AbstractsBiology & Animal Science

Development, characterization, and application of a novel scale-down apparatus for the investigation of the scale-up dependent CO2/HCO3– stimulus in Corynebacterium glutamicum

by Jens Buchholz

Institution: University of Stuttgart
Department: Fakultät Energie-, Verfahrens- und Biotechnik
Degree: PhD
Year: 2015
Record ID: 1103534
Full text PDF: http://elib.uni-stuttgart.de/opus/volltexte/2015/9969/


Industrial biotechnology is regarded as a key technology for the sustainable production of chemicals and biofuels from renewable raw materials. The economy-of-scale principle leads to a more frequent application of large scale, non-ideally mixed bioreactors of several hundred cubic meters. As a result, the cells are subjected to continuously changing micro-environments along their flow path, such as gradients of the CO2 partial pressure (pCO2), which results due to the tank hydrostatics in exceptional amounts of dissolved CO2/HCO3– towards the bottom of the reactor. This thesis covered the first time investigation of “quasi-stationary” ’low’ (< 40 mbar) and ’high’ (> 300 mbar) pCO2 conditions on the growth kinetics and the transcriptional regulation in Corynebacterium glutamicum . Fermentations performed under ’high’ pCO2 levels resulted in initially enhanced biomass to substrate yields and similar total performance parameters as growth rate and substrate uptake, as well as enzyme activities of selected de-/carboxylating enzymes compared to the reference process. A global comparative transcriptional analysis revealed the importance of the global regulator DtxR (51 % of 117 differential genes) for growth in presence of ’high’ CO2/HCO3– levels, which was corroborated by the significantly reduced growth rate of a delta dtxR mutant. ’Low’ pCO2 led to a bi-level growth behavior with a growth rate reduction of 50 % in phase II. A complex regulatory mechanism in response to ’low’ CO2/HCO3– levels was deduced by combining the results of enzyme activity measurements (two-fold increase of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activity), by-product formation (L-alanine and L-valine), and DNA microarray analyses (140 differential genes). It is therefore assumed that an amplified expression of thiamin pyrophosphate (TPP) biosynthesis genes occurs to enhance the activity of TPP-dependent reactions to finally increase the availability of CO2 in the cells. The quantitative analysis of processes via carbon balancing is essential for advanced considerations such as flux analysis, especially under different CO2/HCO3– conditions. A novel balancing approach was established by using a total carbon analyzer that considers all carbon species in the liquid phase and in the biomass. Besides leading to completely matched balances of (96–100) %, it prevented false-negative underestimations and allowed the identification and assignment of balance gaps. In addition, it could be shown that considerable amounts of CO2/HCO3– dissolved in the culture medium, even under reference conditions, which in combination with inaccurate biomass carbon contents derived from the literature led to balance gaps of 24 % in the course of fermentations. The quantitative understanding of large scale production environments is an important factor for the rational improvement of bioprocesses. Experimental approaches focus on the abstraction of real bioreactors using specially designed scale-down devices. For this purpose, a novel…