Microfluidics: a new look at cell migration analysis

This thesis explores the development and employment of microfluidic devices as a tool for studying the effect of the surrounding environment on embryonic stem cells during the migration phenomena. Different single-cell microchips were designed and manufactured to study mouse embryonic fibroblasts (MEFs) migration towards an environmental variation (increase of serum concentration in the culture medium) that was expected to function as a motility stimuli. Considering the experimental, cells were injected into the microchips chambers and individually isolated by dedicated cell traps with view to a single-cell analysis. Once fribroblasts were attached to the surface, culture medium with an increased serum level was subsequently injected in an adjacent chamber to promote the formation of a serum concentration gradient. The gradient established between the chambers could be sensed by the fibroblasts and thus triggered the cells mobilization towards and in the direction of the richer serum medium. Additionally, the experiment allowed the observation of MEFs’ structural reorganization when migrating through micro-tunnels containing widths below the cell size, suggesting a cytoskeleton rearrangement on account of the nutritional stimulus introduced. Furthermore, results indicate that fibronectin promotes MEFs adhesion to the substrate and that MEFs migration is characterized as haptotactic.

Cell functional enviromics: applications to Pichia pastoris

The present PhD thesis develops the cell functional enviromics (CFE) method to investigate the relationship between environment and cellular physiology. CFE may be defined as the envirome-wide cellular function reconstruction through the collection and systems-level analysis of dynamic envirome data. Throughout the thesis, CFE is illustrated by two main applications to cultures of a constitutive P. pastoris X33 strain expressing a scFv antibody fragment. The first application addresses the challenge of culture media development. A dataset was built from 26 shake flask experiments, with variations in trace elements concentrations and basal medium dilution based on the standard BSM+PTM1. Protein yield showed high sensitivity to culture medium variations, while biomass was essentially determined by BSM dilution. High scFv yield was associated with high overall metabolic fluxes through central carbon pathways concomitantly with a relative shift of carbon flux from biosynthetic towards energy-generating pathways. CFE identified three cellular functions (growth, energy generation and by-product formation) that together described 98.8% of the variance in observed fluxes. Analyses of how medium factors relate to identified cellular functions showed iron and manganese at concentrations close to PTM1 inhibit overall metabolic activity. The second application addresses bioreactor operation. Pilot 50 L fed-batch cultivations, followed by 1H-NMR exometabolite profiling, allowed the acquisition of data for 21 environmental factors over time. CFE identified five major metabolic pathway groups that are frequently activated by the environment. The resulting functional enviromics map may serve as template for future optimization of media composition and feeding strategies for Pichia pastoris. The present PhD thesis is a step forward towards establishing the foundations of CFE that is still at its infancy. The methods developed herein are a contribution for changing the culture media and process development paradigm towards a holistic and systematic discipline in the future.

Coupling organic substrate removal to methane production in a fully biological microbial electrolysis cell

Microbial electrolysis cells (MECs) are an innovative and emerging technique based on the use of solid-state electrodes to stimulate microbial metabolism for wastewater treatment and simultaneous production of value-added compounds (such as methane). This research studied the performance of a two-chamber MEC in terms of organic matter oxidation (at the anode) and methane production (at the cathode). MEC‟s anode had been previously inoculated with an activated sludge, whereas the cathode chamber inoculum was an anaerobic sludge (containing methanogenic microorganisms). During the experimentation, the bioanode was continuously fed with synthetic solutions in anaerobic basal medium, at an organic load rate (OLR) of around 1 g L-1 d-1, referred to the chemical oxygen demand (COD). At the beginning (Run I), the feeding solution contained acetate and subsequently (Run II) it was replaced with a more complex solution containing soluble organic compounds other than acetate. For both conditions, the anode potential was controlled at -0.1 V vs. standard hydrogen electrode, by means of a potentiostat. During Run I, over 80% of the influent acetate was anaerobically oxidized at the anode, and the resulting electric current was recovered as methane at the cathode (with a cathode capture efficiency, CCE, accounting around 115 %). The average energy efficiency of the system (i.e., the energy captured into methane relative to the electrical energy input) under these conditions was over 170%. However, reactor‟s performance decreased over time during this run. Throughout Run II, a substrate oxidation over 60% (on COD basis) was observed. The electric current produced (57% of coulombic efficiency) was also recovered as methane, with a CCE of 90%. For this run the MEC‟s average energy efficiency accounted for almost 170 %. During all the experimentation, a very low biomass growth was observed at the anode whereas ammonium was transferred through the cationic membrane and concentrated at the cathode. Tracer experiments and scanning electron microscopy analyses were also carried out to gain a deeper insight into the reactor performance and also to investigate the possible reasons for partial loss of performance. In conclusion, this research suggests the great potential of MEC to successfully treat low-strength wastewaters, with high energy efficiency and very low sludge production.