E.coli prpoS::gfp strain as biosensor of glucose heterogeneity inside industrial bioreactors

• OBJECTIVE: Escherichia coli is a microorganism widely used in the industry for the production of recombinant proteins. The performances obtained at the laboratory level are not reproducible at a large scale. Actually, the mixing operation is not efficient enough: gradients of glucose and oxygen appear when operating in fed-batch mode (addition of glucose during the culture). These gradients cause adverse impacts on the production of biomass and recombinant protein. The aim of this work is to use the microbial population as biocaptor of the encoutered stress inside heterogeneous industrial bioreactors to better scale-up and regulate these reactors. • MATERIALS AND METHODS: A plasmid carrying a stress promoter followed by the coding sequence of the Green Fluorescent Protein (GFP) is introduced in the bacterial host (in our case, the strain E. coli K12 will be considered as a model organism). When the cell is submitted to given stress condition, GFP synthesis is induced and accumulated into the cytoplasm, leading to the increase of the cell's fluorescence. Flow cytometry detection is used in order to quantify the fluorescence at the single cell level. Obtained results are frequency histograms of fluorescenceintensity in the microbial population • RESULTS: The rpoS gene is a gene of the general stress response, mainly induced at the entrance to stationary phase (during a lack of glucose). The tracking of the GFP fluorescence linked to the activation / repression of the rpoS promoter gives good results. Indeed, there is appearance of a segregation at the level of the GFP content among the microbial population. The intensity of the segregation, as well as its time of appearance during the culture can be related to the bioreactor mixing efficiency. • CONCLUSION: prpoS::gfp strains can be used as biosensors of the heterogeneity of glucose encountered inside industrial reactors. • POTENTIAL APPLICATIONS & KEY BENEFITS: These strains could be used to validate a fed-batch regulation (addition of glucose) at the industrial level

Bioreactor mixing efficiency modulates the activity of a prpoS::GFP reporter gene in E. coli

<p>Abstract</p> <p>Background</p> <p>Extensive studies have shown that up-scaling of bioprocesses has a significant impact on the physiology of the microorganisms. Among the factors associated with the fluid dynamics of the bioreactor, concentration gradients induced by loss of the global mixing efficiency associated with the increasing scale is the main phenomena leading to strong physiological modifications at the level of the microbial population. These changes are not fully understood since they involve complex physiological mechanisms. In this work, we intend to investigate, at the single cell level, the expression of the rpoS gene associated with the stress response of <it>E. coli</it>. The cultures of the reporter strain have been performed in a small scale reactor as well as in a series of scaled-down bioreactors able to induce extracellular perturbations with increasing level of magnitude.</p> <p>Results</p> <p>The rpoS level has been monitored by the aim of a transcriptional reporter gene based on the synthesis of the green fluorescent protein (GFP). It has been observed that the level of GFP increases during the transition from batch to fed-batch phase. After this initial increase, the GFP content of the cell drops, primarily due to the dilution by cell division. However, a significant drop of the GFP content has been observed if using a partitioned bioreactor, for which the mixing conditions are very bad, leading to the exposure of the cells to cyclic and stochastic extracellular fluctuations. If considering the flow cytometric profile of the cell to cell GFP content, this drop has to be attributed to the appearance of segregation at the level of the GFP content among the microbial population.</p> <p>Conclusion</p> <p>The generation of extracellular perturbations (in the present case, at the level of the sugar concentration and the dissolved oxygen level) has led to a drop at the level of the rpoS expression level. This drop has to be attributed to a segregation phenomenon in microbial population, with a major sub-population exhibiting a low expression level and a minor sub-population keeping its initial elevated expression level. The intensity of the segregation, as well as its time of appearance during the culture can be related to the bioreactor mixing efficiency.</p

Use of on-line flow cytometry for the characterization of physiological behavior in stress conditions during the bioprocess

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Modified semi-continuous fermentation for resuscitating nongrowing cells during high-temperature gluconic acid production by Acetobacter senegalensis.

peer reviewedAIMS: The formation of metabolically inactive and nongrowing cells is an inevitable by-product of intensive fermentation. This study investigated whether co-feeding can be used to resuscitate nongrowing Acetobacter senegalensis cells to enable them to produce gluconic acid in successive fermentation runs at 38°C. METHODS AND RESULTS: In the first fermentation cycle, 75 g l(-1) of glucose were converted to gluconic acid. Subsequently, however, stationary-phase cells were unable to initiate a new fermentation cycle. The majority of stationary-phase cells (97%) were nonculturable on glucose at 38°C. In addition, 54 and 41% of cells contained non-active cellular dehydrogenases and a compromised cell envelope respectively. Co-feeding stationary-phase cells with a mixture of ethanol, glucose and acetic acid for 7 h enabled these cells to grow on 75 g l(-1) of glucose and produce gluconic acid. Additionally, 74% of cells contained active forms of cellular dehydrogenases after 7 h of co-feeding. However, co-feeding did not improve cell envelope integrity. Quantification of cellular NAD content showed that stationary-phase cells contained moderately reduced levels of total NAD (NADt) as compared with exponential-phase cells. Interestingly, the analysis of stationary-phase cells showed that co-feeding resulted in higher levels of NADt and NADH, suggesting that the regeneration of NADH is one of the limiting factors of glucose consumption. Expression of catalase and superoxide dismutase was increased in stationary-phase cells, but analysis of protein carbonylation and lipid peroxidation did not confirm an extensive oxidative stress. CONCLUSIONS: Co-feeding with favourable nutrients may enable resuscitation of cells and utilization of less-favourable carbon sources in successive cycles. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proposed a unique method for resuscitation of nongrowing cells during high-temperature fermentation. By applying this method, cells can be used for consecutive fermentation cycles

Raman spectroscopy-based measurements of single-cell phenotypic diversity in microbial populations

Microbial cells experience physiological changes due to environmental change, such as pH and temperature, the release of bactericidal agents, or nutrient limitation. This has been shown to affect community assembly and physiological processes (e.g., stress tolerance, virulence, or cellular metabolic activity). Metabolic stress is typically quantified by measuring community phenotypic properties such as biomass growth, reactive oxygen species, or cell permeability. However, bulk community measurements do not take into account single-cell phenotypic diversity, which is important for a better understanding and the subsequent management of microbial populations. Raman spectroscopy is a nondestructive alternative that provides detailed information on the biochemical makeup of each individual cell. Here, we introduce a method for describing single-cell phenotypic diversity using the Hill diversity framework of Raman spectra. Using the biomolecular profile of individual cells, we obtained a metric to compare cellular states and used it to study stress-induced changes. First, in two Escherichia coli populations either treated with ethanol or nontreated and then in two Saccharomyces cerevisiae subpopulations with either high or low expression of a stress reporter. In both cases, we were able to quantify single-cell phenotypic diversity and to discriminate metabolically stressed cells using a clustering algorithm. We also described how the lipid, protein, and nucleic acid compositions changed after the exposure to the stressor using information from the Raman spectra. Our results show that Raman spectroscopy delivers the necessary resolution to quantify phenotypic diversity within individual cells and that this information can be used to study stress-driven metabolic diversity in microbial populations. IMPORTANCE Microbial cells that live in the same community can exist in different physiological and morphological states that change as a function of spatiotemporal variations in environmental conditions. This phenomenon is commonly known as phenotypic heterogeneity and/or diversity. Measuring this plethora of cellular expressions is needed to better understand and manage microbial processes. However, most tools to study phenotypic diversity only average the behavior of the sampled community. In this work, we present a way to quantify the phenotypic diversity of microbial samples by inferring the (bio)molecular profile of its constituent cells using Raman spectroscopy. We demonstrate how this tool can be used to quantify the phenotypic diversity that arises after the exposure of microbes to stress. Raman spectroscopy holds potential for the detection of stressed cells in bioproduction

Utilisation d’un bio-réacteur scale-down pour la production de lipase à partir de Yarrowia lipolytica

Les résultats obtenus au niveau de fermenteur de laboratoire et de volume industriel sont souvent différents à cause de la diminution de l’efficacité d’homogénéisation (substrats, oxygène dissous …etc.) du réacteur lorsque le volume augmente tels que. Dans cette optique, le fermenteur scale-down peut être utilisé. Il s’agit d’un système permettant de représenter à petite échelle les conditions de mélange des réacteurs de taille industrielle. Les lipases microbiennes sont stables dans de nombreux solvants organiques et ne requièrent pas de co-facteur pour être actives. Leurs domaines d'application sont très vastes et variés. Pour la production de la lipase, la levure non conventionnelle présente de nombreuses potentialités. A ce propos, Yarrowia lipolytica est le microorganisme le plus important vu qu’il produit les lipases à l’échelle industrielle. Pour des raisons économiques, les conditions de culture (pH, dispersion liquide-liquide, aération, …etc.) lors de la production de la lipase, doivent être optimalisées. Ce poster présente les résultats de l’effet de l’extrapolation sur la production de lipase à partir de Yarrowia lipolytica en mode batch avec un milieu de production optimisé (Destain et al., 2005). Les effets de la concentration en oxygène dissous, la dispersion liquide-liquide et les gradients de pH, qui sont affectés par le mélange du milieu de culture, sont présentés dans ce poster. Dans cette optique, plusieurs dispositifs scale-down ont été utilisés. Ceux-ci permettent de reproduire à l’échelle du laboratoire les hétérogénéités rencontrées à l’échelle industrielle

Control of phenotypic diversification based on serial cultivations on different carbon sources leads to improved bacterial xylanase production

peer reviewedThermobacillus xylanilyticus is a thermophilic and hemicellulolytic bacterium of interest for the production of thermostable hemicellulases. Enzymes’ production by this bacterium is challenging, because the proliferation of a cheating subpopulation of cells during exponential growth impairs the production of xylanase after serial cultivations. Accordingly, a strategy of successive cultivations with cells transfers in stationary phase and the use of wheat bran and wheat straw as carbon sources were tested. The ratio between subpopulations and their corresponding metabolic activities were studied by flow cytometry and the resulting hemicellulases production (xylanase, acetyl esterase and β-xylosidase) followed. During serial cultivations, the results pointed out an increase of the enzymatic activities. On xylan, compared to the first cultivation, the xylanase activity increases by 7.15-fold after only four cultivations. On the other hand, the debranching activities were increased by 5.88-fold and 57.2-fold on wheat straw and by 2.77-fold and 3.34-fold on wheat bran for β-xylosidase and acetyl esterase, respectively. The different enzymatic activities then stabilized, reached a plateau and further decreased. Study of the stability and reversibility of the enzyme production revealed cell-to-cell heterogeneities in metabolic activities which could be linked to the reversibility of enzymatic activity changes. Thus, the strategy of successive transfers during the stationary phase of growth, combined with the use of complex lignocellulosic substrates as carbon sources, is an efficient strategy to optimize the hemicellulases production by T. xylanilyticus, by preventing the selection of cheaters. © 2022, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature

From diverse origins to specific targets: Role of microorganisms in indirect pest biological control

Integrated pest management (IPM) is today a widely accepted pest management strategy to select and use the most efficient control tactics and at the same time reduce over-dependence on chemical insecticides and their potentially negative environmental effects. One of the main pillars of IPM is biological control. While biological control programs of pest insects commonly rely on natural enemies such as predatory insects, parasitoids and microbial pathogens, there is increasing evidence that plant, soil and insect microbiomes can also be exploited to enhance plant defense against herbivores. In this mini-review, we illustrate how microorganisms from diverse origins can contribute to plant fitness, functional traits and indirect defense responses against pest insects, and therefore be indirectly used to improve biological pest control practices. Microorganisms in the rhizosphere, phyllosphere and endosphere have not only been shown to enhance plant growth and plant strength, but also promote plant defense against herbivores both above-and belowground by providing feeding deterrence or antibiosis. Also, herbivore associated molecular patterns may be induced by microorganisms that come from oral phytophagous insect secretions and elicit plant-specific responses to herbivore attacks. Furthermore, microorganisms that inhabit floral nectar and insect honeydew produce volatile organic compounds that attract beneficial insects like natural enemies, thereby providing indirect pest control. Given the multiple benefits of microorganisms to plants, we argue that future IPMs should consider and exploit the whole range of possibilities that microorganisms offer to enhance plant defense and increase attraction, fecundity and performance of natural enemies. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Combined utilization of metabolic inhibitors to prevent synergistic multi-species biofilm formation.

peer reviewedBiofilm is ubiquitous in industrial water systems, causing biofouling and leading to heat transfer efficiency decreases. In particular, multi-species living in biofilms could boost biomass production and enhance treatment resistance. In this study, a total of 37 bacterial strains were isolated from a cooling tower biofilm where acetic acid and propionic acid were detected as the main carbon sources. These isolates mainly belonged to Proteobacteria and Firmicutes, which occupied more than 80% of the total strains according to the 16S rRNA gene amplicon sequencing. Four species (Acinetobacter sp. CTS3, Corynebacterium sp. CTS5, Providencia sp. CTS12, and Pseudomonas sp. CTS17) were observed co-existing in the synthetic medium. Quantitative comparison of biofilm biomass from mono- and multi-species showed a synergistic effect towards biofilm formation among these four species. Three metabolic inhibitors (sulfathiazole, 3-bromopyruvic acid, and 3-nitropropionic acid) were employed to prevent biofilm formation based on their inhibitory effect on corresponding metabolic pathways. All of them displayed evident inhibition profiles to biofilm formation. Notably, combining these three inhibitors possessed a remarkable ability to block the multi-species biofilm development with lower concentrations, suggesting an enhanced effect appeared in simultaneous use. This study demonstrates that combined utilization of metabolic inhibitors is an alternative strategy to prevent multi-species biofilm formation