Control analysis of mixed populations of gluconobacter oxydans and saccharomyces cerevisiae

Malherbe, Christiaan Johannes (2010-12)

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2010.

Thesis

ENGLISH ABSTRACT: In the last decade a need arose to find a theoretical framework capable of gaining a quantitative understanding of ecosystems. Control analysis was proposed as a suitable candidate for the analysis of ecosystems with various theoretical applications being developed, i.e. trophic control analysis (TCA) and ecological control analysis (ECA). We set out to test the latter approach through experimental means by applying techniques akin to enzyme kinetics of biochemistry on a simple ecosystem between Saccharomyces cerevisiae and Gluconobacter oxydans. However, this exercise was far more complex than we originally expected due to the extra metabolic activities presented by both organisms. Nevertheless, we derived suitable kinetic equations to describe the metabolic behaviour of both organisms, with regards to the activities of interest to us, from pure culture experiments. We developed new techniques to determine ethanol and oxygen sensitivity of G. oxydans based on its obligately aerobic nature. These parameters were then used to build a simple kinetic model and a more complex model incorporating oxygen limited metabolism we observed at higher cell densities of G. oxydans. Our models could predict both situations satisfactorily for pure cultures and especially the more complex model could describe the lack of linearity observed between metabolic activity and cell density at higher cell densities of G. oxydans. Mixed populations of S. cerevisiae and G. oxydans reached quasi-steady states in terms of ethanol concentration and acetate flux, which was a positive indication for the application of control analysis on the ecosystem. However, the theoretical models based on parameters derived from pure culture experiments did not predict mixed culture steady states accurately. Careful analysis showed that these parameters were mostly under-estimated for G. oxydans and overestimated for S. cerevisiae. Hence, we calculated the kinetic parameters for mixed population assays directly from the experimental data obtained from mixed cultures. We could calculate the control coefficients directly from the experimental data of mixed population studies and compare it with those from theoretical models based on 3 different parameter sets. Our analysis showed that the yeast had all the control over the acetate flux while control over the steady-state ethanol was shared. The strength of our approach lies in designing our experiments with a control analysis approach in mind, but we have also shown that even for simple ecosystems this approach is non-trivial. Despite the various experimental challenges, this approach was very rewarding due to the extra information obtained especially regarding control structure with regards to the steady-state ethanol concentration.

AFRIKAANSE OPSOMMING: In die afgelope dekade het daar ’n behoefte ontstaan na ‘n teoretiese raamwerk om tot ‘n kwantitatiewe begrip van ekosisteme te kom. As kandidaat vir so tipe raamwerk is kontrole analise voorgestel gepaardgaande met die ontwikkeling van verskeie teoretiese toepassings, i.e. trofiese kontrole analise en ekologiese kontrole analise. In hierdie tesis het ons laasgenoemde aanslag eksperimenteel ondersoek op ‘n eenvoudige ekosisteem, tussen Saccharomyces cerevisiae en Gluconobacter oxydans, deur gebruik te maak van tegnieke vanuit ensiemkinetika van biochemie. Hierdie strategie was egter baie meer kompleks as wat oorspronklik verwag is as gevolg van verdere metabolise aktiwiteite aanwesig in beide organismes. Ons het egter steeds daarin geslaag om kinetiese vergelykings af te lei, vanuit suiwer kulture, wat die metaboliese gedrag van beide organismes beskryf vir die aktiwiteite van belang vir ons studie. Ons het nuwe tegnieke, gebaseer op die aerobiese natuur van G. oxydans, ontwikkel om die sensitiwiteit van G. oxydans vir etanol en suurstof te bepaal. Hierdie parameters is gebruik om eers ’n eenvoudige model en toe ‘n meer gevorderde model, wat die suurstof-beperkte metabolisme van G. oxydans by hoër biomassa te beskryf, op te stel. Beide modelle was baie effektief in die voorspelling van die situasies waarvoor hulle ontwikkel is vir die suiwer kulture waar veral die meer gevorderde model die gebrek aan ‘n linieêre verband tussen die metabolisme van G. oxydans en biomassa by hoër biomassa kon beskryf. ’n Bemoedigende aanduiding dat kontrole analise toegepas kon word op die ekosisteem was dat mengkulture van S. cerevisiae en G. oxydans het quasi-bestendige toestande bereik het in terme van etanol konsentrasies en asetaat-fluksie. Die teoretiese modelle gebaseer op die parameters afgelei vanaf suiwer kulture kon egter nie die bestendige toestande in mengkulture akkuraat voorspel nie. Nadere ondersoek het aangedui dat die parameters meesal onderskat is vir G. oxydans en oorskat is vir S. cerevisiae. Gevolglik het ons die kinetiese parameters vir mengkulture direk van eksperimentele data van die mengkulture bereken. Verder kon ons die kontrole koeffisiente ook direk vanaf die eksperimentele data van mengkulture bereken en vergelyk met dié bereken vanuit die teoretiese modelle gebaseer op drie verskillende paremeter-stelle. Ons analise het gewys dat die gis alle beheer op die asetaat-fluksie uitoefen en dat die beheer oor die etanol-konsnetrasie gedeel is tussen die twee organismes. Die krag van ons aanslag lê daarin dat die eksperimente ontwerp is met ‘n kontrole analise in gedagte, maar ons het ook bewys dat hierdie aanslag selfs vir eenvoudige ekosisteme nie triviaal is nie. Ten spyte van die eksperimentele uitdagings, was die aanslag baie waardevol as gevolg van die ekstra inligting verkry met spesifieke klem op die kontrole-struktuur met betrekking tot die etanol konsentrasie by bestendige toestand.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/5396
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