Functionality of microbial phenotypic heterogeneity in bioprocessing conditions: an analysis based on the use of on-line flow cytometry

Microbial phenotypic heterogeneity is known to be naturally present in isogenic population and can be attributed to the stochastic nature of the biochemical reactions. An important question at this level was to determine whether such stochastic behavior exhibits some functionality, i.e. how single cell heterogeneity leads to population level strategies. One of this strategies, called bet-hedging, is known to give a competitive advantage to the population, by leading for example to a persistent phenotype able to survive to antibiotics exposure. Among the single cell toolbox available for the analysis of phenotypic heterogeneity, flow cytometry present the advantage of being compatible with bioprocess cultivation tools. In this work, we present an on-line analytical workflow based on automated flow cytometry that can be used to monitor simultaneously several bioreactors, testing different bioreactor operating conditions, in combination with fluorescent transcriptional reporter strategies: promoter involved in metabolism fused to a gene expressing an unstable variant of GFP , and viability fluorescent tagging: propidium iodide uptake, correlated with membrane permeability

Fungal biofilm reactor improves the quality of a fusion protein GLA::GFP produced by Aspergillus oryzae

Fungal biofilm is known to promote the excretion of secondary metabolites, in accordance with solid-state related physiological mechanisms. In this work, the potentialities of fungal biofilm will be investigated in the context of the production of a Gla::GFP fusion protein by Aspergillus oryzae. Since the production of this protein is under the control of the promoter glaB, specifically induced in solid-state fermentation, biofilm mode of culture is expected to enhance the global productivity. However, we found that the glaB promoter is also activated in submerged bioreactor and the fusion protein production is higher in this mode of culture. This result is related to the high shear stress leading to biomass autolysis and leakage of intracellular fusion protein into the extracellular medium. Moreover, 2D-gel electrophoresis highlights preservation of the fusion protein integrity produced in biofilm conditions whereas proteolysis strongly affects fusion protein recovery in the submerged cultures performed at high stirring rate. Fungal biofilm reactor design was then further investigated and the scale-up potentialities were evaluated. Indeed, the specific design investigated in this work involves the use of metal structured packing exhibiting a high specific area and that can be easily expanded to large-scale bioprocessing conditions

Single cell analysis of Escherichia coli outer membrane porin composition in response to nutrient depletion

Characterization of outer membrane integrity into isoclonal population of Escherichia coli using fluorescent probe specific to cell viability: propidium iodide (PI) staining; combined to FACS cytometry and proteomic studies of sorted subpopulations. The results tend to reveal two distincts cellular strategies: Cells positively probed by PI offer a high outer membrane protein (OMP) content, indicating nutrient competence in response to substrate limitative conditions. Unprobed cells, characterized by a low OMPs rate, could suffer from growth arrest and develop stress responses (Crp, Cra, RpoS,RpoN, RpoH-dependent)

Design of a fungal biofilm reactor for recombinant protein production from Aspergillus oryzae

Fungi are microorganisms exhibiting high secretive power of various metabolites and have the ability to perform post-translational modifications during protein synthesis. In the field of fermentation industry, they are ideal hosts for secondary metabolites and recombinant protein production. At the industrial-scale, equipments usually required for solid-state or submerged fermentation of filamentous fungi have demonstrated their limitations in terms of productivity, mass transfers or products recovery (1, 2). Recently, fungal biofilm reactors were designed to combine advantages from submerged and solid-state culture and reveal their usefulness for greater secondary metabolites production relative to submerged culture conditions (3). In our work, we propose the design of a fungal biofilm reactor for a recombinant protein production from an Aspergillus oryzae strain containing a GFP reporter gene system under the control of a promoter specifically induced in solid-state conditions. The fungal biofilm reactor is composed of a metal structured packing, having the function of inert support for biofilm growth, immerged or aspersed by a liquid medium. Whereas recombinant protein production is not significantly different at the flask-scale between submerged and biofilm conditions, productivity is higher in the submerged conditions at the bioreactor-scale. Presence of recombinant proteins entrapped in the biofilm matrix highlights a diffusion constraint and a lower mass transfer in our fungal biofilm reactor. However, persistence of a free liquid biomass of low viscosity and fungal biomass retention on the support are attractive for the implementation of a continuous process in our fungal biofilm reactor. Further studies will consider a 2-D proteomic comparison of the extracellular medium from fungal biofilm reactor and submerged culture conditions in order to better understand proteins secretion and identify over-expressed proteins in biofilm conditions

Study of microbial phenotypic heterogeneity under bioprocess conditions using « single-cell » techniques

Presentation of single-cell techniques in order to characterize microbial phenotypic heterogeneity: fluorescent labelling, biosensors, automated flow cytometry combined to proteomics approach

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::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, 2D-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