Heins, Anna-Lena

Modsat den tidligere traditionelle opfattelse er det i dag velkendt at en population af celler i en bioreaktor er heterogen. Cellerne i populationen er forskellige og derfor finder man fordelinger af enkel celle egenskaber, f.eks. celle størrelse, levedygtighed og metabolisk aktivitet, i modsætning til karakteristikker der kan blive beskrevet af gennemsnitlige værdier. Der vil altid være en vis heterogenicitet i en celle population, men heterogeniciteten bliver mere tydelig på grund af forskel i metabolske og stress responser hos enkelte celler når de bevæger sig igennem reaktoren og udsættes for gradienter af substrat, pH og oxygen, forårsaget af en ikke-ideel omrørning i industri-skala bioreaktorer. Denne afhandling sigter efter at opnå en større forståelse af hvordan mikrobiel fysiologi og celle dynamik er påvirket af spatial heterogenicitet i en bioreaktor. Derfor blev industriskala fermenteringer simuleret i laboratorieskala, hvor der blev anvendt to af industriens mest relevante organismer, E. coli and S. cerevisiae. Enkel celle fordelinger af celle størrelse og fluorescens – udsprunget fra henholdsvis vækst-, cellemembran robusthed- og ethanol- reporter stammer eller fra forskellige fluorescens indfarvninger (for at detektere f.eks. levedygtighed og metabolisk aktivitet) – blev dermed fulgt ved at anvende flødescytometri.Cellernes respons blev studeret i forskellige fermenterings opstillinger; i steady state ved varierende vækst rate og efter glukose pulser i kontinuerlige gæringer, med formål at simulere fodringszonen af en storskala fed-batch fermentering samt i batch for at karakterisere cellepopulationens adfærd i et dynamisk miljø. Dertil blev en to-kammer kemostat opstilling, med hensigt at simulere de forskellige zoner som opstår i storskala reaktorer, udviklet og studeret under forskellige betingelser. Den observerede population heterogenicitets fordelningen, kunde beskrives kvantitativt ved brug af ny-udviklede matematiske parametre, percentil analyse i kombination med multivariat statistik samt ved modellering.Generelt set viste sig de anvendte reporter stammer, såvel som fluorescens indfarvningsmetoderne, i kombination med flødescytometri at være værdifulde værktøjer til at studere populations heterogenicitet i forskellige setups, simulerende storskala fermentering. Strategier brugt i denne afhandling har potentiale til at blive anvendt i udvikling og optimering af storskala processer. Følgende er succesfuldt blevet visualiseret og kvantificeret: Forskelle i vækst og membranrobusthed under varierende vækstbetingelser og mellem langsomt og hurtigt voksende celler; forskellige metabolske aktiviteter på forskellige substrater og fænomener relateret til hurtigt skift mellem vækstbetingelser, som normalt er skjulte i en regulær kemostat. Desuden vil den opnåede fysiologiske indsigt kunne bruges i udformning af fremtidlige proces optimerings strategier.Today it is well known that a population of cells in a bioreactor is heterogeneous, opposite to traditional belief, and thus exhibiting distributions of single cell properties e.g. cell size, viability and metabolic activity rather than having a set of characteristics that can be described by averaged values. Population distributions always exist, but are significantly pronounced due to a combination of metabolic and stress responses of single cells travelling throughout the reactor experiencing gradients of substrate, pH and oxygen caused by non-ideal mixing in industrial scale bioprocesses. This thesis aimed at reaching a deeper understanding of how microbial physiology and cell dynamics are affected by the spatial heterogeneity in a bioreactor. Therefore large scale fermentation was simulated in laboratory scale using two of the most industrially relevant organisms E. coli and S. cerevisiae. Single cell distributions of cell size and fluorescence - originating from growth, cell membrane robustness and ethanol reporter strains or different fluorescence stains (for e.g. viability and metabolic activity) - were thereby followed by applying flow cytometry. Cell responses were studied in different cultivations modes, in steady state at different growth rates and in response to glucose perturbation in continuous culture, simulating the feeding zone of a large scale fed-batch fermentation and in batch culture to characterise the single cell behaviour in a dynamic environment. Furthermore, a two compartment chemostat setup, simulating different zones seen in large scale cultivations, was developed and studied under different compartmentalization conditions. The observed population heterogeneity distributions were, opposite to the common approach using mean values, described and validated in a quantitative manner through newly developed parameters, using percentile analysis followed by multivariate statistics as well as using a modeling approach.In general the applied reporter strains as well as fluorescence stains in combination with flow cytometry showed to be valuable tools to study population heterogeneity in the different setups simulating large scale fermentation that can potentially be used in development and optimisation of industrial scale processes. Differences in growth and membrane robustness due to varying growth conditions and between slow and fast growing cells, different metabolic activities on different substrates and phenomena during compartmentalization which are hidden in a normal chemostat could be successfully visualised and quantified

Online Research Database In Technology

Population heterogeneity in Saccharomyces cerevisiae and Escherichia coli lab scale cultivations simulating industrial scale bioprocesses

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Population heterogeneity in Saccharomyces cerevisiae and Escherichia coli lab scale cultivations simulating industrial scale bioprocesses

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Online Research Database In Technology