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Bioreactors --- Bioreactors --- Hydrodynamics --- Hydrodynamics --- Escherichia coli --- Escherichia coli --- Saccharomyces cerevisiae --- Saccharomyces cerevisiae --- Microbial properties --- Microbial properties --- Physiological regulation --- Physiological regulation --- Models --- Models --- evaluation. --- evaluation

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Fermentation --- Fermentation --- Models --- Models --- foaming --- foaming --- chemicophysical properties --- chemicophysical properties --- Laboratory experimentation --- Laboratory experimentation --- industry --- industry --- Changement d'echelle --- Changement d'echelle

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In the last decade, numerous single-species biofilm reactors of various configurations have been implemented at lab and pilot scale for the production of chemicals and biological products. Compared to their counterparts in submerged cultures, these processes benefit from the specific physiology of biofilms, i.e. high robustness of the microbial system, long-term activity, continuous implementation and low ratio size / productivity. However, the risks of biofouling and the lack of analytical tools for the control and the monitoring of biofilms are obstacles for scale-up strategies. Up to now, single-species biofilm reactors have been mainly confined to the production of metabolites ranging from low (bulk chemicals) to medium (fine chemicals) added values. In this way, there is a need to design efficient single-species biofilm reactors exhibiting good scalability potentials and intended for the production of high added value compounds. In this work, an experimental single-species biofilm reactor has been designed for the production of target molecules derived from metabolic pathways involved in biofilm physiology. On the basis of these criteria, three biological models having good abilities of biofilm formation and secretion performances were selected : - the gram positive bacterium Bacillus subtilis for the production of surfactin, a surface active metabolite involved in biofilm formation. - the filamentous fungus Trichoderma reesei for the production of hydrophobin (HFBII), a surface active protein (7kDa) involved in adhesion process of spores and mycelium on solid surface. - the filamentous fungus Aspergillus oryaze (engineered strain) for the production of a recombinant protein (Gla::GFP) under the control of the glaB promoter specifically activated in solid-state fermentation. The proposed experimental biofilm reactor has the configuration of a trickle-bed bioreactor. The agitation axis of a stirred tank reactor has been removed and replaced by a stainless steel structured packing filling the top of the vessel. The liquid medium, located in the bottom of the vessel is continuously recirculated on the packing element thanks to a peristaltic pump. An ascending air flow is performed above the liquid phase just under the packing element. This thesis reports the screening of the three biological models in the experimental biofilm reactor. The results include the characterization of process performances in terms of biofilm formation and secretion of the target molecule under different operating conditions. An original methodology based on high energy X-ray tomography has been developed to non-invasively visualize and quantify the biofilm colonization inside the packing element. This technique has highlighted that biofilm colonization and liquid phase distribution across the packing are strongly interrelated phenomena. The biofilm of B. subtilis occurring by cell aggregation preferentially developed on solid areas wetted by the liquid. Accordingly, optimal operating conditions improving liquid phase distribution have been defined for biofilm colonization. The fungal biofilm of A. oryzae and T. reesei occuring by cell filamentation equally colonize submerged and aerial surfaces of the packing element. Consequently, another configuration of biofilm reactor comprising a packing element totally immersed in the liquid medium has been investigated. The production yields of surfactin and hydrophobin in the experimental biofilm reactor are respectively 1.25 and 2.64 times greater than those of a submerged culture in a stirred tank reactor. This suggests that surface-active molecules involved in biofilm formation have a real interest for the design of single-species biofilm reactors. Although the Gla::GFP fusion protein is greater produced in the stirred tank culture, its integrity was preserved in the biofilm reactor despite the presence of proteases. This suggests that the quality and the stability of heterologous proteins produced in a fungal biofilm reactor are improved compared with a submerged culture. Finally, the implementation of the biofilm reactor has led to technological progresses including low energy consumption, no foam formation, continuous processing and simplification of downstream process operations. Further experiments should deepen the understanding of structured phenotypic heterogeneity impact on secretion performances in the biofilm reactor. These experiments should consider development of operating conditions allowing for the growth of a thin biofilm homogeneously distributed on the whole surface provided by the packing element in order to optimize nutrients and metabolites mass transfers. The scale-up and the continuous implementation of the process should be also investigated.

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Bioprocess technologies involving mixed culture display, high socio-economic interest. From the production of various chemicals, biological compounds to several environmental applications, such bioprocesses are implemented in many ways. However, despite widely use, complexity of the biological mechanisms involved in is still not unresolved. Nowadays, most of the biotechnological processes involving microorganisms are controlled and steer based on the monitoring of physico-chemical parameters. Microbial communities and underlying biological reactions, main actors of the processes, are considered as a "black box". In order to improve the management of such bioprocesses, it is necessary to elucidate these biological "black boxes". Particularly, key questions such as "Who is there ?", "Who is doing what ?" and "Who is doing what with whom ?" should be answered for better understanding of communities' activities. Microbial resource management, a concept previously developed by Verstraete et al. (2007), aims to control and/or steer microbial community capacities through answering these questions. In this work, microbial resource management has been considered for improving cellulose anaerobic digestion capacities of communities involved in industrial / environmental bioprocesses. Bioaugmentation of two communities with a cellulolytic community isolated from compost has been carried out. Hydrolysis step is indeed recognized as the limiting step in global anaerobic digestion process of cellulosic substrates. For both cases, management of the microbial communities led to an increase of biogas production from (ligno-)cellulosic substrates. Furthermore, two culture independent techniques have been considered for the assessment of the bioaugmentation treatment applied to leachate communities. Community structure has been monitored through 16s rRNA gene sequencing and flow cytometry. Flow cytometry fits particularly well for a fast and routine monitoring of microbial communities. However, data treatment/transformation is required for an efficient comparison of flow cytometric patterns. Based on this statement, we developed a flow cytometric fingerprinting method allowing the transformation of cytogram into vector of values for further statistical analyses. Both implemented fingerprinting methods give same evidence about microbial dynamics throughout cellulose anaerobic digestion assay. There are however a lack of significant correlation between cytometric and amplicon sequencing fingerprint at the genus or species level. Same phenotypic profiling of microbiota during assays matched to several 16s rRNA gene sequencing ones. Flow cytometry fingerprinting can thus be considered as a promising routine on-site method, suitable for the detection of stability/variation/disturbance of complex microbial communities involved in bioprocesses. Finally, flow cytometry fingerprinting has been applied for the monitoring of metabolic heterogeneities among monospecies biofilm. Indeed, biofilm is the main form of microbial communities encountered in natural and engineered environment. The development of specific technique aiming at analyzing function of microbial population must then take into account this kind of structure. High degree of specialization can be observed among the cells embedded in biofilm. In order to simplify the approach, the efforts have been focused on single species biofilm in the context of this thesis. Indeed, even isogenic population of cells can take advantage from phenotypic heterogeneities through a division of labor strategy. Such multicellular communities must be considered as a heterogeneous, "multi phenotypes", communities and not anymore as a homogeneous community. Therefore, similar key questions than for multispecies communities must be answered for elucidating their organization and allowing further efficient management/steering of biofilm based bioprocesses. The efficiency of flow cytometry fingerprint for monitoring metabolic heterogeneities has been proved through a set of experimentations carried out with Bacillus amyloliquefaciens as reference organism. Metabolic flow cytometry fingerprinting allows for evaluating the impact of genetic mutation (B. amyloliquefaciens mutants were constructed in order to obtain strains with deficient in lipopeptide synthesis) on metabolic profiles. In conclusion, the management of microbial communities, through bioaugmentation treatment, leads to an improvement of the performances of anaerobic digestion bioprocesses. Moreover, a fast, cheap, culture and operator independent monitoring technique, able to provide crucial information about dynamics of heterogeneous microbial communities has been developed. Efficient for the monitoring of multispecies communities such as the ones involved in anaerobic digestion processes, this approach also reveals its potentialities for the monitoring of phenotypic heterogeneities among isogenic biofilm communities.