Project description:In large-scale production processes, metabolic control is typically achieved by limited supply of essential nutrients like ammonia. With increasing bioreactor dimensions, microbial producers such as Escherichia coli are exposed to changing substrate availabilities due to limited mixing. In turn, cells sense and respond to these dynamic conditions leading to frequent activation of their regulatory programs which result in production yield losses. This study is focused on transcriptional changes due to fluctuating ammonia supply, while sampling a continuously running two-compartment bioreactor system comprising a stirred tank reactor (STR) and a plug flow reactor (PFR). A previously created mutant E.coli SR was used to limit the reaction to environmntal influences via knock-out of stringent response. E. coli WT revealed highly diverging short-term transcriptional responses in ammonia fluctuation compared E. coli SR.
Project description:In large-scale production processes, metabolic control is typically achieved by limited supply of essential nutrients like glucose or ammonia. With increasing bioreactor dimensions, microbial producers such as Escherichia coli are exposed to changing substrate availabilities due to limited mixing. In turn, cells sense and respond to these dynamic conditions leading to frequent activation of their regulatory programs. Previously, we characterized short- and long-term strategies of cells to adapt to glucose fluctuations. Here, we focused on fluctuating ammonia supply, while studying a continuously running two-compartment bioreactor system comprising a stirred tank reactor (STR) and a plug flow reactor (PFR). Genes were repeatedly switched on/off when E. coli returned to the STR. Moreover, E. coli revealed highly diverging long-term transcriptional responses in ammonia compared to glucose fluctuations. The identification of target genes may help to create robust cells and processes for large-scale application.
Project description:Polyhydroxyalkanoates (PHAs) are bio-based, biodegradable polyesters that can be produced from organic-rich waste streams using mixed microbial cultures. To maximize PHA production, mixed microbial cultures may be enriched for PHA-producing bacteria with a high storage capacity through the imposition of cyclic, aerobic feast-famine conditions in a sequencing batch reactor (SBR). Though enrichment SBRs have been extensively investigated a bulk solutions-level, little evidence at the proteome level is available to describe the observed SBR behavior to guide future SBR optimization strategies. As such, the purpose of this investigation was to characterize proteome dynamics of a mixed microbial culture in an SBR operated under aerobic feast-famine conditions using fermented dairy manure as the feedstock for PHA production. At the beginning of the SBR cycle, excess PHA precursors were provided to the mixed microbial culture (i.e., feast), after which followed a long duration devoid of exogenous substrate (i.e., famine). Two-dimensional electrophoresis was used to separate protein mixtures during a complete SBR cycle, and proteins of interest were identified.
Project description:Human embryonic stem cells (hESC) have an enormous potential as a source for cell replacement therapies, tissue engineering and in vitro toxicology applications. The lack of standardized and robust bioprocesses for hESC expansion in relevant quantities while maintaining their pluripotency has hindered the application of hESC and their derivatives in clinical setting. Here, we developed a scalable and well-characterized bioprocess for hESC expansion under fully-defined conditions and explored the potential of transcriptomic and metabolomic tools to evaluate the impact of culture system on hESC phenotype. Two different hESC lines (feeder-dependent and feeder-free lines) were efficiently expanded on xeno-free microcarriers in stirred culture systems. Moreover, both hESC lines maintained the expression of stemness markers such as Oct-4, Nanog, SSEA-4 and TRA1-60, and the ability to spontaneously differentiate into the three germ layers. Whole-genome transcriptome profiling revealed a phenotypic convergence between both hESC lines along the expansion process in stirred-tank bioreactor cultures, providing strong evidence on the robustness of the cultivation process to homogenize cellular phenotype. Under low oxygen tensions, results showed a metabolic rearrangement with the up-regulation of the glycolytic machinery favoring an anaerobic glycolysis Warburg-effect like phenotype, with no evidence of hypoxic stress response, in contrast to 2-dimensional culture. Overall, we report a scalable and fully-defined bioprocess for the propagation of hESC while guaranteeing product quality. Furthermore, the “omics” tools herein used provided relevant findings on the physiological/metabolic changes during the hESC expansion in environmentally-controlled stirred-tank bioreactors, which can contribute for more standardized production systems.
Project description:Comparison of gene expression profile of human pluripotent stem cells (hHSC_F1285T_iPS2) expanded in perfused stirred tank bioreactors after 3 and 7 days compared to 2D precultures (day 0)
