Anissa Delepierre

A fungal biofilm reactor based on metal structured packing improves the quality of a Gla::GFP fusion protein produced by Aspergillus oryzae

Fungal biofilm is known to promote the excretion of secondary metabolites in accordance with solid-state-related physiological mechanisms. This work is based on the comparative analysis of classical submerged fermentation with a fungal biofilm reactor for the production of a Gla::green fluorescent protein (GFP) fusion protein by Aspergillus oryzae. The biofilm reactor comprises a metal structured packing allowing the attachment of the fungal biomass. Since the production of the target protein is under the control of the promoter glaB, specifically induced in solid-state fermentation, the biofilm mode of culture is expected to enhance the global productivity. Although production of the target protein was enhanced by using the biofilm mode of culture, we also found that fusion protein production is also significant when the submerged mode of culture is used. This result is related to high shear stress leading to biomass autolysis and leakage of intracellular fusion protein into the extracellular medium. Moreover, 2-D gel electrophoresis highlights the preservation of fusion protein integrity produced in biofilm conditions. Two fungal biofilm reactor designs were then investigated further, i.e. with full immersion of the packing or with medium recirculation on the packing, and the scale-up potentialities were evaluated. In this context, it has been shown that full immersion of the metal packing in the liquid medium during cultivation allows for a uniform colonization of the packing by the fungal biomass and leads to a better quality of the fusion protein.

Phenotypic variability in bioprocessing conditions can be tracked on the basis of on-line flow cytometry and fits to a scaling law

Noise in gene and protein expression is a major cause for bioprocess deviation. However, this phenomenon has been only scarcely considered in real bioprocessing conditions. In this work, a scaling-law derived from genome-scale studies based on GFP reporter systems has been calibrated to an on-line flow cytometry device, allowing thus to get an insight at the level of promoter activity and associated noise during a whole microbial culture carried out in bioreactor. We show that most of the GFP reporter systems investigated and thus corresponding genes could be included inside the area covered by the scaling-law. The experimental results suggest that this scaling-law could be used to predict the dynamics of promoter activity, as well as the associated noise, in bioprocessing conditions. The knowledge acquired throughout this work could be used for the design of more robust expression systems.

Characterisation of phosphate accumulating organisms and techniques for polyphosphate detection : a review

Phosphate minerals have long been used for the production of phosphorus-based chemicals used in many economic sectors. However, these resources are not renewable and the natural phosphate stocks are decreasing. In this context, the research of new phosphate sources has become necessary. Many types of wastes contain non-negligible phosphate concentrations, such as wastewater. In wastewater treatment plants, phosphorus is eliminated by physicochemical and/or biological techniques. In this latter case, a specific microbiota, phosphate accumulating organisms (PAOs), accumulates phosphate as polyphosphate. This molecule can be considered as an alternative phosphate source, and is directly extracted from wastewater generated by human activities. This review focuses on the techniques which can be applied to enrich and try to isolate these PAOs, and to detect the presence of polyphosphate in microbial cells.

Microbial population heterogeneity versus bioreactor heterogeneity: evaluation of Redox Sensor Green as an exogenous metabolic biosensor

Microbial heterogeneity in metabolic performances has attracted a lot of attention, considering its potential impact on industrial bioprocesses. However, little is known about the impact of extracellular perturbations (i.e. bioreactor heterogeneity) on cell‐to‐cell variability in metabolic performances (i.e. microbial population heterogeneity). In this work, we have evaluated the relevance of Redox Sensor Green (RSG) as an exogenous biosensor of metabolic activity at the single‐cell level. RSG signal is proportional to the activity of the electron transport chain and its signal is strongly affected by metabolic burden, availability of electron final acceptor, and side metabolisms (i.e. overflow and mixed acid fermentation). RSG can also be used for the estimation of the impact of scale‐down conditions on microbial metabolic robustness. The relationship linking averaged RSG activity and its cell‐to‐cell variability (noise) has been highlighted but seems unaffected by environmental perturbations.