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Februar 2017
Katrin Rosenthal
Single cell analysis and quantitative physiology of industrially relevant microorganisms
The analysis of single cell behavior is currently an important research topic in industrial biotechnology for studying functions and mechanisms of microorganisms without relying on average values of a population. Lab-on-a-chip (LOC) devices provide the basis for controlled single cell cultivations enabling the analysis of cellular physiology and extracellular environment in biotechnological contexts.
This thesis focuses on the technological development of a new microfluidic LOC device for single cell cultivation and on analytical methods for investigating the cellular physiology at a single cell level. A new manufacturing process was established to generate a versatile, chemically inert Envirostat for contactless single cell cultivation. The new Envirostat provides exceptional operational stability and a defined surface chemistry offering new analytical opportunities as single cell secretome analyses. Various analytical technologies for single cell secretome analyses were evaluated in terms of sensitivity to amino acids, the main biotechnological products of the industrially relevant bacterium Corynebacterium glutamicum. Furthermore, methodologies based on optical fluorescence readouts were applied. Individual medium components, typically used in L-lysine production processes, were identified to either hamper or support L-lysine production by C. glutamicum. Finally, a regulatory influence of glucose availability on substrate uptake rate was found constituting a basis for further studies of mechanisms controlling the metabolism of C. glutamicum. In this thesis, new technologies and methods were developed and applied for investigating key parameters of the cellular physiology aiming towards a holistic description of the smallest biological unit, the single cell.
This thesis focuses on the technological development of a new microfluidic LOC device for single cell cultivation and on analytical methods for investigating the cellular physiology at a single cell level. A new manufacturing process was established to generate a versatile, chemically inert Envirostat for contactless single cell cultivation. The new Envirostat provides exceptional operational stability and a defined surface chemistry offering new analytical opportunities as single cell secretome analyses. Various analytical technologies for single cell secretome analyses were evaluated in terms of sensitivity to amino acids, the main biotechnological products of the industrially relevant bacterium Corynebacterium glutamicum. Furthermore, methodologies based on optical fluorescence readouts were applied. Individual medium components, typically used in L-lysine production processes, were identified to either hamper or support L-lysine production by C. glutamicum. Finally, a regulatory influence of glucose availability on substrate uptake rate was found constituting a basis for further studies of mechanisms controlling the metabolism of C. glutamicum. In this thesis, new technologies and methods were developed and applied for investigating key parameters of the cellular physiology aiming towards a holistic description of the smallest biological unit, the single cell.
Schlagwörter: single cell analysis; lab-on-a-chip; Corynebacterium glutamicum; microfluidics; fluorescence microscopy
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