Production of poly(3-hydroxybutyrate) (PHB) was done in a cyclone bioreactor using various culture methods; including batch (lab and pilot scale) fed-batch and self-cycling fermentation with and without starvation periods. Alcaligenes eutrophus DSM 545 was used to accumulate about 87% (wt/wt) PHB to a total of 6.2 g L$ sp{-1}$ PHB at the end of a 48 hour batch. Similar pilot scale experiments contained a maximum of 96% (wt/wt) PHB with 4.9 g L$ sp{-1}$ accumulated. Fed-batch culture of A. eutrophus produced 96% (wt/wt) PHB with a final PHB concentration of 22.2 g L$ sp{-1}$ after 54 h. Self-cycling fermentation (SCF) production of PHB resulted in an average of 35% (wt/wt) PHB without starvation periods with production rates reaching 0.24 g L$ rm sp{-1} hr sp{-1}.$ With starvation periods of 4, 6 and 8 h extended on the cycle times, production of PHB decreased except in the 8 hour starvation period which was 59% (wt/wt). However, the rates of production all decreased to below 0.13 g $ rm L sp{-1} h sp{-1}$ as the lengths of the starvation periods were increased.

Copies of author's previously published articles inserted. / Bibliography: leaves 175-194. / xiii, 195 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Eikenella corrodens, a Gram-negative rod, is a normal inhabitant of the human oral cavity. It is one of the most commonly detected cultivable bacteria from sub- and supra-gingival plaque and is often isolated in elevated proportions from sites exhibiting destructive periodontal disease compared with healthy sites. The primary aim of this study was to investigate the metabolism of E. corrodons, with particular reference to energy generation, and to determine the effect of physical environmental parameters on its growth. / Thesis (Ph.D.)--University of Adelaide, Dept. of Dentistry, 2000

Copies of author's previously published articles inserted. / Bibliography: leaves 175-194. / xiii, 195 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Eikenella corrodens, a Gram-negative rod, is a normal inhabitant of the human oral cavity. It is one of the most commonly detected cultivable bacteria from sub- and supra-gingival plaque and is often isolated in elevated proportions from sites exhibiting destructive periodontal disease compared with healthy sites. The primary aim of this study was to investigate the metabolism of E. corrodons, with particular reference to energy generation, and to determine the effect of physical environmental parameters on its growth. / Thesis (Ph.D.)--University of Adelaide, Dept. of Dentistry, 2000

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 113-117).

Orientador: Maria Helena Andrade Santana / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-14T16:51:23Z (GMT). No. of bitstreams: 1 Pires_AlineMaraBarbosa_D.pdf: 5667585 bytes, checksum: 99be814ed0f4b52809cad017f91235eb (MD5) Previous issue date: 2009 / Resumo: Neste trabalho, estudou-se a otimização da produção de ácido hialurônico (HA) por cultivo de Streptococcus zooepidemicus em batelada, com base nas alterações metabólicas ao longo do cultivo. Ás condições ambientais estudadas foram a concentração inicial de glicose, controle do pH, íons minerais e fonte de nitrogênio orgânico. Nos cultivos em frascos, a concentração inicial de glicose não alterou nem o crescimento celular nem a produção de HA. Entretanto, no cultivo em biorreator sem o controle do pH, ambos foram fortemente dependentes da concentração inicial de glicose, com maior produção de HA (1,21 g.L-1) no cultivo realizado em meio com 25 g.L-1 glicose. Tal condição nutricional foi a única que apresentou maior conversão de glicose em HA (YHA/S) do que conversão de glicose em massa celular (YX/S). O controle do pH ao longo do cultivo com 25 g.L-1 glicose resultou em maior produtividade de células (0,21 g.L-1.h-1) e de HA (0,10 g.L-1.h-1). apesar dos menores rendimentos em relação à glicose. A combinação desses resultados relaciona o maior direcionamento da fonte de carbono para HA do que para células a uma resposta do microrganismo ao stress ácido ocorrido no cultivo sem controle do pH. Uma análise da distribuição dos fluxos metabólicos nas condições ambientais estudadas demonstrou que as alterações na via de produção de HA foram mais relacionadas à distribuição dos fluxos para os açúcares precursores da síntese do polímero que à disponibilidade de energia (ATP) ou potencial redutor (NADH/NAD+) das células. A total suplementação do meio de cultura com íons minerais (K+, Mg++, Na+, Fe++, Ca++, Mn++, Zn++ e Cu++) foi benéfica para o crescimento celular, porém não alterou a produção de HA de forma significativa. O estudo demonstrou ainda que a qualidade do polímero produzido pode ser modulada pela suplementação do meio com íons minerais. As propriedades reológicas do HA com baixo teor de proteína (0.44 g.g-1) e massa molar média de 4.0 x 106 Da demonstraram elevada densidade de emaranhamento das cadeias devido à alta dependência do módulo elástico com a concentração e desvios da viscosidade complexa com relação à regra de Cox-Merz. O estudo de meios alternativos contendo derivados agroindustriais demonstrou maiores concentrações de HA em meios contendo extrato de levedura como fonte de nitrogênio. Este conjunto de resultados contribui para a otimização da produção de HA, assim como para um melhor entendimento do metabolismo do Streptococcus zooepidemicus. / Abstract: In this work, h was studied the optimization of HA production by hatch culture of Streptococcus zooepidemicus, with focus on the metabolic changes along cultivation. The environmental conditions studied were the initial glucose concentration, pH control, mineral ions and organic nitrogen source. In flask cultivations, the initial glucose concentration had no influence on the amounts of either the biomass or the MA produced. However, in bioreactor cultivations, at non-controlled pH. both were strongly dependent on the initial glucose concentration. The highest HA concentration (1.21 g.L-1) was obtained from 25 g.L-1 glucose, which was the only cultivation where the conversion of glucose to HA (YHA/S) was higher than the one of glucose to biomass (YX/S). Not only did the pH control along cultivation result in higher cell productivity (0.21 g.L-1.h-1), but also in the HA productivity (0.10 g.L-1.h-1), However, the HA and cell yields from glucose were lower. The combination of these results relates the higher direction of the carbon source to the HA synthesis at the expenses of the cell growth to a microbial response to the acid stress observed in non-controlled pH. An analysis of the metabolic flux distribution in the environmental conditions studied shows that the changes in the HA production pathway were more related to die distributions of duxes 10 the precursors of HA synthesis than to the energy availability (ATP) or redox slate (NADH/NAD+) of the cells. The total supplementation of the culture medium with ions was beneficial to die cell growth. However, if did not have any influence on the HA production. Moreover, the results showed that the HA quality may be modulated through the mineral ion supplementation. The rheological properties of HA with low protein content (0.44 g.g-1) and average molecular weight of 4.0 x 106 Da showed the high entanglements density of the HA chains due to the high storage modulus concentration dependence as well as to the complex viscosity deviations with respect to the Cox - Merz rule. Alternative media containing agricultural resources derivates were studied. The higher HA concentrations were produced in media whose organic nitrogen source was yeast extract. This set of results contributes not only to the optimization of the HA production, but also to a better understanding of the Streptococcus zooepidemicus metabolism. / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutor em Engenharia Química

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Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The interests in understanding the metabolic processes of microbial systems are numerous. The interest in the species Lactococcus lactis (L. lactis) lies in applications to the food industry and in studies comparing the metabolism of related organisms. The aim of this study was to perform in vivo supply-demand analysis on anaerobically fermenting L. lactis. This was done by perturbing both the supply and demand pathways, then measuring glycolytic flux (by means of 13C NMR spectroscopy) and intracellular ATP/ADP (by means of 31P NMR spectroscopy) at steady state – where the central metabolite, ATP, is produced at the same rate as it is consumed and its concentration thereby remains constant. The L. lactis reference strain MG1363 was supplemented with glucose and analysed “online” by 13C-NMR under anaerobically-fermentative conditions. The rates of glucose consumption and lactate production were determined from this 13C flux. Due to experimental difficulties with the online detection of 31P (possibly due to the low biomass yield and the choice of growth medium), ATP/ADP levels had to be determined offline: from the same batch cultures as the 13C samples, fermentations were performed and halted at time points when the cells had attained a steady state. These fermentation cultures were then subjected to cell lysis and centrifugation in order to extract intracellular metabolites. These cell extracts were analysed offline by 31P NMR in order to determined levels of phosphate metabolites, specifically ATP and ADP. Perturbation of the supply pathway was achieved by utilising a genetically modified strain (the CS8 strain with over-expressed las operon) and comparing it to the reference strain MG1363. This resulted in a slight increase in ATP/ADP, but also yielded a slightly reduced flux, which is contrary to expectations from a mutant with over expressed glycolytic enzymes. The demand pathway was perturbed by two methods: 1) utilisating a genetically modified strain (the BK1506 strain with over-expressed F1-ATPase) and comparing it to the reference strain MG1363, and 2) by treating wild-type MG1363 with sodium acetate and comparing flux and ATP/ADP values to the untreated wild-type. Sodium acetate dissociates in the cytoplasm and causes dissipation of the transmembrane proton motive force, which is re-established by upregulation of membrane-bound H+-translocating ATPases. While the use of genetically modified strains provided only one flux-vs-ATP/ADP data point to compare to the wild-type (not sufficient for complete supply-demand analysis), the treatment of the wild type with uncoupler yielded several data points where flux and ATP/ADP values differed according to the concentration of uncoupler added. The CS8 strain demonstrated a 19 % reduced glucose flux (24 % reduced lactate flux) with respect to the wild type MG1363. The BK1506 strain demonstrated a 72 % increase in glucose flux (33 % increase in lactate flux) with respect to the wild type. The treatment with 2 mM acetate resulted in a 72 % increase in glucose flux (123 % increase in lactate flux), whereas treatment with 4 mM acetate resulted in a 107 % and a 126 % increase in glucose and lactate fluxes, respectively. The treatment with different concentrations of acetate provided several data points with corresponding flux and ATP/ADP, enabling the calculation of the elasticity coefficient of the supply pathway to changes in ATP/ADP (εsupply ) which was found to be -5.6 and -6.3 for glucose and lactate, respectively. ATP/ADP The elasticity coefficient was high compared with values obtained in similar studies on other organisms. Considering that at steady state the supply and demand fluxes are equal, the high supply elasticity (which is easier to measure), when incorporated into control coefficient summation theorems, gives the indication that: 1) a greater amount of control may reside in the ATP demand pathway (the elasticity of which is more difficult to determine experimentally, but which may well be lower than the supply elasticity), and 2) ATP/ADP homeostasis is good, as indicated by a high elasticity of the supply pathway to ATP/ADP. This study represents a basis for further supply-demand analysis with non-growing batch cultures of L. lactis. / AFRIKAANSE OPSOMMING: Daar is groot belangstelling daarin om die metaboliese prosesse van mikrobiese sisteme beter te verstaan. Die belang van die spesie Lactococcus lactis (L. lactis) lê beide in die toepassing in die voedselbedryf en in studies wat die metabolisme van verskeie organismes vergelyk. Die doel van hierdie studie was om in vivo vraag-aanbod analise uit te voer op anaerobies-fermenterende L. lactis. Dit was gedoen deur beide die aanbod en vraag reaksie-blokke te moduleer en dan die glikolitiese fluksie (d.m.v. 13C KMR spektroskopie) en die intrasellulêre ATP/ADP (d.m.v. 31P KMR spektroskopie) in ’n bestendige toestand te meet (wanneer die sentrale metaboliet, ATP, teen dieselfde tempo geproduseer en verbruik word en sy konsentrasie daardeur konstant bly). Die L. lactis verwysing-stam MG1363 is met glukose aangevul en 13C fluksie is aanlyn onder anaerobies-fermenterende kondisies gemeet. Die tempo van glukose verbruik en laktaat produksie is vanaf die 13C fluksie bereken. Eksperimentele probleme met die aanlyn bepaling van 31P (dalk as gevolg van lae biomassa en/of die keuse van groeimedium) moes ATP/ADP vlakke af-lyn indirek bepaal word: fermentasies van dieselfde lot-kulture as die 13C monsters is opgestel en by sekere tydpunte gestop wanneer ‘n bestendige toestand bereik was (waar ATP/ADP konstant bly). Hierdie fermenterende kulture is blootgestel aan sel-lise en sentrifugasie om intrasellulêre metaboliete te onttrek. Dié sel-ekstrakte is deur 31P KMR geanaliseer om die vlakke van fosfaat metaboliete, spesifiek ATP en ADP, te bepaal. Die aanbod blok is gemoduleer deur gebruik te maak van ‘n geneties-gemodifiseerde stam (die CS8 stam met ‘n ooruitgedrukte las operon) en met die verwysing stam MG1363 te vergelyk. Dié gemuteerde stam het ’n effense toename in ATP/ADP getoon, maar het gelyktydig ook ’n afname in glikolitiese fluksie getoon, wat onverwags is vir ’n stam met ooruitgedrukte glikolitiese ensieme. Die vraag blok is met twee metodes gemoduleer: 1) deur gebruik te maak van ‘n geneties-gemodifiseerde stam (die BK1506 stam met ‘n ooruitgedrukte F1-ATPase), en 2) deur die wildetipe MG1363 met natrium asetaat te behandel en daardeur ATP verbruik van biomassa produksie te ontkoppel en die vraag na ATP te vermeerder. Daarna word die fluksie en ATP/ADP waardes met die onbehandelde wildetipe vergelyk. Natrium asetaat dissosieer in die sitoplasma en verswak die transmembraan elektriese potensiaal, wat dan weer versterk word deur membraan-gekoppelde H+-ATPase ensieme wat protone uit die sitoplasma uit pomp. Terwyl die gebruik van geneties-gemodifiseerde stamme net een fluksie-tot-ATP/ADP datapunt voorsien om met die wildetipe te vergelyk (wat nie voldoende is vir totale vraag-aanbod analise nie), het die behandeling van die wildetipe met ontkoppelaar meerdere datapunte voorsien waar fluksie en ATP/ADP waardes verskil volgens die konsentrasie van ontkoppelaar wat bygevoeg is. Die CS8 stam het ’n 19 % verminderde glukose fluskie getoon, asook ’n 23 % verminderde laktaat fluksie, in vergelyking met die wilde tipe MG1363. Die BK1506 stam het ’n 73 % toename in glukose fluskie getoon, asook ’n 34 % toename in laktaat fluksie, in vergelyking met die wilde tipe. Behandeling met 2 mM natrium asetaat het ’n 64 % toename in glukose fluksie veroorsaak, sowel as ’n 124 % toename in laktaat fluksie, en behandeling met 4 mM natrium asetaat het 108 % toename in glukose fluksie en 127 % toename in laktaat fluskie veroorsaak. Behandeling met verskillende konsentrasies natrium asetaat het genoeg data punte (fluksies met toepaslike ATP/ADP waardes) verskaf om die berekening van elastisiteits-koëffisiënt van die aanbod reaksie-blok tot veranderinge in ATP/ADP (εsupply ) te bereken. Die waardes was -5.6 vir glukose fluksie en -6.3 vir laktaat fluksie. ATP/ADP Die elastisiteits koëffisiënt was relatief hoog in vergelyking met waardes wat in soorteglyke studies op ander organismes bepaal is. Aangesien die fluksies van die aanbod en vraag reaksie blokke by ’n bestendige toestand dieselfde tempo het, kan die hoë waarde van die aanbod elastisiteits-koëffisiënt (wat die makliker een is om te meet) na die volgende afleidings lei: 1) meer kontrole mag in die ATP verbruikende reaksie-blok geleë wees (die elastisiteits-koëffisiënt is moeiliker om eksperimenteel te bepaal maar mag wel laer as die aanbod-elastisiteit wees), en 2) ATP/ADP homeostase word goed gehandhaaf, soos aangetoon deur die hoë aanbod-elastisiteit. Hierdie studie dien as ’n basis vir verdere vraag-aanbod analise in nie-groeiende L. lactis lot-kulture.

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).