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Among hemicellulases, xylanases are catalysts of considerable interest so as fundamental than applied point of view. However,it is paradoxical to note that the high cost of their production limits their use on a large scale. The use of purified xylan as culturesubstrate increases the production cost of the enzyme. Consequently, for commercial applications, it is advisable to developprocesses starting from inexpensive substrates. The purpose of this study is to optimise xylanases production in solid-statefermentation based on agricultural residues. The strain is Penicillium canescens 10-10c, selected in our laboratory for his abilityto produce xylanase activity free of cellulase. Assays concern optimization of different culture parameters in order to developin the future a solid-state fermentation reactor with soya oil cake. These parameters are: medium composition, temperatureincubation, induction and repression mechanisms. Soya oil cake in pellets (size > 10 mm) gave a higher enzymatic activity.Great volume of culture medium reduced the enzymatic production. The presence of lactose, saccharose or starch of corn hasa positive effect on the production of xylanase while the presence of xylose, mannose, galactose, arabinose, cellobiose andpectin or methylcellulose reduces the production of xylanase. The sources of phosphorus (di-potassic and di-sodic) enhancexylanase production. The enzymatic production obtained in Erlenmeyer flasks (250 ml) after 7 days incubation at 30°C isabout 14 000 U.g-1 of carbon source. The nature of inoculum affects the enzymatic productivity. Indeed, better productivitywas obtained with inoculation by solid preculture (956 U.g-1 per day) than liquid preculture (473 U.g-1 per day) and sporessuspension (383 U.g-1 per day). These observed enzymatic activity levels are higher than those related in the literature, whichshows all the potentialities of this strain and this technique for the production of xylanase and allow to develop our solid-statefermentation bioreactor.

Aims: 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. [ABSTRACT FROM AUTHOR]

Mangrove ecosystems are areas prone to various types of pollution, especially hydrocarbons. These hydrocarbons mostly stem from human activities such as spills coming from offshore oil operations, runoff from surrounding urban areas or atmospheric deposition. This pollution causes the decline of mangroves, which results in an imbalance in the functioning of this particular ecosystem with damages to the microbiota. Biodegradation allows to restore these ecosystems. This biodegradation can only be effective in specific environmental conditions. The presence of nutrients, which stimulate bacterial growth and promote biodegradation, is a key parameter to be considered. During this experiment, we achieved biodegradation tests to assess the effect of nitrogen and phosphorus on the process. The results showed that the biodegradation rates were strongly bound to the presence of nutrients. The degradation rates depended on the medium. The treatment that reached the best rate of degradation of diesel after 10 days was the one using 20% of a nutrient solution (MSM) containing nitrogen and phosphorus. This treatment led to a maximal degradation of 84.7% ± 4.7% obtained in the flasks containing 20% of a nutrient solution (MSM) containing nitrogen and phosphorus. [ABSTRACT FROM AUTHOR]

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Abstract: The potentiality of a stochastic approach is examined in the case of a mixing model for stirred vessels. This model is interesting due to the probabilistic and discrete properties that can be used further to facilitate the implementation of a hydrodynamic model into complex reacting systems, such as those encountered in bioprocesses. Stochastic model performances are compared to well known deterministic compartment mixing models (CM). It appears that parameters coming from CM can be used in the stochastic approach and that they give equivalent results. A methodology is elaborated that simplifies the determination procedure of the adjustable parameters of the stochastic model. The most important parameter to determine is the time step of a simulation performed by the aim of the stochastic model. Indeed, the time step is not explicitly given by the model and a correlation is necessary to translate simulation intervals into real time increments. After an appropriate analysis of several mixing systems (single or multi-agitated), it appears that a simple correlation involving circulation time could be used to perform this translation. The correlation contains an adjustable parameter, which has been quantified for the operating conditions covered in the study. The circulation of micro-organisms was also simulated simply by using the transition matrix coming from the stochastic model, which shows the potentiality of this kind of model in the field of complex reacting systems, such as those encountered in bioprocesses. [Copyright &y& Elsevier]

A primary cDNA library of Aspergillus niger H1 was constructed using the switching mechanism at the 5′ end of the RNA transcript (SMART) technique. A total of 169 clones exhibited halos when grown on tricalcium phosphate medium, and the H-46 clone displayed a clear halo. The full-length cDNA of the clone H-46 clone was 1 407 bp in length with a complete open reading frame (ORF) of 816 bp, and it encoded a protein that contained 272 amino acids. Multiple alignment analysis revealed a high degree of homology between the ORFs of the H-46 clone and the Bax inhibitor family (BI-1-like) proteins of other fungi. Acetic acid was secreted by Escherichia coli DH5α that express the BI-1-like gene. The level attained was 492.52 mg L−1, which was associated with the release of 0.212 mg mL−1 of soluble phosphate at 28 h. These results showed that the heterologous expression of BI-1-like genes in Eschericha coli DH5α increased the secretion of acetic acid by altering the membrane permeability and enhancing the solubility of phosphate (P).

Abstract Background 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 E. coli. 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. Results 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. Conclusion 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.

Introduction. Over the centuries, bio-sourced compounds isolated from plants, insects and microorganisms have been a potent source of drugs for the treatment of human diseases. Literature. Bio-sourced extracts offer a wide diversity of compounds with a large number of potentially beneficial effects in humans. Serendipity has frequently played a key role in the discovery of new medicines. The canonical discovery of penicillin required both chance and a prepared mind to understand and exploit its potential for the treatment of human infections. Nowadays, most anti-cancer drugs currently in clinical use were at least partly discovered by a "fortunate happenstance". Conclusions. In this review, we recapitulate the story of one of these compounds, 2-propylpentanoic acid, derived from the Valeriana officinalis flowering plant and its path to validation as a cancer treatment.

The production of fungal metabolites and conidia at an industrial scale requires an adequate yield at relatively low cost. To this end, many factors are examined and the design of the bioreactor to be used for the selected product takes a predominant place in the analysis. One approach to addressing the issue is to integrate the scaling-up procedure according to the biological characteristics of the microorganism considered, i.e. in our case filamentous fungi. Indeed, the scaling-up procedure is considered as one of the major bottlenecks in fermentation technology, mainly due to the near impossibility of reproducing the ideal conditions obtained in small reactors designed for research purposes when transposing them to a much larger production scale. The present review seeks to make the point regarding the bioreactor design and its implementation for cultivation of filamentous fungi and the production of fungal metabolites according to different developmental stages of fungi of industrial interest. Solid-state (semi-solid), submerged, fermentation and biofilm reactors are analyzed. The different bioreactor designs used for these three processes are also described at the technological level.

Overview of current knowledge on management of hydrocarbon pollution in mangroves. Mangrove forests are vital in terms of biomass production and maintenance of the natural balance in coastal areas in the tropics. However, mangroves are subject to pollution from human activities. Oil pollution is one of the causes leading to the decline of mangroves, which represent only 1% of the area of all the world's tropical forests. Several techniques are available for the remediation of oil contaminated areas. Biodegradation appears to be the best suited to mangrove ecosystems. However this technique remains confined to the laboratory. Field tests in situ need to be conducted in order to evaluate the results obtained in the laboratory.

Bioreactor scale-up and design on the basis of physiologically relevant parameters: application to the production of lipase by Yarrowia lipolytica. Bioreactor scale-up often poses a serious issue during the industrial development of a bioprocess considering the numerous physical and biological phenomena occurring in the reacting volume. The basic principles of scale-up coming from the traditional chemical and process engineering approaches will be first reviewed and will be then compared to a new one involving recent development at the level of microbial strain manipulation. This "physiological" approach of scale-up involves directly a biological component of the system (by comparison with the traditional approach for scaling-up involving physical parameters indirectly linked to the physiological phenomena occurring in the bioreactor), i.e. the synthesis of a reporter fluorescent protein when microbial cells are exposed to stress. It will be shown how this principle can be used for a better understanding of the relationship between bioreactor hydrodynamics and microbial stress.

The impact of the fluctuations in dissolved oxygen tension (DOT) on the lipase production by Yarrowia lipolytica has been investigated in a scale-down reactor (SDR). This bioreactor comprises a 20 l agitated vessel with an automatic valve controlling the opening and closure of the air flow line. This kind of scale-down apparatus is used in order to generate DOT gradients encountered in large-scale, while maintaining the other environmental conditions constant. The impact of DOT fluctuations has been estimated at three levels of the lipase synthesis machinery: lipase gene expression, lipase translation, lipase excretion to the extracellular medium. Among these levels, the performance of lipase production under oscillating DOT was significantly affected at the lipase gene expression level.

Understanding and modeling the complex interactions between biological reaction and hydrodynamics are a key problem when dealing with bioprocesses. It is fundamental to be able to accurately predict the hydrodynamics behavior of bioreactors of different size and its interaction with the biological reaction. CFD can provide detailed modeling about hydrodynamics and mixing. However, it is computationally intensive, especially when reactions are taken into account. Another way to predict hydrodynamics is the use of "Compartment" or "Multi-zone" models which are much less demanding in computation time than CFD. However, compartments and fluxes between them are often defined by considering global quantities not representative of the flow. To overcome the limitations of these two methods, a solution is to combine compartment modeling and CFD simulations. Therefore, the aim of this study is to develop a methodology in order to propose a compartment model based on CFD simulations of a bioreactor. The flow rate between two compartments can be easily computed from the velocity fields obtained by CFD. The difficulty lies in the definition of the zones in such a way they can be considered as perfectly mixed. The creation of the model compartments from CFD cells can be achieved manually or automatically. The manual zoning consists in aggregating CFD cells according to the user's wish. The automatic zoning defines compartments as regions within which the value of one or several properties are uniform with respect to a given tolerance. Both manual and automatic zoning methods have been developed and compared by simulating the mixing of an inert scalar. For the automatic zoning, several algorithms and different flow properties have been tested as criteria for the compartment creation.

Trehalose is a reserve carbohydrate produced by Saccharomyces cerevisiae under stress conditions and used as a cryoprotectant during freeze drying. So the cellular content of trehalose is an important parameter to control in industrial context. Scale-down reactors have been used to produce Saccharomyces cerevisiae. These reactors permit to mimic the hydrodynamic conditions experienced by cells during large scale production. Three types of scale-down reactors have been tested, differing by the geometry of the non-mixed part and the recirculation flow rate. The results show that cells cultivated in scale-down reactors produced less trehalose compared to the reference reactor. These results are completed by the study of the expression of the TPS2 promoter coupled with a green fluorescent protein (GFP). TPS2 is a gene coding for a subunit of the enzymatic complex responsible of trehalose synthesis. This strain was produced in mini-bioreactors which are shake flasks equipped with a dissolved oxygen probe. This approach allows using trehalose, or related enzymes, as a cellular marker of the stress encountered by yeast in industrial process.

Heterogeneities caused by deficient mixing in large-scale bioreactors have been identified and described in literature. These heterogeneities affect physiological changes of microorganisms through its passage in different zones of concentrations. Consequently the differences in terms of productivities, qualities and/or yields of products of interest have been observed during scaling-up from laboratory to larger scales. For this reason, large-scale process improvement depends on the understanding of dynamic interactions between microbial responses and physical phenomena inside bioreactors. The dynamic responses of microorganisms are used as a tool for gaining insight into the fundamentals of microbial changes under a mixing-well controlled environment. Our research group has not only applied scale-down methodology to study the microbial responses at molecular, microscopic and macroscopic levels of observation, but also has used innovative strains and process engineering tools to evaluate fast dynamic responses of microorganism at time scales from seconds to minutes. This presentation focuses on the application of rational strategies in order to characterize distributed relaxation times of microorganisms which are considered to be constant at all fermentation scales. Experiments were conducted with reporter bioluminescent strains of Escherichia coli in which the luxCDABE operon was fused to promoters responding to different selected environmental stresses (dissolved oxygen, pH, temperature and substrate, etc.). Such strains allow real-time recording of the expression of genes involved in stress responses. Kinetic analyses of biomass, extracellular metabolites, inlet/exhaust gas, were carried out in order to determine mass balances and biological kinetic parameters. We will present our approach and the results concerning continuous culture of E. coli DPD2417 (nirB::luxCDABE) to monitor the microbial responses to oxygen limitation.

Much attention has focused on understanding microbial interactions leading to stable co-cultures. In this work, substrate pulsing was performed to promote better control of the metabolic niches (MNs) corresponding to each species, leading to the continuous co-cultivation of diverse microbial organisms. We used a cell-machine interface, which allows adjustment of the temporal profile of two MNs according to a rhythm, ensuring the successive growth of two species, in our case, a yeast and a bacterium. The resulting approach, called 'automated adjustment of metabolic niches' (AAMN), was effective for stabilizing both cooperative and competitive co-cultures. AAMN can be considered an enabling technology for the deployment of co-cultures in bioprocesses, demonstrated here based on the continuous bioproduction of p-coumaric acid. The data accumulated suggest that AAMN could be used not only for a wider range of biological systems, but also to gain fundamental insights into microbial interaction mechanisms., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Cell-to-cell heterogeneity presents challenges across various fields, from biomedicine to bioproduction, where precise cellular responses are vital. While single cell technologies have significantly enhanced our understanding of population heterogeneity, the predominant focus has been on monitoring intracellular compounds. Recognizing the added complexity introduced by the secretion system, in this review, we first provide a systematic overview of the distinct steps necessary for driving protein secretion. We discuss the various sources of noise acting from the synthesized preprotein to the secretory protein released based on a Gram-positive cellular system as a model. We next explore the applicability of single cell technologies for monitoring protein secretion throughout these functional stages. We also emphasize the importance of applying these single cell technologies for monitoring protein secretion during bioproduction., Competing Interests: Declaration of interests None declared by authors., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Decoupling cell formation from recombinant protein synthesis is a potent strategy to intensify bioprocesses. Escherichia coli strains with mutations in the glucose uptake components lack catabolite repression, display low growth rate, no overflow metabolism, and high recombinant protein yields. Fast growth rates were promoted by the simultaneous consumption of glucose and glycerol, and this was followed by a phase of slow growth, when only glucose remained in the medium. A glycerol-repressible genetic circuit was designed to autonomously induce recombinant protein expression. The engineered strain bearing the genetic circuit was cultured in 3.9 g L -1 glycerol + 18 g L -1 glucose in microbioreactors with online oxygen transfer rate monitoring. The growth was fast during the simultaneous consumption of both carbon sources (C-sources), while expression of the recombinant protein was low. When glycerol was depleted, the growth rate decreased, and the specific fluorescence reached values 17% higher than those obtained with a strong constitutive promoter. Despite the relatively high amount of C-source used, no oxygen limitation was observed. The proposed approach eliminates the need for the substrate feeding or inducers addition and is set as a simple batch culture while mimicking fed-batch performance., (© 2024 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

"Giving the cells exactly what they need, when they need it" is the core idea behind the proposed bioprocess control strategy: operating bioprocess based on the physiological behavior of the microbial population rather than exclusive monitoring of environmental parameters. We are envisioning to achieve this through the use of genetically encoded biosensors combined with online flow cytometry (FCM) to obtain a time-dependent "physiological fingerprint" of the population. We developed a biosensor based on the glnA promoter (glnAp) and applied it for monitoring the nitrogen-related nutritional state of Escherichia coli. The functionality of the biosensor was demonstrated through multiple cultivation runs performed at various scales-from microplate to 20 L bioreactor. We also developed a fully automated bioreactor-FCM interface for on-line monitoring of the microbial population. Finally, we validated the proposed strategy by performing a fed-batch experiment where the biosensor signal is used as the actuator for a nitrogen feeding feedback control. This new generation of process control, -based on the specific needs of the cells, -opens the possibility of improving process development on a short timescale and therewith, the robustness and performance of fermentation processes., (© 2024 Wiley Periodicals LLC.)

The genetic stability and metabolic robustness of production strains is one of the key criteria for the production of bio-based products by microbial fermentation on an industrial scale. These criteria were here explored in an industrial ethanol-producer strain of Saccharomyces cerevisiae able to co-ferment D-xylose and L-arabinose with glucose through the chromosomal integration of several copies of pivotal genes for the use of these pentose (C5) sugars. Using batch sequential cultures in a controlled bioreactor that mimics long-term fermentation in an industrial setting, this strain was found to exhibit significant fluctuations in D-xylose and L-arabinose consumption as early as the 50th generation and beyond. These fluctuations seem not related to the few low-consumption C5 sugar clones that appeared throughout the sequential batch cultures at a frequency lower than 1.5% and that were due to the reduction in the number of copies of transgenes coding for C5 sugar assimilation enzymes. Also, subpopulations enriched with low or high RAD52 expression, whose expression level was reported to be proportional to homologous recombination rate did not exhibit defect in C5-sugar assimilation, arguing that other mechanisms may be responsible for copy number variation of transgenes. Overall, this work highlighted the existence of genetic and metabolic instabilities in an industrial yeast which, although modest in our conditions, could be more deleterious in harsher industrial conditions, leading to reduced production performance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Duperray, Delvenne, François, Delvigne and Capp.)

Isogenic cell populations can cope with stress conditions by switching to alternative phenotypes. Even if it can lead to increased fitness in a natural context, this feature is typically unwanted for a range of applications (e.g., bioproduction, synthetic biology, and biomedicine) where it tends to make cellular response unpredictable. However, little is known about the diversification profiles that can be adopted by a cell population. Here, we characterize the diversification dynamics for various systems (bacteria and yeast) and for different phenotypes (utilization of alternative carbon sources, general stress response and more complex development patterns). Our results suggest that the diversification dynamics and the fitness cost associated with cell switching are coupled. To quantify the contribution of the switching cost on population dynamics, we design a stochastic model that let us reproduce the dynamics observed experimentally and identify three diversification regimes, i.e., constrained (at low switching cost), dispersed (at medium and high switching cost), and bursty (for very high switching cost). Furthermore, we use a cell-machine interface called Segregostat to demonstrate that different levels of control can be applied to these diversification regimes, enabling applications involving more precise cellular responses., (© 2023. Springer Nature Limited.)

Automated flow cytometry (FC) has been initially considered for bioprocess monitoring and optimization. More recently, new physical and software interfaces have been made available, facilitating the access to this technology for labs and industries. It also comes with new capabilities, such as being able to act on the cultivation conditions based on population data. This approach, known as reactive FC, extended the range of applications of automated FC to bioprocess control and the stabilization of cocultures, but also to the broad field of synthetic and systems biology for the characterization of gene circuits. However, several issues must be addressed before automated and reactive FC can be considered standard and modular technologies., Competing Interests: Declaration of Competing Interest No conflict is to be declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Human T-cell leukemia virus type-1 (HTLV-1) is the first pathogenic retrovirus discovered in human. Although HTLV-1-induced diseases are well-characterized and linked to the encoded Tax-1 oncoprotein, there is currently no strategy to target Tax-1 functions with small molecules. Here, we analyzed the binding of Tax-1 to the human homolog of the drosophila discs large tumor suppressor (hDLG1/SAP97), a multi-domain scaffolding protein involved in Tax-1-transformation ability. We have solved the structures of the PDZ binding motif (PBM) of Tax-1 in complex with the PDZ1 and PDZ2 domains of hDLG1 and assessed the binding of 10 million molecules by virtual screening. Among the 19 experimentally confirmed compounds, one systematically inhibited the Tax-1-hDLG1 interaction in different biophysical and cellular assays, as well as HTLV-1 cell-to-cell transmission in a T-cell model. Thus, our work demonstrates that interactions involving Tax-1 PDZ-domains are amenable to small-molecule inhibition, which provides a framework for the design of targeted therapies for HTLV-1-induced diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The current plastic pollution throughout the world is a rising concern that demands the optimization of biodegradation processes. One avenue for this is to identify plastic-degrading bacteria and associated enzymes from the gut bacteria of insect models such as Tenebrio molitor, Plodia interpunctella or Galleria mellonella that have the ability to ingest and rapidly degrade polyethylene. Therefore, this study takes part in understanding the role of the gut bacteria by investigating G. mellonella as a biological model feeding with a diet based on honeybee wax mixed or not with low-density polyethylene. Gut microbiome was analyzed by high throughput 16S rRNA sequencing, and Enterococcaceae and Oxalobacteraceae were found to be the major bacterial families. Compared to the control, the supplementation of low-density polyethylene did not cause significant modification of the bacterial microbiota at community and taxa levels, suggesting bacterial microbiome resilience. The bacterial proteome analysis of gut contents was encouraging for the identification of plastic degrading enzymes such as the phenylacetaldehyde dehydrogenase which participate in styrene degradation. This study allowed a better characterization of the gut bacteria of G. mellonella and provided a basis for the further study of biodegradation of polyethylene based on the bacterial microbiota from insect guts., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Different expression vectors are available for the effective production of recombinant proteins by bacterial populations. However, the productivity of such systems is limited by the inherent noise of the gene circuits used for the synthesis of recombinant products. An extreme case of cell-to-cell heterogeneity that has been previously reported for the ara- and lac-based expression systems in E. coli is the all-or-none response. According to this mode of response, two subpopulations of cells are generated, i.e., a "low-" subpopulation exhibiting a shallow expression level and a "high-" subpopulation exhibiting a high-expression level. The "low-" subpopulation can be considered as a cluster of non-producing cells contributing to the loss of productivity. Here we describe the setup, design, and operation of a continuous culture where inducer addition is operated based on microbial population dynamics. The determination of population dynamics is done based on an automated flow cytometry (FC) procedure previously denoted as segregostat. We illustrate how this setup can be used to control the activation of an ara-based expression system and avoid phenotypic diversification leading to an all-or-none response. Upon the determination of the natural frequency of the gene circuit used as an expression system, our current protocol can be set up without the requirement of a feedback controller., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Microbial consortia are an exciting alternative for increasing the performances of bioprocesses for the production of complex metabolic products. However, the functional properties of microbial communities remain challenging to control, considering the complex interaction mechanisms occurring between co-cultured microbial species. Indeed, microbial communities are highly dynamic and can adapt to changing environmental conditions through complex mechanisms, such as phenotypic diversification. We focused on stabilizing a co-culture of Saccharomyces cerevisiae and Escherichia coli in continuous cultures. Our preliminary data pointed out that transient diauxic shifts could lead to stable co-culture by providing periodic fitness advantages to the yeast. Based on a computational toolbox called MONCKS (for MONod-type Co-culture Kinetic Simulation), we were able to predict the dynamics of diauxic shift for both species based on a cybernetic approach. This toolbox was further used to predict the frequency of diauxic shift to be applied to reach co-culture stability. These simulations were successfully reproduced experimentally in continuous bioreactors with glucose pulsing. Finally, based on a bet-hedging reporter, we observed that the yeast population exhibited an increased phenotypic diversification process in co-culture compared with mono-culture, suggesting that this mechanism could be the basis of the metabolic fitness of the yeast., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Martinez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The modification of glucose import capacity is an engineering strategy that has been shown to improve the characteristics of Escherichia coli as a microbial factory. A reduction in glucose import capacity can have a positive effect on production strain performance, however, this is not always the case. In this study, E. coli W3110 and a group of four isogenic derivative strains, harboring single or multiple deletions of genes encoding phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent transporters as well as non-PTS transporters were characterized by determining their transcriptomic response to reduced glucose import capacity., Results: These strains were grown in bioreactors with M9 mineral salts medium containing 20 g/L of glucose, where they displayed specific growth rates ranging from 0.67 to 0.27 h -1 , and specific glucose consumption rates (qs) ranging from 1.78 to 0.37 g/g h. RNA-seq analysis revealed a transcriptional response consistent with carbon source limitation among all the mutant strains, involving functions related to transport and metabolism of alternate carbon sources and characterized by a decrease in genes encoding glycolytic enzymes and an increase in gluconeogenic functions. A total of 107 and 185 genes displayed positive and negative correlations with qs, respectively. Functions displaying positive correlation included energy generation, amino acid biosynthesis, and sugar import., Conclusion: Changes in gene expression of E. coli strains with impaired glucose import capacity could be correlated with qs values and this allowed an inference of the physiological state of each mutant. In strains with lower qs values, a gene expression pattern is consistent with energy limitation and entry into the stationary phase. This physiological state could explain why these strains display a lower capacity to produce recombinant protein, even when they show very low rates of acetate production. The comparison of the transcriptomes of the engineered strains employed as microbial factories is an effective approach for identifying favorable phenotypes with the potential to improve the synthesis of biotechnological products., (© 2022. The Author(s).)

Thermobacillus 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.)

It has been more than a decade since Acetobacter senegalensis was isolated, identified and described as a thermotolerant strain of acetic acid bacteria. It was isolated from mango fruits in Senegal and used for industrial vinegar production in developing countries, mainly in sub-Saharan Africa. The strain was tested during several spirit vinegar fermentation processes at relatively high temperatures in accordance with African acclimation. The upstream fermentation process had significant stress factors, which are highlighted in this review so that the fermentation process can be better controlled. Due to its high industrial potential, this strain was extensively investigated by diverse industrial microbiologists worldwide; they concentrated on its microbiological, physiological and genomic features. A research group based in Belgium proposed an important project for the investigation of the whole-genome sequence of A. senegalensis. It would use a 454-pyrosequencing technique to determine and corroborate features that could give this strain significant diverse bio-industrial applications. For instance, its application in cocoa bean fermentation has made it a more suitable acetic acid bacterium for the making of chocolate than Acetobacter pasteurianus. Therefore, in this paper, we present a review that summarizes the current research on A. senegalensis at its microbial and genomic levels and also its specific bio-industrial applications, which can provide economic opportunities for African agribusiness. This review summarizes the physiological and genomic characteristics of Acetobacter senegalensis, a thermotolerant strain isolated from mango fruits and intended to be used in industrial vinegar fermentation processes. It also explores other bio-industrial applications such as cocoa fermentation. Vinegar fermentation is usually performed with mesophilic strains in temperate regions of the world. Developing countries, such as Senegal, import vinegar or make 'fake' vinegar by diluting acetic acid obtained from petrochemicals. The use of a thermotolerant Acetobacter senegalensis strain as a solid functional starter culture, as well as the design of a new adapted bioreactor, has significantly contributed to food security and the creation of small- to medium-sized enterprises that produce mango vinegar in West Africa., (© 2022 Society for Applied Microbiology.)

Competence for DNA transformation is a major strategy for bacterial adaptation and survival. Yet, this successful tactic is energy-consuming, shifts dramatically the metabolism, and transitory impairs the regular cell-cycle. In streptococci, complex regulatory pathways control competence deactivation to narrow its development to a sharp window of time, a process known as competence shut-off. Although characterized in streptococci whose competence is activated by the ComCDE signaling pathway, it remains unclear for those controlled by the ComRS system. In this work, we investigate competence shut-off in the major human gut commensal Streptococcus salivarius. Using a deterministic mathematical model of the ComRS system, we predicted a negative player under the control of the central regulator ComX as involved in ComS/XIP pheromone degradation through a negative feedback loop. The individual inactivation of peptidase genes belonging to the ComX regulon allowed the identification of PepF as an essential oligoendopeptidase in S. salivarius. By combining conditional mutants, transcriptional analyses, and biochemical characterization of pheromone degradation, we validated the reciprocal role of PepF and XIP in ComRS shut-off. Notably, engineering cleavage site residues generated ultra-resistant peptides producing high and long-lasting competence activation. Altogether, this study reveals a proteolytic shut-off mechanism of competence in the salivarius group and suggests that this mechanism could be shared by other ComRS-containing streptococci., Competing Interests: The authors of this manuscript have the following competing interests: PH is a Research Director at FNRS.

Cocultures have been widely explored for their use in deciphering microbial interaction and its impact on the metabolisms of the interacting microorganisms. In this work, we investigate, in different liquid coculture conditions, the compatibility of two microorganisms with the potential for the biocontrol of plant diseases: the fungus Trichoderma harzianum IHEM5437 and the bacterium Bacillus velezensis GA1 (a strong antifungal lipopeptide producing strain). While the Bacillus overgrew the Trichoderma in a rich medium due to its antifungal lipopeptide production, a drastically different trend was observed in a medium in which a nitrogen nutritional dependency was imposed. Indeed, in this minimum medium containing nitrate as the sole nitrogen source, cooperation between the bacterium and the fungus was established. This is reflected by the growth of both species as well as the inhibition of the expression of Bacillus genes encoding lipopeptide synthetases. Interestingly, the growth of the bacterium in the minimum medium was enabled by the amendment of the culture by the fungal supernatant, which, in this case, ensures a high production yield of lipopeptides. These results highlight, for the first time, that Trichoderma harzianum and Bacillus velezensis are able, in specific environmental conditions, to adapt their metabolisms in order to grow together.

Microbial populations can adapt to adverse environmental conditions either by appropriately sensing and responding to the changes in their surroundings or by stochastically switching to an alternative phenotypic state. Recent data point out that these two strategies can be exhibited by the same cellular system, depending on the amplitude/frequency of the environmental perturbations and on the architecture of the genetic circuits involved in the adaptation process. Accordingly, several mitigation strategies have been designed for the effective control of microbial populations in different contexts, ranging from biomedicine to bioprocess engineering. Technically, such control strategies have been made possible by the advances made at the level of computational and synthetic biology combined with control theory. However, these control strategies have been applied mostly to synthetic gene circuits, impairing the applicability of the approach to natural circuits. In this review, we argue that it is possible to expand these control strategies to any cellular system and gene circuits based on a metric derived from this information theory, i.e., mutual information (MI). Indeed, based on this metric, it should be possible to characterize the natural frequency of any gene circuits and use it for controlling gene circuits within a population of cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Henrion, Delvenne, Bajoul Kakahi, Moreno-Avitia and Delvigne.)