bagasse. Sweet sorghum bagasse is clearly a very effective and cheap biomass that can be used to produce bioethanol, since very high yields of fermentable sugars were obtained from the feedstock. / Thesis (MSc (Engineering Sciences in Chemical Engineering))--North-West University, Potchefstroom Campus, 2013.
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