Publications

Abstract Spontaneous biological oscillations are typically attributed to specific architectures within metabolic or gene regulatory networks. Here, we uncover a more general mechanism arising from the interplay between cellular growth, burdensome gene expression, and nutrient availability, which can generate oscillations in both growth and gene expression. Focusing on sporulation dynamics in Bacillus subtilis , we developed a minimal model that captures these coupled processes and analytically identified the range of continuous culture conditions that give rise to oscillatory behavior. These predictions were experimentally validated in chemostat cultures. Our results demonstrate that oscillations can emerge independently of specific genetic circuit architectures, and without external forcing. More broadly, they reveal that feedback between environmental conditions and cellular states is sufficient to drive oscillatory dynamics, suggesting that such behavior may be widespread in long-term cultivation systems where gene expression, growth, and resource availability are tightly coupled.

The fall armyworm (FAW), Spodoptera frugiperda, is a highly polyphagous pest that has rapidly expanded across Africa, Asia, and Oceania, threatening food security and agricultural productivity. Reliance on synthetic insecticides for FAW management is increasingly challenged by insecticide resistance, environmental contamination, and adverse effects on non-target organisms and human health. This review synthesizes current knowledge on the potential of endophytic fungi as a sustainable strategy for FAW management. Following a systematic literature search, 59 original research articles published over the past 41 years were evaluated. These studies investigated 44 fungal species across 37 host crops, with Zea mays representing the most extensively studied system. The fungal species most frequently assessed were Beauveria bassiana, Metarhizium anisopliae sensu lato (s.l.), and Epichloe coenophialum. Overall, several fungal species successfully colonized plant tissues and reduced FAW performance through increased larval mortality, delayed development, and reduced feeding. These effects were associated with the production of bioactive secondary metabolites, induction of plant defense responses, and alteration of volatile organic compound emissions. Despite these promising findings, important knowledge gaps remain. Studies assessing effects on non-target organisms and natural enemies are scarce, and field validation remains limited. Research efforts are also geographically biased, with relatively few studies conducted in Africa despite the continent experiencing some of the highest FAW-related losses. Future research should prioritize field evaluations, multitrophic interaction studies, investigations in underrepresented regions, and socio-economic analyses to support farmer adoption. Addressing these gaps will be critical for determining the practical role of endophytic fungi in sustainable FAW management.

Rapid, low-cost ethanol quantification is vital for beverage quality control, biofuel production, and pharmaceutical applications, yet current approaches are costly, reagent- or label-dependent, or rely on spectroscopy with substantial sample preparation. We introduce a purely cell-based, label-free biosensor that exploits temperature-gradient-induced spontaneous detachment of Saccharomyces cerevisiae from a chip surface. The readout is the detachment half-time, td50, derived from time-resolved changes in interfacial thermal resistance, Rth, at the solid–liquid interface. Cells were pre-exposed to ethanol (0–70% v/v) and the detachment kinetics monitored using the heat transfer method (HTM). Under these conditions, cells display a pronounced non-monotonic td50 response with a peak around 20% v/v ethanol. Overall, the td50 rises from ~45 min (0% ethanol) to ≳10 h (20%) and then decreases, with no detachment at 60% and beyond. Critically, cell quality gates the detachment window. Fresh yeast responds up to ~50%, whereas aged yeast ceases to detach above ~8%, demonstrating a dual-function assay. Complementary measurements show that ethanol decreases surface tension monotonically, as expected, while optical/SEM imaging reveals aggregation above the detachment window. Requiring only a heater and a temperature probe, this platform offers a compact and low-cost strategy for ethanol sensing. Its applicability in a complex matrix is further demonstrated using whiskey diluted to selected alcohol concentrations, which produced responses consistent with the ethanol calibration trend. Potentially, it also offers a thermal assay for real-time monitoring of microbial cell quality across biotechnology and bioengineering applications. Considering ethanol as a proxy for drugs, the strategy may also support label-free drug screening on cells. At a fundamental level, the non-monotonic effect of ethanol, and especially the sharp maximum at 20%, remains unresolved and invites further studies.

Abstract How microbial populations respond to repeated environmental transitions determines both their ecological fitness and their utility in biotechnological applications. Using Pseudomonas putida KT2440 equipped with fluorescent biosensors and monitored by automated flow cytometry in the Segregostat platform, we show that exposure to benzoate, a plastic-derived aromatic feedstock, progressively reduces cellular responsiveness, defined as the fraction of cells that successfully activate a gene circuit following an environmental transition. Unlike classical switching costs, which promote phenotypic diversification, benzoate suppresses responsiveness without increasing population entropy, in a concentration-dependent and circuit-independent manner tightly correlated with fitness loss. A resource allocation model incorporating the competing demands of benzoate assimilation, toxicity, and tolerance reveals that this impairment emerges from a three-way competition for limited cellular resources. Above a critical benzoate load, insufficient resources remain available to sustain the adaptive reallocation required for circuit activation. In continuous culture, a non-responsive subpopulation accumulates as a leading indicator of population collapse. Exploiting this signal, we implement a two-stage connected bioreactor system in which benzoate feeding is autonomously regulated based on real-time population structure, enabling complete substrate consumption and stable operation at otherwise destabilizing concentrations. These results establish cellular responsiveness as a quantitative population variable and demonstrate that structure-aware feedback control, acting on population composition rather than bulk physiology, provides a principled route toward autonomous bioprocesses on challenging substrates.

ABSTRACT Glucose uptake through the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) enables rapid growth of Escherichia coli but promotes overflow metabolism that limits recombinant protein production. To define how glucose transport capacity shapes cellular physiology and bioprocess performance, we compared wild‐type W3110 with two PTS‐deficient derivatives, VH33 (Δ ptsHIcrr , constitutive galP expression) and WHIC (Δ ptsHIcrr Δ mglABC ), in controlled stirred‐tank bioreactors containing 20 g L − 1 glucose. PTS inactivation reduced specific growth and glucose uptake rates by up to 63% and decreased acetate accumulation by more than 95%, while final biomass concentrations remained comparable. Transcriptomic analysis revealed metabolic rewiring in the PTS − strains, which displayed highly similar global transcriptional profiles. However, the upregulation of alternative carbon metabolism and fimbrial genes in VH33 indicates subtle regulatory differences that may result in differential allocation of cellular resources. When expressing GFP, VH33, and WHIC achieved 2.5‐ and 4.4‐fold higher titers than W3110, respectively, with WHIC exhibiting the highest biomass‐specific yield (37.5 mg g − 1 ). Flow cytometry demonstrated strain‐dependent population heterogeneity and enrichment of recombinant protein in filamentous cells. Across strains, GFP synthesis rates approached growth dilution rates, indicating sustained intracellular accumulation. These results demonstrate that severe restriction of glucose import improves recombinant protein production by reshaping metabolic and physiological states.

Stabilizing microbial cocultures is a central challenge for bioproduction. While division of labor between strains can enhance efficiency, it often results in population instability over time. Classical strategies, including cross-feeding, quorum sensing, and toxin-antitoxin modules, often rely on complex ecological interactions that are difficult to predict or maintain under bioprocess conditions. We report the first implementation, to our knowledge, of a synthetic-niche-based coculture operated in a continuous bioreactor, using genetic toggle switches that couple growth to defined phenotypic states. We engineered two auxotrophic strains, TOGGLE_green and TOGGLE_yellow, in which growth is linked to either GFP- or YFP-expressing states and assessed their behavior under continuous bioreactor conditions using automated and reactive flow cytometry. Unexpectedly, the introduction of auxotrophic pressure reshaped circuit function, i.e., rather than maintaining bistability as typically reported in batch or microfluidic systems, toggle strains behaved as unidirectional inducible systems that reverted upon inducer withdrawal. This emergent behavior enabled simplified single-input control at the bioreactor scale, but also revealed a critical limitation for long-term operation, namely rapid mutational escape of the growth-impaired strain occurring within fewer than 10 generations in coculture, a markedly shorter time scale than the ∼50 generations typically reported in the literature. A simple repression-based ODE model recapitulated the reversible dynamics, capturing the effective inducible behavior observed under continuous cultivation, whereas deviations under prolonged operation highlighted the rapid evolutionary erosion of synthetic control. Our findings demonstrate both the potential and the limitations of synthetic niches as a scalable coculture control strategy.

Inferring protein production kinetics in dividing cells is complicated by protein inheritance from the mother cell. For instance, fluorescence measurements commonly used to assess gene activation may reflect not only newly produced proteins but also those inherited through successive cell divisions. In such cases, observed protein levels in any given cell are shaped by its division history. As a case study, we examine the activation of the glc3 gene in yeast involved in glycogen synthesis and expressed under nutrient-limiting conditions. We monitor this activity using snapshot fluorescence measurements via flow cytometry, where green fluorescent protein (GFP) expression reflects glc3 promoter activity. A naïve analysis of flow cytometry data ignoring cell division suggests many cells are active at low expression levels. Explicitly accounting for the (inherently non-Markovian) effects of cell division and protein inheritance makes it impossible to write down a tractable likelihood, namely the probability of observing data given a model—a key ingredient in physics-inspired inference. The dependence on a cell’s division history breaks the assumptions of standard (Markovian) master equations, rendering traditional likelihood-based approaches inapplicable. In order to generate a method for inference in arbitrary non-Markovian dynamics, we adapt conditional normalizing flows (a class of neural network models designed to learn probability distributions) to approximate otherwise intractable likelihoods from simulated data. In doing so, we find that glc3 is mostly inactive under stress, showing that while cells occasionally activate the gene, expression is brief and transient.

Controlling cell population dynamics and phenotypic diversification is a key objective in systems and synthetic biology, particularly for ensuring uniform responses from engineered gene circuits. While cell-machine interfaces have been employed to modulate host-gene circuit interactions, environmental perturbations typical of industrial bioreactor conditions remain underexplored. In this study, we investigate the impact of such perturbations on the general stress response in Escherichia coli and Saccharomyces cerevisiae. Using scale-down bioreactor experiments, we evaluate the performance of the Segregostat, a real-time control system that leverages automated flow cytometry to induce dynamic nutrient shifts. The Segregostat achieves robust stress response control, even under severe perturbations such as extended residence times in a two-compartment reactor. We hypothesise that this robustness arises from the system's ability to amplify host-compatible fluctuations beyond bioreactor-induced perturbations. Our findings highlight the importance of integrating environmental factors into control strategies for reliable gene circuit behaviour in industrial bioprocessing environments.

Microplastics (MPs) contaminate terrestrial, freshwater, and marine ecosystems worldwide, yet the mechanisms linking their ingestion, biological effects, and ecological redistribution by organisms remain poorly integrated across taxa and environments. Although many organisms ingest MPs, existing evidence is often fragmented by ecosystem or species group, limiting our ability to identify broader patterns. This review addresses this gap by examining fish and insects, two ecologically distinct and influential groups that collectively span all major ecosystems, to reveal cross-taxon insights in MP exposure and impacts. We synthesize current knowledge on MP sources, environmental distribution, and diversity, and compare the mechanistic pathways through which organisms are exposed to MPs and how such exposure affects physiology, behavior, development, reproduction, and gut microbiota. Despite their contrasting anatomies and life histories, fish and insects exhibit convergent responses to MPs and play key roles in their redistribution through trophic transfer, movement, and cross-ecosystem life cycles. Some species from both groups demonstrated the ability to alter or degrade polymers, likely mediated by their microbiota, with potential implications for MP fate. This cross-taxon perspective clarifies how individual-level effects scale to ecosystem processes and highlights uneven research efforts across taxa, which hinder accurate comparisons. This underscores the need for harmonized, standardized, and ecologically realistic approaches to advance global assessments of MP pollution.

The plastic-degrading capacity of some insects has been investigated over the past decade, with the aim of identifying gut microorganisms potentially involved in plastic degradation. However, plastic-only diets impose severe nutritional constraints, potentially driving microbial selection independently of plastic exposure. Here, we examined how nutritional stress influences gut bacterial community and the identification of plastic-associated bacteria in two plastivorous insects, Galleria mellonella and Tenebrio molitor, using polyurethane (PU) as a representative polymer. Bacterial communities were characterized by 16S rRNA gene sequencing under contrasted dietary conditions, including starvation, and complemented by a culture-dependent isolation approach using PU as the sole carbon source. In both species, gut bacterial communities under plastic-only feeding closely resembled those observed under starvation, whereas they differed from nutritionally balanced conditions. Differential abundance analyses reflected this pattern, as taxa enriched under plastic feeding were also enriched under starvation. This convergence was strong and structured in T. molitor, but weaker and more variable in G. mellonella. In addition, bacterial strains were isolated from the gut of T. molitor under both PU-amended and carbon-free conditions. Overall, our results demonstrate that nutritional stress is a driver of gut bacterial community restructuring under plastic-based diets and can bias the identification of candidate plastic-associated bacteria.

Archaea comprise deeply rooted and phylogenetically diverse lineages that inhabit a wide range of environments and play essential roles in global biogeochemical cycles. However, the diversity of MATE (Multidrug and Toxic Compound Extrusion) family transporters in archaea, which are presumably involved in habitat adaptation, remains poorly understood. Here, we systematically analyzed archaeal MatE transporters using large-scale phylogenetic and comparative genomic analyses, combined with structure-based clustering and molecular docking. Our results show that MatE transporters are significantly enriched in archaea from host-associated and hypersaline environments compared with those from other habitats. Specific MatE transporters are strongly associated with particular habitats, and their copy numbers are positively correlated with genome size. Moreover, MatE transporters in archaea exhibit structural diversity and can be classified into four structural classes, among which Class I is predominant in both abundance and phylogenetic distribution compared with Classes II, III, and IV. Overall, these findings indicate that the successful adaptation of archaea to specific habitats is related to the acquisition and maintenance of MatE transporters, which may be critical for their survival in these environments.

Abstract We present findings on a Fitness–Entropy Compensation (FEC) mechanism which offsets the activation of gene circuits that compromise survival. It counteracts the resulting fitness reduction by increasing the diversity in gene expression among individual cells within the population. This diversity, quantified by the Shannon entropy, enables cells with lower expression levels to support the survival of the entire population. We investigated the presence of FEC in a range of synthetic and stress-related genetic circuits in continuous culture. Our results reveal that it effectively stabilizes cell populations by mitigating the detrimental trade-offs between growth and gene expression. This stabilization is due to the reduced growth rate of the induced phenotype that leads to environmental changes, decreases induction strength, and promotes escape from unfit states. These findings suggest that the FEC mechanism may be a universal strategy for stabilization in various cellular systems and set the basis for a quantitative description of the trade-off between growth and gene expression and its consequences at the population level.

Publisher of over 50 scientific journals across the life, physical, earth, and health sciences, both independently and in partnership with scientific societies including Cell, Neuron, Immunity, Current Biology, AJHG, and the Trends Journals.

Methoxylated aromatic compounds, are abundant in subsurface ecosystems. Recently, it was discovered that Methermicoccus shengliensis can convert methoxylated aromatics to methane. Specifically, the MATE family transporters (MatE) and transduction-like protein (Tlp) were hypothesized to play a crucial role in substrate transport. However, their biological function and the transporting model remained unclear. To address this knowledge gap, we employed bacterial two-hybrid and structural model assays to investigate the interaction between Tlp and MatE. Our results revealed that Tlp senses 2-methoxybenzoate and interacts with MatE to facilitate substrate transport. Furthermore, we observed that the matE knock-out mutant significantly impaired the growth and methane production of M. shengliensis when using 2-methoxybenzoate as a substrate, highlighting the essential role of MatE in methoxydotrophic methanogenesis. Overall, our findings suggest that the MatE-Tlp system regulates substrate uptake and methane metabolism in M. shengliensis, providing new avenues for reducing global methane emissions caused by methanogens.

Abstract The activation of gene circuits can impose a significant burden on cells, leading to heterogeneous expression and reduced productivity. In this work, we focused on the T7 production system in E. coli BL21, a prime example of a burdensome gene circuit, to investigate the main cause for this gene expression heterogeneity and methods to mitigate it. Based on continuous cultivation analyzed and control by automated flow cytometry, we quantified the trade-off between cellular growth and gene expression and tracked the cell-to-cell heterogeneity in gene expression (measured as entropy). We concluded that the growth reduction associated to the activation of the burdensome gene circuit, i.e., the switching cost, is at the origin of the population heterogeneity. The loss of growth rate imposed by the burdensome activation of the gene is compensated at the population level by the overgrowth of less induced cells that safeguard the population by generating entropy. We tried to homogenize the population by pulsing the inducer with increasing frequency but found that the population escapes control through promoter mutation, leading to a genotype exhibiting reduced gene expression, but also, reduced entropy. To engineer a more homogeneous population without sacrificing gene expression, we decreased the switching cost associated to the induction by lowering the quality of the main carbon source. This strategy successfully led to a more homogeneous and productive population. Our approach allows for a precise quantification of the trade-off between growth and gene expression in cell population cultivated under dynamic conditions and highlights the importance of the switching cost for designing efficient approaches of cell population control.

Abstract Oscillators are fundamental to biological systems, underpinning essential processes such as cell division, circadian rhythms, and developmental cycles. While both natural and synthetic genetic oscillators have been extensively studied, oscillatory behaviors in cells can also emerge without dedicated genetic circuits. In earlier work, we uncovered sustained oscillations in phenotypic switching across diverse cellular systems and gene circuits, occurring spontaneously, without external forcing and linked them to the induction of slow-growing phenotypes. In this study, we identify the conditions that give rise to such intrinsic phenotypic instabilities, leading to population-level oscillations. We develop and analytically solve a simplified mathematical model of a stress-induced phenotype, mapping the range of continuous culture conditions that trigger oscillatory gene expression. This instability range, predicted by the model, was experimentally validated in Bacillus subtilis cultures. Our findings reveal that oscillations can arise in the complete absence of genetic oscillators or external perturbations. Although demonstrated here for a stress response in continuous culture, this phenomenon may occur in any long-term cultivation where environmental feedback links an inducer to the cellular system, broadening the landscape of possible oscillatory behaviors in microbiology and synthetic biology.

Abstract A lot of attention has been given to the understanding of microbial interactions leading to stable co-cultures, but the resulting technologies have been rarely challenged in dynamic cultivation conditions. In this work, substrate pulsing was performed to promote better control of the metabolic niches corresponding to each species, leading to the continuous co-cultivation of diverse microbial organisms. For this purpose, we used a cell-machine interface relying on automated flow cytometry, allowing to adjust the temporal profile of two metabolic niches according to a rhythm ensuring the successive growth of two species i.e., in our case a yeast and a bacterium. The resulting approach, called Automated Adjustment of Metabolic Niches (AAMN), was successfully employed for stabilizing both cooperative and competitive co-cultures. Additionally, AAMN can be considered as an enabling technology for the deployment of co-cultures in bioprocesses, demonstrated here based on the continuous bioproduction of p-coumaric acid. Taken altogether, the data accumulated suggest that AAMN could be used for a wider range of biological systems, but also to gain fundamental insights about microbial interaction mechanisms.

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.

Abstract 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.

"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.

Withdrawal Statement The authors have withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2021/430776. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The correct preprint can be found at doi: 10.1101/2021.02.11.430776

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.

Abstract Background The fall armyworm (FAW),Spodoptera frugiperda(J. E. Smith) (Lepidoptera: Noctuidae), poses a threat to the food security of populations in sub-Saharan Africa because of its damage to maize crops. As alternative to the use of hazardous pesticides, microbial control is one of the most promising sustainable approaches adopted to limit the damages caused byS. frugiperda. The sampling targeted mainly larvae ofS. frugiperda; however, during the survey, cadavers of earwig found on the same sampling sites were also collected and involved in the study. Cadavers of targeted insects, with and without sign of fungal infection, were sampled from 3 localities in eastern DR Congo. Culture of fungal isolates was performed in selective Sabouraud dextrose agar media. Results Morphological study of fungal features such as conidia (shape and size) and conidiophores showed that the isolates were from the genusBeauveria. Conidial measurements were highly variable and ranged from 2.4 to 3.6 µm in length and from 1.75 to 3.0 µm in width. Molecular characterization and phylogenetic analysis of the 2Beauveriaisolates based on DNA sequencing of ITS-5.8S region confirmed that both isolates belong toBeauveria bassiana. The 2 isolates ofB. bassianaP5E (OP419735.1) and KA14 (OP419734.1) were isolated from cadavers of FAW and earwig, respectively. The alignment with different sequences ofB. bassianafrom different continent showed that P5E belonged to the same clade of previous isolates reported from Iran and Mexico, while KA14 was with the same clade as isolates from Kenya and China. Conclusion To our knowledge, this is the first study reporting the occurrence ofB. bassianainfecting FAW and earwig in eastern DR Congo and in Africa.

Abstract 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, biomedicine…) where it tends to decrease the controllability of the cellular response. However, little is known about the diversification profiles that can be adopted by a cell population. We characterized 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). Interestingly, our results suggest that the diversification dynamics and the fitness cost associated with cell switching are coupled. For quantifying the contribution of the switching cost on population dynamics, we built a stochastic model that allowed us to reproduce the dynamics observed experimentally and identified 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 used a cell-machine interface that we call the Segregostat to demonstrate that different levels of control can be applied to these diversification regimes, enabling applications involving more precise cellular responses.

Abstract 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 analysed 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.

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.

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: , 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.

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.

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.

Carbon limitation is a common feeding strategy in bioprocesses to enable an efficient microbiological conversion of a substrate to a product. However, industrial settings inherently promote mixing insufficiencies, creating zones of famine conditions. Cells frequently traveling through such regions repeatedly experience substrate shortages and respond individually but often with a deteriorated production performance. A priori knowledge of the expected strain performance would enable targeted strain, process, and bioreactor engineering for minimizing performance loss. Today, computational fluid dynamics (CFD) coupled to data-driven kinetic models are a promising route for the in silico investigation of the impact of the dynamic environment in the large-scale bioreactor on microbial performance. However, profound wet-lab datasets are needed to cover relevant perturbations on realistic time scales. As a pioneering study, we quantified intracellular metabolome dynamics of Saccharomyces cerevisiae following an industrially relevant famine perturbation. Stimulus-response experiments were operated as chemostats with an intermittent feed and high-frequency sampling. Our results reveal that even mild glucose gradients in the range of 100 μmol·L−1 impose significant perturbations in adapted and non-adapted yeast cells, altering energy and redox homeostasis. Apparently, yeast sacrifices catabolic reduction charges for the sake of anabolic persistence under acute carbon starvation conditions. After repeated exposure to famine conditions, adapted cells show 2.7% increased maintenance demands.

BACKGROUND: The microbial production of hemicellulasic cocktails is still a challenge for the biorefineries sector and agro-waste valorization. In this work, the production of hemicellulolytic enzymes by Thermobacillus xylanilyticus has been considered. This microorganism is of interest since it is able to produce an original set of thermostable hemicellulolytic enzymes, notably a xylanase GH11, Tx-xyn11. However, cell-to-cell heterogeneity impairs the production capability of the whole microbial population. RESULTS: Sequential cultivations of the strain on xylan as a carbon source has been considered in order to highlight and better understand this cell-to-cell heterogeneity. Successive cultivations pointed out a fast decrease of xylanase activity (loss of ~ 75%) and Tx-xyn11 gene expression after 23.5 generations. During serial cultivations on xylan, flow cytometry analyses pointed out that two subpopulations, differing at their light-scattering properties, were present. An increase of the recurrence of the subpopulation exhibiting low forward scatter (FSC) signal was correlated with a progressive loss of xylanase activity over several generations. Cell sorting and direct observation of the sorted subpopulations revealed that the low-FSC subpopulation was not sporulating, whereas the high-FSC subpopulation contained cells at the onset of the sporulation stage. The subpopulation differences (growth and xylanase activity) were assessed during independent growth. The low-FSC subpopulation exhibited a lag phase of 10 h of cultivation (and xylanase activities from 0.15 ± 0.21 to 3.89 ± 0.14 IU/mL along the cultivation) and the high-FSC subpopulation exhibited a lag phase of 5 h (and xylanase activities from 0.52 ± 0.00 to 4.43 ± 0.61 over subcultivations). Serial cultivations on glucose, followed by a switch to xylan led to a ~ 1.5-fold to ~ 15-fold improvement of xylanase activity, suggesting that alternating cultivation conditions could lead to an efficient population management strategy for the production of xylanase. CONCLUSIONS: Taken altogether, the data from this study point out that a cheating behavior is responsible for the progressive reduction in xylanase activity during serial cultivations of T. xylanilyticus. Alternating cultivation conditions between glucose and xylan could be used as an efficient strategy for promoting population stability and higher enzymatic productivity from this bacterium.

Biofilm 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.

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.

Abstract 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.

ABSTRACT Human T-cell leukemia virus type-1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL). Although ATL is a well-characterized T-cell neoplasm, linked to intermittent expression of the viral Tax-1 protein, there is currently no strategy to target Tax-1 functions using small molecules. Here, we report a comprehensive interaction map between Tax-1 and human PDZ domain-containing proteins (hPDZome). We show that Tax-1 interacts with more than one-third of hPDZome components, including proteins involved in cell cycle, cell-cell junctions, cytoskeleton organization, and membrane complex assembly. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the structural basis of the interaction between the C -terminal PDZ binding motif (PBM) of Tax-1, and the PDZ domains of syntenin-1, an evolutionary conserved hub that controls exosome trafficking. Finally, we have used confocal imaging, molecular modelling, NMR and mammalian cell-based assays to demonstrate that the Tax-1/syntenin-1 interaction is amenable to small-molecule inhibition. Altogether, our study highlights the biological significance of Tax-PDZ interactome and its interplay with exosome formation. It shows a direct link between extracellular vesicles and HTLV-1 transmission, providing a novel framework for the design of targeted therapies for HTLV-1-induced diseases.

Predicting the fate of individual cells among a microbial population (i.e., growth and gene expression) remains a challenge, especially when this population is exposed to very dynamic environmental conditions, such as those encountered during continuous cultivation. Indeed, the dynamic nature of a continuous cultivation process implies the potential diversification of the microbial population resulting in genotypic and phenotypic heterogeneity. The present work focused on the induction of the arabinose operon in Escherichia coli as a model system to study this diversification process in continuous cultivations. As a preliminary step, the green fluorescent protein (GFP) level triggered by an arabinose-inducible ParaBAD promoter was tracked by flow cytometry in chemostat cultivations with glucose-arabinose co-feeding. For a wide range of glucose-arabinose co-feeding concentrations in the chemostats, the simultaneous occurrence of GFP positive and negative subpopulation was observed. In the second set of experiments, continuous cultivation was performed by adding glucose continuously and arabinose based on the capability of individual cells to switch from low GFP to high GFP expression states, performed with a technology setup called segregostat. In the segregostat cultivation mode, on-line flow cytometry analysis was used for adjusting the arabinose/glucose transitions based on the phenotypic switching profiles of the microbial population. This strategy allowed finding an appropriate arabinose pulsing frequency, leading to prolonged maintenance of the induction level with a limited increase in the phenotypic diversity for more than 60 generations. The results suggest that the steady forcing of individual cells into a given phenotypic trajectory may not be the best strategy for controlling cell populations. Instead, allowing individual cells to switch periodically around a predefined threshold seems to be a more robust strategy leading to oscillations, but within a predictable cell population behavior range.

Abstract The current plastic pollution throughout the world implies a crucial optimization of its (bio)degradation processes. In order to identify plastic degrading bacteria and associated enzymes, the gut microbiota of insects has raised interest. Some entomological models such as Tenebrio molitor (L. 1758), Plodia interpunctella (Hübner 1813) or Galleria mellonella (L. 1758) have the ability to ingest and degrade polyethylene. Then, it is promising to identify the composition and the role of the gut microbiota in this process. This study takes part in this issue by investigating G. mellonella as a biological model feeding with a polyethylene diet. Gut microbiome samples were processed by high throughput 16S rRNA sequencing, and Enterococcaceae and Oxalobacteraceae were found to be the major bacterial families. At low polyethylene dose, we detect no bacterial community change and no amplicon sequence variant associated with the polyethylene diet suggesting microbiome resilience. The functional analysis of insects gut content was promising for the identification of plastic degrading enzymes such as the phenylacetaldehyde dehydrogenase which participate in styrene degradation. This study allows a better characterization of the gut microbiota of G. mellonella and provides a basis for the further biodegradation study of polyethylene based on the microorganism valorization from insect guts.

Predicting the fate of a microbial population (i.e., growth, gene expression…) remains a challenge, especially when this population is exposed to very dynamic environmental conditions, such as those encountered during continuous cultivation. Indeed, the dynamic nature of continuous cultivation process implies the potential deviation of the microbial population involving genotypic and phenotypic diversification. This work has been focused on the induction of the arabinose operon in Escherichia coli as a model system. As a preliminary step, the GFP level triggered by an arabinose-inducible ParaBAD promoter has been tracked by flow cytometry in chemostat with glucose-arabinose co-feeding. For a large range of glucose-arabinose co-feeding, the simultaneous occurrence of GFP positive and negative subpopulation was observed. In a second set of experiments, continuous cultivation was performed by adding either glucose or arabinose, based on the ability of individual cells for switching from low GFP to high GFP states, according to a technology called segregostat. In segregostat mode of cultivation, on-line flow cytometry analysis was used for adjusting the arabinose/glucose transitions based on the phenotypic switching capabilities of the microbial population. This strategy allowed finding an appropriate arabinose pulsing frequency, leading to a prolonged maintenance of the induction level with limited impact on phenotypic diversity for more than 60 generations. This result suggests that constraining individual cells into a given phenotypic trajectory is maybe not the best strategy for directing cell population. Instead, allowing individual cells switching around a predefined threshold seems to be a robust strategy leading to oscillating, but predictable, cell population behavior.

Abstract The different steps involved in biofilm formation have been the subjects of intensive researches. However, the very early cell decision-making process related to the switch from planktonic to sessile state still remains uncharacterized. Based on the use of Pseudomonas putida KT2440 and derivatives with varying biofilm-forming capabilities, we observed a subpopulation of cells bound to extracellular DNA (eDNA) in the planktonic phase, as indicated by propidium iodide (PI) staining. Strikingly, the size of this eDNA-bound/PI-positive subpopulation correlated with the overall biofilm forming capability of the bacterial population. This finding challenges the conventional view of phenotypic switching and suggests that, in Pseudomonas , biofilm switching is determined collectively based on the quantity of eDNA released in the supernatant. The whole process can be followed based on automated flow cytometry, and the appearance of PI-positive cells was considered as an early-warning indicator for biofilm formation. For this purpose, automated glucose pulsing was used successfully to interfere with the proliferation of PI-positive cells, resulting in a reduction of biofilm formation. This study provides insights into the collective determinants of biofilm switching in Pseudomonas species and introduces a potential strategy for controlling biofilm formation.

Abstract Biofilm is ubiquitous in natural environments, causing biofouling in industrial water systems and leading to liquidity and heat transfer efficiency decreases. In particular, multi-species coexistence in biofilms can provide the synergy needed to boost biomass production and enhance treatment resistance. In this study, a total of 37 bacterial strains were isolated from a cooling tower where acetic acid and propionic acid were used as the primary 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 to co-exist in the synthetic medium, showing a synergistic effect towards biofilm formation. Three metabolic inhibitors (sulfathiazole, 3-Bromopyruvic acid, and 3-Nitropropionic acid) were employed as possible treatments against biofilm formation due to their inhibition effect on c-di-GMP biosynthesis or assimilation of volatile fatty acids. All of them displayed evident inhibition profiles to biofilm formation. Notably, the combination of these three inhibitors possessed a remarkable ability to block the development of a multi-species biofilm with lower concentrations, suggesting an enhanced effect with their simultaneous use. This study demonstrates that targeting cellular metabolism is an effective way to inhibit biofilm formation derived from multi-species.

Semi-continuous production of xanthan gum using self-immobilized Xanthomonas campestris cells in biofilm reactors was studied. Fermentation was carried out using two different designs of biofilm reactor equipped with a) stainless-steel support (SSS) and b) polyethylene support (PES). Fermentation was performed in three cycles with refreshing the media at the beginning of each: cycle 1, 0-27 h; cycle 2, 27-54 h; and cycle 3, 54-78.5 h. Results showed that the glucose consumption and the pH reduction in the PES biofilm reactor was faster compared to the SSS biofilm reactor. Scanning electron microscopy showed that the SSS was capable to immobilize more cells during the growth of X. campestris. The maximum concentration of xanthan gum in the SSS biofilm reactor obtained after 27 h (3.47 ± 0.71 g/L), while the maximum concentration of xanthan in the PES biofilm reactor obtained after 78.5 h (3.21 ± 0.68 g/L). Thermal stability analysis of xanthan using differential scanning calorimetry showed the presence of two fractures attributed to dehydration and degradation of polymer. The thermogram represented both endothermal and exothermal behaviour of xanthan polymer. Furthermore, the functional groups and molecular structure of the xanthan produced in this study was evaluated using Fourier transform infrared spectrometry and also proton nuclear magnetic resonance. in addition, the surface tension of (0.2 %, w/v) xanthan gum solution was in a range of 52.16-56.5 mN/m. Rheological analysis of xanthan showed that the G' values were higher than the G″ in all frequencies demonstrating a relatively high elasticity of the produced xanthan gum.

Abstract Predicting the fate of a microbial population (i.e., growth, gene expression…) remains a challenge, especially when this population is exposed to very dynamic environmental conditions, such as those encountered during continuous cultivation. Indeed, the dynamic nature of continuous cultivation process implies the potential deviation of the microbial population involving genotypic and phenotypic diversification. This work has been focused on the induction of the arabinose operon in Escherichia coli as a model system. As a preliminary step, the GFP level triggered by an arabinose-inducible P araBAD promoter has been tracked by flow cytometry in chemostat with glucose-arabinose co-feeding. For a large range of glucose-arabinose co-feeding, the simultaneous occurrence of GFP positive and negative subpopulation was observed. In a second set of experiments, continuous cultivation was performed by adding either glucose or arabinose, based on the ability of individual cells for switching from low GFP to high GFP states, according to a technology called segregostat. In segregostat mode of cultivation, on-line flow cytometry analysis was used for adjusting the arabinose/glucose transitions based on the phenotypic switching capabilities of the microbial population. This strategy allowed finding an appropriate arabinose pulsing frequency, leading to a prolonged maintenance of the induction level with limited impact on phenotypic diversity for more than 60 generations. This result suggests that constraining individual cells into a given phenotypic trajectory is maybe not the best strategy for directing cell population. Instead, allowing individual cells switching around a predefined threshold seems to be a robust strategy leading to oscillating, but predictable, cell population behavior.

There is a growing interest in the use of post-fermentation mycelial waste to obtain cell wall chitin as an added-value product. In the pursuit to identify suitable production strains that can be used for post-fermentation cell wall harvesting, we turned to an Aspergillus niger strain in which the kexB gene was deleted. Previous work has shown that the deletion of kexB causes hyper-branching and thicker cell walls, traits that may be beneficial for the reduction in fermentation viscosity and lysis. Hyper-branching of ∆kexB was previously found to be pH-dependent on solid medium at pH 6.0, but was absent at pH 5.0. This phenotype was reported to be less pronounced during submerged growth. Here, we show a series of controlled batch cultivations at a pH range of 5, 5.5, and 6 to examine the pellet phenotype of ΔkexB in liquid medium. Morphological analysis showed that ΔkexB formed wild type-like pellets at pH 5.0, whereas the hyper-branching ΔkexB phenotype was found at pH 6.0. The transition of phenotypic plasticity was found in cultivations at pH 5.5, seen as an intermediate phenotype. Analyzing the cell walls of ΔkexB from these controlled pH-conditions showed an increase in chitin content compared to the wild type across all three pH values. Surprisingly, the increase in chitin content was found to be irrespective of the hyper-branching morphology. Evidence for alterations in cell wall make-up are corroborated by transcriptional analysis that showed a significant cell wall stress response in addition to the upregulation of genes encoding other unrelated cell wall biosynthetic genes.

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.

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.

Continuous cultivation with E. coli has several benefits compared to classical fed-batch cultivation. The economic benefits would be a stable process, which leads to time independent quality of the product, and hence ease the downstream process. However, continuous biomanufacturing with E. coli is known to exhibit a drop of productivity after about four to five days of cultivation depending on dilution rate. These cultivations are generally performed on glucose, being the favorite carbon source for E. coli and used in combination with IPTG for induction. In recent works harsh induction with IPTG was changed to softer induction using lactose for the T7-based plasmids, with the result of reducing the metabolic stress and tunability of productivity. These mixed feed systems based on glucose and lactose result in high amounts of correctly folded protein. In this study we used different mixed feed systems with glucose/lactose and glycerol/lactose to investigate productivity of E. coli based chemostats. We tested three strains producing different model proteins, with the final aim of a stable long-time protein expression. While glucose fed chemostats showed the well-known drop in productivity after a certain process time, glycerol fed cultivations recovered productivity after about 200 h of induction, which corresponds to around 30 generation times. We want to further highlight that also the cellular response upon galactose utilization in BL21 might be a key parameter in process design to achieve stable productivity soon. This “Lazarus” phenomenon has not been described in literature before and may enable a stabilization of continuous cultivation with E. coli using different carbon sources.

In this study, conjugated linoleic acid (CLA) was produced using Lactobacillus plantarum in biofilm reactors with different carbon sources (glucose, lactose or a combination of glucose-lactose (1:1)). Presence of metal support in reactors resulted in high biofilm formation by L. plantarum. Two isomers of CLA were identified in fermentation broths; a) cis-9, trans-11 C18:2 (CLA1) as the predominant isomer (22.12 μg/mL), and b) trans-9, trans-11 C18:2 (CLA2) (14.96 μg/mL). Further analyses showed that depending on the carbon source used for the growth of L. plantarum in biofilm reactors, the amount of biomass and CLA production changed. Results indicated that by using combined lactose-glucose (1:1) as carbon source in biofilm reactors, the highest biomass (3.66 g/L) and the maximum concentration of CLA (37.08 μg/mL) were obtained. Scanning electron microscopy of the support used within the fermentation showed that the highest biofilm accumulation on the metal support was associated with the broth supplemented with combined glucose-lactose. This study suggested a high capacity of immobilized L. plantarum on the metal support to produce high quantity of CLA isomers with the use of combined glucose-lactose.

Abstract 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 other processes such as stress tolerance, virulence or cell physiology. Metabolic stress is one such physiological changes and is typically quantified by measuring community phenotypic properties such as biomass growth, reactive oxygen species or cell permeability. However, community measurements do not take into account single-cell phenotypic diversity, important for a better understanding and management of microbial populations. Raman spectroscopy is a non-destructive alternative that provides detailed information on the biochemical make-up 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 non-treated. 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 composition 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 communities. Importance Microbes that live in the same community respond differently to stress. This phenomemon is known as phenotypic diversity. Describing this plethora of expressions can help to better understand and manage microbial processes. However, most tools to study phenotypic diversity only average the behaviour of the community. In this work, we present a way to quantify the phenotypic diversity of single cells using Raman spectroscopy - a tool that can describe the molecular profile of microbes. We demonstrate how this tool can be used to quantify the phenotypic diversity that arises after the exposure of microbes to stress. We also show its potential as an ‘alarm’ system to detect when communities are changing into a ‘stressed’ type.

Two fungi, i.e., Aspergillus flavus Link and Aspergillus oryzae (Ahlb.) E. Cohn, were cultivated according to two methodologies, namely submerged and biofilm cultures with the primary aim to use their secondary metabolites the supernatant CL50, and CL90 varied between 1.3% (v/v) to 12.7% (v/v) for incubation times from 24 to 72 h. While the A. flavus supernatant entomotoxicity was higher than this of A. oryzae, the biofilm culture application increased the efficiency of the former. Proteomic analysis of the supernatants revealed discrepancies among the two species and modes of cultivation. Furthermore, the secondary metabolite profiles of both Aspergillus cultures were verified. Aspergillic acid, beta-cyclopiazonic acid, cyclopiazonic acid, ferrineospergillin, flavacol, and spermadin A were most predominant. Generally, these secondary metabolites were present in higher concentrations in the supernatants of A. flavus and biofilm cultures. These molecular identifications correlated positively with entomotoxic activity. Noteworthy, the absence of carcinogenic aflatoxins was remarkable, and it will allow further valorization to produce A. flavus to develop potential biopesticides.

Biofilm bioreactors are promising systems for continuous biosurfactant production since they provide process stability through cell immobilization and avoid foam formation. In this work, a two-compartment biofilm bioreactor was designed consisting of a stirred tank reactor and a trickle-bed reactor containing a structured metal packing for biofilm formation. A strong and poor biofilm forming B. subtilis 168 strain due to restored exopolysaccharides (EPS) production or not were cultivated in the system to study the growth behavior of the planktonic and biofilm population for the establishment of a growth model. A high dilution rate was used in order to promote biofilm formation on the packing and wash out unwanted planktonic cells. Biofilm development kinetics on the packing were assessed through a total organic carbon mass balance. The EPS+ strain showed a significantly improved performance in terms of adhesion capacity and surfactin production. The mean surfactin productivity of the EPS+ strain was about 37% higher during the continuous cultivation compared to the EPS- strain. The substrate consumption together with the planktonic cell and biofilm development were properly predicted by the model (α = 0.05). The results show the efficiency of the biofilm bioreactor for continuous surfactin production using an EPS producing strain.

The goal of the research was to assess the inhibitory effects of hop extracts, iso-α and β acids, against Pediococcus pentosaceus bacteria, during a short incubation period, both in liquid selective media (high pH values) and beer wort fermentation (low pH values) and testing if the identified iso-α acid stress changes the activity of S. cerevisiae boulardii yeast and ethanol production. Flow cytometry analysis was used for bacterial and yeast cell viability. In relation to the antibacterial activity of β-acids, a lower viability of Pediococcus pentosaceus cells was observed after a short incubation period in selective media, under iso-α acid stress. In beer wort, for a mixed culture with P. pentosaceus bacteria and S. cerevisiae boulardii yeast, under iso-α acid stress conditions at pH 4.0-5.0, Pediococcus pentosaceus exhibited lower cell viability (20.7%) than in selective media (61.4%). Regarding iso-α hop acid on S. cerevisiae boulardii yeast, the results showed that iso-α does not change the S. cerevisiae activity but prevents the culture from being contaminated by Pediococcus pentosaceus. The results highlighted reliable inhibitory effects of iso-α and β-acids against P. pentosaceus, both at pH 6.0-7.0 and pH 4.0-5.0, which open the possibility of hops being used as a supplement to prevent beverage contamination with spoilage microorganisms. ********* In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue. *********

Abstract To locate mating partners and essential resources such as food, oviposition sites and shelter, insects rely to a large extent on chemical cues. While most research has focused on cues derived from plants and insects, there is mounting evidence that indicates that micro‐organisms emit volatile compounds that may play an important role in insect behaviour. In this study, we assessed how volatile compounds emitted by phylogenetically diverse bacteria affected the olfactory response of the primary parasitoid Aphidius colemani and one of its secondary parasitoids, Dendrocerus aphidum . Olfactory responses were evaluated for volatile blends emitted by bacteria isolated from diverse sources from the parasitoid's habitat, including aphids, aphid mummies and honeydew, and from the parasitoids themselves. Results revealed that A. colemani showed a wide variation in response to bacterial volatiles, ranging from significant attraction over no response to significant repellence. Our results further showed that the olfactory response of A. colemani to bacterial volatile emissions was different from that of D. aphidum . Gas chromatography‐mass spectrometry analysis of the volatile blends revealed that bacterial strains repellent to A. colemani produced significantly higher amounts of esters, organic acids, aromatics and cycloalkanes than attractive strains. Strains repellent to D. aphidum produced significantly higher amounts of alcohols and ketones, whereas the strains attractive to D. aphidum produced higher amounts of the monoterpenes limonene, linalool and geraniol. Overall, our results indicate that bacterial volatiles can have an important impact on insect olfactory responses, and should therefore be considered as an additional, so far often overlooked factor in studying multitrophic interactions between plants and insects. A free Plain Language Summary can be found within the Supporting Information of this article.

Production of Cambodian rice wine involves complex microbial consortia. Indeed, previous studies focused on traditional microbial starters used for this product revealed that three microbial strains with complementary metabolic activities are required for an effective fermentation, i.e., filamentous fungi (Rhizopus oryzae), yeast (Saccharomyces cerevisiae), and lactic acid bacteria (Lactobacillus plantarum). Modulating the ratio between these three key players led to significant differences, not only in terms of ethanol and organic acid production, but also on the profile of volatile compounds, in comparison with natural communities. However, we observed that using an equal ratio of spores/cells of the three microbial strains during inoculation led to flavor profile and ethanol yield close to that obtained through the use of natural communities. Compartmentalization of metabolic tasks through the use of a biofilm cultivation device allows further improvement of the whole fermentation process, notably by increasing the amount of key components of the aroma profile of the fermented beverage (i.e., mainly phenylethyl alcohol, isobutyl alcohol, isoamyl alcohol, and 2-methyl-butanol) and reducing the amount of off-flavor compounds. This study is a step forward in our understanding of interkingdom microbial interactions with strong application potential in food biotechnology.

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. 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. Co‐feeding with favourable nutrients may enable resuscitation of cells and utilization of less‐favourable carbon sources in successive cycles. 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.

used traditional microbial starters revealed that effective fermentation requires three microbial strains with complementary metabolic activities: filamentous fungi (Rhizopus oryzae), yeast (Saccharomyces cerevisiae), and lactic acid bacteria (Lactobacillus plantarum). Relative to natural communities, modulation of the ratio of these three microorganisms led to significant differences not only in terms of ethanol and organic acid production, but also with the profile of volatile compounds. However, inoculation of an equal ratio of spores/cells of the three aforementioned microbial strains led to a flavor profile and ethanol yield similar to that obtained with natural communities. Compartmentalization of metabolic tasks through the use of a biofilm cultivation device allowed further improvement of the entire fermentation process, notably by increasing the amount of key components of the aroma profile of the fermented beverage (i.e., mainly phenylethyl alcohol, isobutyl alcohol, isoamyl alcohol, and 2-methyl-butanol) and reducing the amount of off-flavor compound. This study represents an initial step toward understanding interkingdom microbial interactions with a strong potential for application in the food biotechnology.

The fungal endophyte Cyanodermella asteris (C. asteris) has been recently isolated from the medicinal plant Aster tataricus (A. tataricus). This fungus produces astin C, a cyclic pentapeptide with anticancer and anti‐inflammatory properties. The production of this secondary metabolite is compared in immobilized and planktonic conditions. For immobilized cultures, a stainless steel packing immersed in the culture broth is used as a support. In these conditions, the fungus exclusively grows on the packing, which provides a considerable advantage for astin C recovery and purification. C. asteris metabolism is different according to the culture conditions in terms of substrate consumption rate, cell growth, and astin C production. Immobilized‐cell cultures yield a 30% increase of astin C production, associated with a 39% increase in biomass. The inoculum type as spores rather than hyphae, and a pre‐inoculation washing procedure with sodium hydroxide, turns out to be beneficial both for astin C production and fungus development onto the support. Finally, the influence of culture parameters such as pH and medium composition on astin C production is evaluated. With optimized culture conditions, astin C yield is further improved reaching a five times higher final specific yield compared to the value reported with astin C extraction from A. tataricus (0.89 mg g−1 and 0.16 mg g−1 respectively).

Controlling and managing the degree of phenotypic diversification of microbial populations is a challenging task. This task not only requires detailed knowledge regarding diversification mechanisms but also advanced technical set-ups for the real-time analyses and control of population behaviour on single-cell level. In this work, set-up, design and operation of the so called segregostat are described which, in contrast to a traditional chemostat, allows the control of phenotypic diversification of microbial populations over time. Two exemplary case studies will be discussed, i.e. phenotypic diversification dynamics of Eschericia coli and Pseudomonas putida based on outer membrane permeabilization, emphasizing the applicability and versatility of the proposed approach. Upon nutrient limitation, cell population tends to diversify into several subpopulations exhibiting distinct phenotypic features (non-permeabilized and permeabilized cells). Online analysis leads to the determination of the ratio between cells in these two states, which in turn triggers the addition of glucose pulses in order to maintain a predefined diversification ratio. These results prove that phenotypic diversification can be controlled by means of defined pulse-frequency modulation within continuously running bioreactor set-ups. This lays the foundation for systematic studies, not only of phenotypic diversification but also for all processes where dynamics single-cell approaches are required, such as synthetic co-culture processes.

Acetic acid bacteria are very vulnerable to environmental changes; hence, they should get acclimated to different kinds of stresses when they undergo downstream processing. In the present study, Acetobacter senegalensis, a thermo-tolerant strain, was acclimated sequentially to different carbon sources including glucose (condition Glc), a mixture of glucose and ethanol (condition EtOH) and a mixture of glucose and acetic acid (condition GlcAA). Then, the effects of acclimation on the cell proteome profiles and some phenotypic characteristics such as growth in culture medium containing ethanol, and tolerance to freeze-drying process were evaluated. Based on the obtained results, despite the cells acclimated to Glc or EtOH conditions, 86% of acclimated cells to GlcAA condition were culturable and resumed growth with a short lag phase in a culture medium containing ethanol and acetic acid. Interestingly, if A. senegalensis had been acclimated to condition GlcAA, 92% of cells exhibited active cellular dehydrogenases, and 59% of cells were culturable after freeze-drying process. Proteome profiles comparison by 2D-DiGE and MS analysis, revealed distinct physiological status between cells exposed to different acclimation treatments, possibly explaining the resulting diversity in phenotypic characteristics. Results of proteome analysis by 2D-DiGE also showed similarities between the differentially expressed proteins of acclimated cells to EtOH condition and the proteome of acclimated cells to GlcAA condition. Most of the differentially regulated proteins are involved in metabolism, folding, sorting and degradation processes. In conclusion, acclimation under appropriate sub-lethal conditions can be used as a method to improve cell phenotypic characteristics such as viability, growth under certain conditions, and tolerance to downstream processes.

Summary Controlling and managing the degree of phenotypic diversification of microbial populations is a challenging task. This task not only requires detailed knowledge regarding diversification mechanisms but also advances technical setups for the real-time analyses and control of population behavior on single-cell level. In this work, setup, design and operation of the so called segregostat is described which, in contrast to a traditional chemostats, allows the control of phenotypic diversification of microbial populations over time. Two exemplary case studies will be discussed, emphasizing the applicability and versatility of the proposed approach. In detail the phenotypic diversification of Eschericia coli or Pseudomonas putida based on monitoring membrane permeability will be controlled. We show that upon nutrient limitation, cell population tends to diversify into several subpopulations exhibiting distinct phenotypic (non-permeablized and permeablized cells). On-line analysis leads to the determination of the ratio between cells in these two states, which in turn trigger the addition of glucose pulses in order to maintain a pre-defined diversification ratio. These results prove that phenotypic diversification can be controlled by means of defined pulse-frequency modulation within continuously running bioreactor setups. This lays the foundation for systematic studies, not only of phenotypic diversification but also for all processes where dynamics single cell approaches are required, such as synthetic co-culture processes.

BACKGROUND: Escherichia coli W3110 and a group of six isogenic derivatives, each displaying distinct specific rates of glucose consumption were characterized to determine levels of GFP production and population heterogeneity. These strains have single or combinatory deletions in genes encoding phosphoenolpyruvate:sugar phosphotransferase system (PTS) permeases as PtsG and ManX, as well as common components EI, Hpr protein and EIIA, also the non-PTS Mgl galactose/glucose ABC transporter. They have been transformed for expressing GFP based on a lac-based expression vector, which is subject to bistability. RESULTS: , respectively. The rate of acetate production was strongly reduced in all mutant strains when compared with W3110/pV21. In bioreactor cultures, wild type W3110/pV21 produced 50.51 mg/L GFP, whereas strains WG/pV21 with inactive PTS IICBGlc and WGM/pV21 with the additional inactivation of PTS IIABMan showed the highest titers of GFP, corresponding to 342 and 438 mg/L, respectively. Moreover, we showed experimentally that bistable expression systems, as lac-based ones, induce strong phenotypic segregation among microbial populations. CONCLUSIONS: We have demonstrated that reduction on glucose consumption rate in E. coli leads to an improvement of GFP production. Furthermore, from the perspective of phenotypic heterogeneity, we observed in this case that heterogeneous systems are also the ones leading to the highest performance. This observation suggests reconsidering the generally accepted proposition stating that phenotypic heterogeneity is generally unwanted in bioprocess applications.

Nowadays, vinegar is industrially produced by mesophilic acetic acid bacteria (AAB). However, temperature fluctuation during acetous fermentation is inevitable, and may cause process disturbances. This can be mostly avoided using thermo-tolerant AAB. The main purpose of the present study was to isolate thermo-tolerant AAB. Fermentation performances were then evaluated. Twenty-eight different isolates were isolated from Iranian traditional vinegar. One of the isolates was able to grow and produce acetic acid in minimal culture media containing 5% (w/v) ethanol at 30-42 oC. 16s rRNA gene analysis showed that the selected thermo-tolerant isolate was Acetobacter tropicalis (L31). Acetous fermentation in Lab-bioreactor showed that Acetobacter tropicalis (L31) grew in minimal culture medium, and produced 5% (w/v) acetic acid at 37°C in batch and semi-continuous fermentation mode. Fermentation time was significantly dependent on dissolved oxygen (DO) concentration and acclimation of cells to low pH and acid stress. In average, acclimated cells produced 2.3-2.7 g L-1h-1 acetic acid during production phase. The final yield was 87% at 37oC in low and high DO concentrations. Low DO concentration (15%) during acetous fermentation caused longer fermentation time, but a large part of cells (91%) grown under such condition was viable if oxygen flow was interrupted. In contrast, cells grown under high DO concentration were not able to tolerate oxygen deficiency. In conclusion, since Acetobacter tropicalis (L31) grew well in minimal culture medium, and showed tolerance to high temperature and low DO, it seems that it is a potential isolate for vinegar starter production.

Bioprocess deviations are likely to occur at different operating scales, leading in most of the case to substrate deviation from main metabolic routes and impact product synthesis. Correlating qS and qP is of utmost importance for bioprocess observability and control and can be modeled actually by advanced metabolic flux models. However, if most of these models are able to make prediction about metabolic switches, they still do not incorporate deviation due to biological noise, i.e. phenotypic and genotypic heterogeneity. These limitations impair observability and thus the use of fundamental knowledge about biological network for practical application, i.e. metabolic engineering or bioprocess scale-up.

The oleaginous yeast Yarrowia lipolytica has the ability to use oils and fats as carbon source, making it a promising cell factory for the design of alternative bioprocesses based on renewable substrates. However, such a multiphasic bioreactor design is rather complex and leads to several constraints when considering emulsification of the oil-in-water mixture, foaming and cell growth/physiology on hydrophobic substrate. This study aims to shed light on the effect of pH changes on the physico-chemical properties of the cultivation medium and on cell physiology. It was indeed observed that at a pH value of 6, cell growth rate and intracellular lipid accumulation were optimized. Additionally, foaming was significantly reduced. In order to avoid over foaming in bioreactor, without impairing cell physiology, the use of alternative processes that can only act on the physical structure of culture medium, seems to be an effective alternative to usual chemical anti-foam agents.

In this study, new thermotolerant Acetobacter pasteurianus CV01 strain recently isolated from local product of Morocco has been investigated for its ability to perform efficient acetous fermentation at a large-scale. Firstly, the thermotolerance basis bioconversion of CV01 strain was compared to other mesophilic and thermotolerant acetic acid bacteria. Subsequently, CV01 strain was assessed for its ability to produce and tolerate high amount of acetic acid at optimal and thermal stress conditions in lab-scale bioreactor. It was found that the studied strain exhibited thermotolerant properties compared to reference strains and could withstand the increase in temperature during acetous fermentation in fermenter. Furthermore, gas chromatography-mass spectrometry (GC-MS) was used on the samples prepared with solid phase microextraction (SPME) to determine the volatile compounds of the pilot plant produced apple vinegar based on developed start-up and semi-continuous fermentation protocol. The operation strategy in the 500-L pilot plant scale acetator allowed achieving 7.3% (w/v) of final acetic acid concentration recording high yield and acetification rate. The aroma profile of experimentally produced vinegar was found different from that of the commercial reference one. According to the literature, the results obtained show that major volatile compounds found in pilot-plant produced apple vinegar are related to good aromatic note descriptors which could have a positive impact on the organoleptic quality of industrial vinegar. Consequently, it can be concluded that CV01 Acetobacter strain is well suited for large-scale production of high quality fruit vinegar.

Dombae is a traditional ferment starter which has been used for starchy based wine production in Cambodia. However, the production technology of rice wine in Cambodia is not optimized. The current study aimed to investigate the microbiota associated in five ferment starters and the effect of a traditional fermentation process using a metagenomics sequencing analysis and HS-SPME-GCMS for the characterization of the aromatic profiles at the end of fermentation. Most of bacteria identified in this study were lactic acid bacteria including Weissella cibaria, Pediococcus sp. MMZ60A, Lactobacillus fermentum and Lactobacillus plantarum. Saccharomyces cerevisiae and Saccharomycopsis fibuligera were found to be abundant yeasts while the only amylolytic filamentous fungus was Rhizopus oryzae. A total of 25 aromatic compounds were detected and identified as esters, alcohols, acids, ketones and aldehydes. The alcohol group was dominant in each rice wine. Significant changes were observed at the level of microbial communities during fermentation, suggesting microbial succession for the assimilation of starch and subsequently assimilation of fermentation by-products leading to the production of flavor compounds. At this level, the presence of Weissella, Pediococcus and Lactobacillus genus was strongly correlated with most of the flavor compounds detected.

Efficient optimization of microbial processes is a critical issue for achieving a number of sustainable development goals, considering the impact of microbial biotechnology in agrofood, environment, biopharmaceutical and chemical industries. Many of these applications require scale-up after proof of concept. However, the behaviour of microbial systems remains unpredictable (at least partially) when shifting from laboratory-scale to industrial conditions. The need for robust microbial systems is thus highly needed in this context, as well as a better understanding of the interactions between fluid mechanics and cell physiology. For that purpose, a full scale-up/down computational framework is already available. This framework links computational fluid dynamics (CFD), metabolic flux analysis and agent-based modelling (ABM) for a better understanding of the cell lifelines in a heterogeneous environment. Ultimately, this framework can be used for the design of scale-down simulators and/or metabolically engineered cells able to cope with environmental fluctuations typically found in large-scale bioreactors. However, this framework still needs some refinements, such as a better integration of gas-liquid flows in CFD, and taking into account intrinsic biological noise in ABM.

Lipopeoptides are amphiphilic compounds combining interesting physicochemical properties and biological activities. Due to their high foaming capacity in aerated bioreactor, the development of scalable bioprocesses for their production is a major bottleneck. In addition, the genes involved in the biosynthesis of these lipopeptides are mainly regulated by the quorum sensing, a global regulatory mechanism depending on cell density and known to be activated in biofilms. Several approaches have thus been considered in literature taking into account two criteria, on one side, to favor, control or avoid foam formation and on the other side, to use planktonic or immobilized (biofilm) cells. These different bioprocesses are discussed in the present review along with the purification strategies proposed for extracting and concentrating these biosurfactants.

The bio-economy is in transit from innovation to commercialization. The bioprocess industry is expected to increasingly deliver bio-products to the market, in large amounts, at high quality and at competitive cost levels. This requires flawless start-up of new large-scale bioprocesses and continuous improvement of running processes. Fermentation scale-up and operation can benefit from recent advances in three areas: 1. computation-driven design of scale-down simulators, 2. omics-driven metabolic engineering and 3. sensing and understanding of population heterogeneity. Integration of these fields requires a unified computational approach, linked to big data and simulated reality frameworks, of which the contours are becoming visible today.

Phenotypic plasticity of microbial cells has attracted much attention and several research efforts have been dedicated to the description of methods aiming at characterizing phenotypic heterogeneity and its impact on microbial populations. However, different approaches have also been suggested in order to take benefit from noise in a bioprocess perspective, e.g. by increasing the robustness or productivity of a microbial population. This review is dedicated to outline these controlling methods. A common issue, that has still to be addressed, is the experimental identification and the mathematical expression of noise. Indeed, the effective interfacing of microbial physiology with external parameters that can be used for controlling physiology depends on the acquisition of reliable signals. Latest technologies, like single cell microfluidics and advanced flow cytometric approaches, enable linking physiology, noise, heterogeneity in productive microbes with environmental cues and hence allow correctly mapping and predicting biological behavior via mathematical representations. However, like in the field of electronics, signals are perpetually subjected to noise. If appropriately interpreted, this noise can give an additional insight into the behavior of the individual cells within a microbial population of interest. This review focuses on recent progress made at describing, treating and exploiting biological noise in the context of microbial populations used in various bioprocess applications.

This study aims to understand and better control the main biological mechanisms and parameters modulating the various phenomena affecting Yarrowia lipolytica JMY775 and its lipids accumulation. The results obtained in this study stress forward that the use of an original tool, consisting of coupling bioreactors to online flow cytometry, is highly efficient. Throughout 48 h of culturing, this emerging process allowed an online continuous observation of the effects of pH and/or aeration on the cell growth and dimorphism and lipid accumulation by Y. lipolytica . This present study showed clearly that online flow cytometry is an advantageous tool for the real-time monitoring of microbial culture at a single-cell level. Indeed, the present investigation showed for the first time that profiling of the various phenomena and their monitoring upon culture time is now possible by coupling online cytometry with culture bioreactors.

BACKGROUND: In recent years, the non-conventional model yeast species Yarrowia lipolytica has received much attention because it is a useful cell factory for producing recombinant proteins. In this species, expression vectors involving LIP2 and POX2 promoters have been developed and used successfully for protein production at yields similar to or even higher than those of other cell factories, such as Pichia pastoris. However, production processes involving these promoters can be difficult to manage, especially if carried out at large scales in fed-batch bioreactors, because they require hydrophobic inducers, such as oleic acid or methyl oleate. Thus, the challenge has become to reduce loads of hydrophobic substrates while simultaneously promoting recombinant protein production. One possible solution is to replace a portion of the inducer with a co-substrate that can serve as an alternative energy source. However, implementing such an approach would require detailed knowledge of how carbon sources impact promoter regulation, which is surprisingly still lacking for the LIP2 and POX2 promoters. This study's aim was thus to better characterize promoter regulation and cell metabolism in Y. lipolytica cultures grown in media supplemented with different carbon sources. RESULTS: pPOX2 induction could be detected when glucose or glycerol was used as sole carbon source, which meant these carbon source could not prevent promoter induction. In addition, when a mixture of glucose and oleic acid was used in complex medium, pPOX2 induction level was lower that that of pLIP2. In contrast, pLIP2 induction was absent when glucose was present in the culture medium, which meant that cell growth could occur without any recombinant gene expression. When a 40/60 mixture of glucose and oleic acid (w/w) was used, a tenfold increase in promoter induction, as compared to when an oleic-acid-only medium was observed. It was also clear that individual cells were adapting metabolically to use both glucose and oleic acid. Indeed, no distinct subpopulations that specialized on glucose versus oleic acid were observed; such an outcome would have led to producer and non-producer phenotypes. In medium containing both glucose and oleic acid, cells tended to directly metabolize oleic acid instead of storing it in lipid bodies. CONCLUSIONS: This study found that pLIP2 is a promoter of choice as compared to pPOX2 to drive gene expression for recombinant protein production by Y. lipolytica used as cell factory.

La présente étude a pour but d’isoler, d’identifier et de caractériser de nouvelles souches de levures d’intérêt industriel. Un total de 54 levures ont été isolées et identifiées à partir de produits agricoles brutes et de sous-produits de l’industrie agro-alimentaire. Parmi ces isolats, quatre souches ont été retenues, purifiées et testées pour leur performance de fermentation sur un jus de dattes de variété Bouslikhène. D’après le séquençage du gène 18S de l’ADN ribosomal, les deux souches YS-DN1 et YS-M isolées respectivement à partir des dattes et de la mélasse ont été identifiées appartenant à l’espèce Saccharomyces cerevisiae. Par ailleurs, les deux autres souches YS-OPM et YS-G isolées des olives et des raisins appartiennent respectivement aux espèces Kluyveromyces marxianus, avec 78 % d’homologie et Candida utilis à 89% d’homologie. D’autre part, la souche YS-DN1, s’est identifiée comme la plus performante dans la production d’éthanol en comparaison à deux autres souches industrielles de référence. La levure YS-DN1 était l’unique souche capable de croître à une température entre 35 et 40°C en présence d’un taux alcoolique élevé et une pression osmotique agressive. Finalement, la production de la biomasse cellulaire de la souche YS -DN1 a été améliorée par optimisation des facteurs température, Brix et pH fixés respectivement à 29.75°C, 15.7% et 4.15. Ces valeurs ont permis d’obtenir un maximum de cellules de l’ordre de 8.4x10 8 UFC/ml. Il a été conclu que cette souche pourrait bien s’adapter à des usages industriels pour la production du vinaigre de fruits à grande échelle. Mots clés: Isolement, levures, Fermentation, Vinaigre, Optimisation, Dattes, Stress

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.

The literature presents a growing body of evidence demonstrating the positive effect of probiotics on health. Probiotic consumption levels are rising quickly in the world despite the fluctuation of their viability and functionality. Technological methods aiming at improving probiotic characteristics are thus highly wanted. However, microbial metabolic engineering toolbox is not available for this kind of application. On the other hand, basic microbiology teaches us that bacteria are able to exhibit adaptation to external stresses. It is known that adequately applied sub-lethal stress, i.e., controlled in amplitude and frequency at a given stage of the culture, is able to enhance microbial robustness. This property could be potentially used to improve the viability of probiotic bacteria, but some technical challenges still need to be overcome before any industrial implementation. This review paper investigates the different technical tools that can be used in order to define the proper condition for improving viability of probiotic bacteria and their implementation at the industrial scale. Based on the example of Bifidobacterium bifidum, potentialities for simultaneously improving viability, but also functionality of probiotics will be described.

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.

High Pichia pastoris biomass density could be obtained using high co-feeding rate of methanol and sorbitol in a fed-batch or continuous culture, while further higher feeding rate finally leads to oxygen limitation in bioreactor. In the literature, there is lack of report about AOX1 promoter regulation with regard to dissolved oxygen level (DO). Therefore, in this work, chemostat cultures were performed to investigate the cell growth, metabolism and regulation of the AOX1 promoter (pAOX1) regarding co-feeding rate of optimized methanol/sorbitol mixture (methanol fraction 0.60 C-mol/C-mol) using a P. pastoris Mut+/pAOX1-lacZ strain. The oxygen transfer rates (OTR) in bioreactor were kept in the range of typical values of large bioreactor, i.e., 4-8 g/(L h) if DO equals 30 % saturation or 5-10 g/(L h) if DO nears zero. For DO >0, an increase of the carbon fed led to an increase of pAOX1 induction. By contrast, when dissolved oxygen was completely depleted, methanol accumulated, causing a 30 % decrease of pAOX1 induction. However, this decrease is more likely to be lined to methanol accumulation than to low level of dissolved oxygen (<4 % DO). Methanol/sorbitol co-feeding allowed cells to adapt to oxygen transient limitations that often occur at industrial scale with reduced effect on pAOX1 induction. The optimal feeding rate tested here was 6.6 mmol C (DCW h)(-1) at an OTR of 8.28 g O2(L h)(-1) with over fivefold pAOX1 induction (probably directly associated with target protein productivity) compared with previous work.

The spore productivity and insecticidal activity of two opportunistic insect pathogenic Aspergillus species (namely: Aspergillus clavatus Desmazieres and Aspergillus flavus Link (Ascomycota: Eurotiales, Trichocomaceae)) were compared to Metarhizium anisopliae sensu lato (Metchnikoff) Sorokin (Ascomycota: Hypocreales, Clavicipitaceae) for mosquito (Diptera: Culicidae) control. The production of aerial spores on wheat bran and white rice was investigated in solid-, semi-solid-, and liquid-state media supplemented with a nutritive solution. Wheat bran-based media increased the spore yield in solid-state from three to sevenfold: A. clavatus produced 48.4 ± 5.2 and 15.7 ± 1.6 × 108 spores/g, A. flavus produced 22.3 ± 4.1 and 3.1 ± 2.5 × 108 spores/g, and M. anisopliae produced 39.6 ± 6.5 and 13.1 ± 2.6 × 108 spores/g of wheat bran or white rice, respectively. A. clavatus, A. flavus and M. anisopliae spores harvested from wheat bran-based solid-state media showed lethal concentrations (LC50) of 1.1, 1.8, and 1.3 × 108 spores/ml against Culex quinquefasciatus Say larvae in 72 h. Because A. clavatus and M. anisopliae displayed similar features when cultured under these conditions, our results suggest that insect pathogenic Aspergillus species may be as productive and virulent against mosquito larvae as a well-recognised entomopathogenic fungus.

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.

Additional file 1: Table S1. Composition of the medium used in this study.

Additional file 1: Table S1. Composition of the medium used in this study.

The use of genetically encoded fluorescent reporters allows speeding up the initial optimization steps of microbial bioprocesses. These reporters can be used for determining the expression level of a particular promoter, not only the synthesis of a specific protein but also the content of intracellular metabolites. The level of protein/metabolite is thus proportional to a fluorescence signal. By this way, mean expression profiles of protein/metabolites can be determined non-invasively at a high-throughput rate, allowing the rapid identification of the best producers. Actually, different kinds of reporter systems are available, as well as specific cultivation devices allowing the on-line recording of the fluorescent signal. Cell-to-cell variability is another important phenomenon that can be integrated into the screening procedures for the selection of more efficient microbial cell factories.

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.

The application of sub-lethal stresses is known to be an efficient strategy to enhance survival of probiotic bacteria during drying processes. In this context, we previously showed that the application of heat stress upon the entry into stationary phase increased significantly the viability of Bifidobacterium bifidum. However, this heat shock has been considered only in small-scale bioreactor and no information is available for a possible scaling-up strategy. Five different operating scales (0.2, 2, 20, 200 and 2000 L) have thus been tested and the results showed that the viability of B. bifidum increases from 3.15 to 6.57 folds, depending on the scale considered. Our observations pointed out the fact that the heat stress procedure is scalable according to the main outcome, i.e., increases in cell viability, but other factors have to be taken into account. Among these factors, dissolved carbon dioxide seems to play a significant role, since it explains the differences observed between the test performed at laboratory scale and in industrial conditions.

In this work, the effect of iron oxide particles Fe2O3 and iron nanoparticles encapsulated in a porous silica matrix (xerogel Fe/SiO2) was investigated on biphenyl biodegradation by the strain Rhodococcus erythropolis T902.1. After 18 days of incubation biodegradation yields of 75% and 85% were achieved respectively in presence of non-autoclaved or autoclaved xerogel Fe/SiO2at 10-5 M iron. These results are 42 and 60% higher than in standard conditions without nanoparticles. They suggest that the autoclave procedure lead to the release of some iron less anchored in the silica matrix. This study highlights that siderophore production byRhodococcus erythropolis T902.1 would be related to the presence of iron nanoparticles in the culture. It suggests that the production of these strong chelating compounds decreases with increase of iron release from xerogel Fe/SiO2. Moreover, most of the surfactants synthesized by Rhodococcus erythropolis T902.1 which are glycolipids containing trehalose (hexose),would be linked to cell surface and not excreted in the culture medium; the biomass hexose content also increased by 85% in presence of iron nanoparticles. Dans ce travail, l’effet de particules d’oxyde de fer Fe2O3 et de nanoparticules de fer encapsulées dans des particules poreuses de silice (xérogel Fe/SiO2) a été étudié sur la biodégradation du biphényle par la souche Rhodococcus erythropolis T902.1. Les pourcentages de biodégradation après 18 jours d’incubation en présence du xérogel Fe/SiO2 à 10-5 M en fer atteignent 75 % et 85 % respectivement pour les conditions sans ou après autoclavage ; c’est-à-dire 42 et 60 % de dégradation supplémentaire par rapport aux conditions standards sans xérogel Fe/SiO2. L’impact dû à l’autoclavage serait directement associé à la libération d’une certaine quantité de fer moins bien ancré dans la matrice de silice. Cette étude a jeté la lumière sur la liaison entre la sécrétion de sidérophores par Rhodococcus erythropolis T902.1 et la présence de nanoparticules de fer dans le milieu de culture. Elle suggère une diminution de la sécrétion de sidérophores avec l’augmentation de la quantité d’ions ferriques mobilisés à partir du xérogel Fe/SiO2. Finalement, la majorité des surfactants glycolipidiques contenant du trehalose (hexose) produits par Rhodococcus erythropolis T902.1sont liés à la surface de la cellule et ne sont pas excrétés dans le milieu de culture ; leur concentration a augmenté de 85 % en présence de nanoparticules de fer.

Zinc, lead and cadmium are metallic trace elements (MTEs) that are widespread in the environment and tend to accumulate in soils because of their low mobility and non-degradability. The purpose of this work is to evaluate the applicability of biosensors as tools able to provide data about the bioavailability of such MTEs in contaminated soils. Here, we tested the genetically-engineered strain Escherichia coli pP(ZntA)gfp as a biosensor applicable to the detection of zinc, lead and cadmium by the biosynthesis of green fluorescent protein (GFP) accumulating inside the cells. Flow cytometry was used to investigate the fluorescence induced by the MTEs. A curvilinear response to zinc between 0 and 25 mg/L and another curvilinear response to cadmium between 0 and 1.5 mg/L were highlighted in liquid media, while lead did not produce exploitable results. The response relating to a Zn2+/Cd2+ ratio of 10 was further investigated. In these conditions, E. coli pP(ZntA)gfp responded to cadmium only. Several contaminated soils with a Zn2+/Cd2+ ratio of 10 were analyzed with the biosensor, and the metallic concentrations were also measured by atomic absorption spectroscopy. Our results showed that E. coli pP(ZntA)gfp could be used as a monitoring tool for contaminated soils being processed.

Steam explosion is a thermo-mechanicochemical pretreatment which allows the breakdown of lignocellulosic structural components by the action of heating, formation of organic acids during the process, and shearing forces resulting in the expansion of the moisture. Two distinct stages compose the steam-explosion process: vapocracking and explosive decompression which include modification of the material components: hydrolysis of hemicellulosic components (mono- and oligosaccharides released), modification of the chemical structure of lignin, and modification of the cellulose crystallinity index, etc. These effects allow the opening of lignocellulosic structures and influence the enzymatic hydrolysis yield of the material.

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.

Pectinolytic enzymes are used in the food industry for the extraction, clarification and filtration of fruit juice and wine. Depending on their mode of action, these enzymes are classified into two major groups, namely: esterases (methylesterase) and depolymerases (polygalacturonase and lyase). Among the methods for their preparation, fermentation is the most used, and its application depends upon knowledge of the strain’s requirements; many parameters are taken into consideration most of which relate to the strain used. Knowledge and control of these parameters are required for optimal production of these enzymes. Many microorganisms (Aspergillus niger; Kluyveromyces marxianus; Trichoderma viride BITRS-1001; Bacillus licheniformis; Saccharomyces pastorianus etc.) have already been studied and we suggested that there is a possibility of producing these enzymes using the microorganisms employed for the retting of cassava. This review provides a wealth of knowledge on the production of pectinolytic enzymes, using different substrates and microorganisms.

status: Published

status: Published

Abstract BACKGROUND Single cell biology has attracted a lot of attention in recent years and has led to numerous fundamental results pointing out the heterogeneity of clonal cell populations. In this context, microbial phenotypic heterogeneity under bioprocessing conditions needs to be further investigated. In this study, yeast based processes have been investigated by using on‐line flow cytometry (FC) in combination with a fluorescent transcriptional reporter (GFP) and viability fluorescence tags (propidium iodide, PI). Methods aiming at expressing the dispersion of these fluorescence tags among the yeast populations have been investigated for different bioreactor operating conditions. RESULTS Yeast viability was determined on the basis of PI uptake. Segregation between PI negative and positive subpopulations could be efficiently quantified on the basis of the mean‐to‐median ratio or the amplitude of the interquartile range. On the other hand, the same quantification could not be made for the segregation occurring at the level of GFP synthesis. Indeed, when cells were exposed to sub‐lethal or mild stresses (such as in scale‐down reactors) two GFP subpopulations could be visualized by real‐time FC, but quantification by one of the above‐mentioned methods was not possible. CONCLUSIONS Yeast population heterogeneity was observed in representative bioreactor operating conditions. Difficulties for the determination of segregation at the level of GFP synthesis point out the fact that one needs to understand the segregation mechanisms for the applied fluorescent reporters, to judge whether simple mathematical tools may be applied or if more sophisticated computational tools are needed for quantification of the microbial population segregation. © 2014 Society of Chemical Industry

status: Published

BACKGROUND: Loss of viability is one of the most important problems during starter culture production. Previous research has mostly focused on the production process of bacterial starters, but there are few studies about cellular protein deterioration causing cell defectiveness during storage. In the present study, we investigated the influence of storage temperature (-21, 4, 35°C) on the cellular protein modifications which may contribute to the senescence of freeze-dried Acetobacter senegalensis. RESULTS: Heterogeneous populations composed of culturable cells, viable but non-culturable cells (VBNC) and dead cells were generated when freeze-dried cells were kept at -21 and 4°C for 12 months whereas higher storage temperature (35°C) mainly caused death of the cells. The analysis of stored cell proteome by 2D-DiGE demonstrated a modified pattern of protein profile for cell kept at 4 and 35°C due to the formation of protein spot trains and shift of Isoelectric point (pI). Quantification of carbonylated protein by ELISA showed that the cells stored at 4 and 35°C had higher carbonylated protein contents than fresh cells. 2D-DiGE followed by Western blotting also confirmed the carbonylation of cellular proteins involved in translation process and energy generation. The auto-fluorescent feature of cells kept at 35°C increased significantly which may be an indication of protein glycation during storage. In addition, the percentage of cellular unsaturated fatty acid and the solubility of cellular proteins decreased upon storage of cells at higher temperature suggesting that peroxidation of fatty acids and possibly protein lipidation and oxidation occurred. CONCLUSIONS: High storage temperature induces some deteriorative reactions such as protein oxidation, lipidation and glycation which may cause further protein modifications like pI-shift, and protein insolubility. These modifications can partly account for the changes in cell viability. It can also be deduced that even moderate carbonylation of some critical cellular proteins (like ribosomal proteins) may lead to VBNC formation or death of freeze-dried bacteria. Moreover, it seems that other mechanisms of biomolecule deterioration preceding protein carbonylation lead to VBNC formation under very low storage temperature.

Many entomopathogenic fungi have been demonstrated to be potential agents for efficiently controlling mosquito populations. In the present study, we investigated a bioreactor system to produce metabolites and conidia by combining technological advantages of submerged and solid-state fermentations. The efficiency of fungal products was tested toward mosquitoes. Aspergillus clavatus (Eurotiales: Trichocomaceae) was grown by semi-solid-state fermentation in a bioreactor for up to 7 days. Depending on conidial doses (2.5×107, 5×107, 7.5×107, 10×107 and 12.5×107 conidia/mL), mortality ranged from 37.2±15.0 to 86.3±5.0% toward larvae and from 35.8±2.0 to 85.2±1.5% toward adults. The metabolites (10, 20, 40, 60, 80 and 100% v/v) yielded mortality from 23.7±15.0 to 100.0±0.1% toward larvae, and two sprayed volumes (5 and 10 mL) reached 45.5±1.4 and 75.6±2.6% mortality, respectively, toward adults.

peer reviewed

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.

Topical application of insecticidal compounds allows directly exposing these substances on insect tissues and measuring their toxicity while ignoring many factors. However, this technique remains difficult to apply on mosquito larvae considering their aquatic lifestyle. Micro-injection could be used for the direct deposition of toxic compounds in the larvae. Capillaries exhibiting an injection tip with an external diameter of 0.5 mm have been designed from silica tubes. For each treatment, a capillary is mounted on a pump connected to a flow rate regulator. Culex quinquefasciatus larvae were injected with 10^7 spores/ml of entomopathogenic fungi (Aspergillus clavatus, Metarhizium anisopliae, Metarhizium sp.). Mortalities were recorded daily during 72h. The distribution of spores stained with methylene blue and injected into the body of larvae was also observed according to the system described. Results showed that spores were distributed over the whole body. The injection of Aspergillus clavatus, Metarhizium anisopliae and Metarhizium sp spores induced corrected mortalities of 62%, 53% and 57% after 72h, and differed statistically from control groups. Finally, post-mortem emergences of filaments from dead larvae were observed in the case of the three fungal strains confirming spore viability. Injection of inactivated spores (or inert bodies of similar size) could help to reject the hypothesis of a response due to the presence of foreign bodies.

BACKGROUND: Microbial cell population heterogeneity is now recognized as a major source of issues in the development and optimization of bioprocesses. Even if single cell technologies are available for the study of microbial population heterogeneity, only a few of these methods are available in order to study the dynamics of segregation directly in bioreactors. In this context, specific interfaces have been developed in order to connect a flow cytometer directly to a bioreactor for automated analyses. In this work, we propose a simplified version of such an interface and demonstrate its usefulness for multiplexed experiments. RESULTS: A low-cost automated flow cytometer has been used in order to monitor the synthesis of a destabilized Green Fluorescent Protein (GFP) under the regulation of the fis promoter and propidium iodide (PI) uptake. The results obtained showed that the dynamics of GFP synthesis are complex and can be attributed to a complex set of biological parameters, i.e. on the one hand the release of protein into the extracellular medium and its uptake modifying the activity of the fis promoter, and on the other hand the stability of the GFP molecule itself, which can be attributed to the protease content and energy status of the cells. In this respect, multiplexed experiments have shown a correlation between heat shock and ATP content and the stability of the reporter molecule. CONCLUSION: This work demonstrates that a simplified version of on-line FC can be used at the process level or in a multiplexed version to investigate the dynamics of complex physiological mechanisms. In this respect, the determination of new on-line parameters derived from automated FC is of primary importance in order to fully integrate the power of FC in dedicated feedback control loops.

Heterogeneity or segregation of microbial populations has been the subject of much research, but the real impact of this phenomenon on bioprocesses remains poorly understood. The main reason for this lack of knowledge is the difficulty in monitoring microbial population heterogeneity under dynamic process conditions. The main concepts resulting in microbial population heterogeneity in the context of bioprocesses have been summarized by two distinct hypotheses. The first involves the individual history of microbial cells or the "path" followed during their residence time inside the process equipment. The second hypothesis involves a coordinated response by the microbial population as a bet-hedging strategy, in order to cope with process-related stresses. The respective contribution of each hypothesis to microbial heterogeneity in bioprocesses is still unclear. This illustrates the fact that, although microbial phenotypic heterogeneity has been thoroughly investigated at a fundamental level, the implications of this phenomenon in the context of microbial bioprocesses are still subject to debate. At this time, automated flow cytometry is the best technique for investigating microbial heterogeneity under process conditions. However, dedicated software and relevant biomarkers are needed for the proper integration of flow cytometry as a bioprocess control tool.

status: Published

When ingested in sufficient numbers, probiotics are expected to confer one or more proven health benefits on the consumer. Theoretically, the effectiveness of a probiotic food product is the sum of its microbial quality and its functional potential. Whereas the latter may vary much with the body (target) site, delivery mode, human target population, and health benefit envisaged microbial assessment of the probiotic product quality is more straightforward. The range of stakeholders that need to be informed on probiotic quality assessments is extremely broad, including academics, food and biotherapeutic industries, healthcare professionals, competent authorities, consumers, and professional press. In view of the rapidly expanding knowledge on this subject, the Belgian Superior Health Council installed Working Group "8651 Probiotics" to review the state of knowledge regarding the methodologies that make it possible to characterize strains and products with purported probiotic activity. This advisory report covers three main steps in the microbial quality assessment process, i.e. (i) correct species identification and strain-specific typing of bacterial and yeast strains used in probiotic applications, (ii) safety assessment of probiotic strains used for human consumption, and (iii) quality of the final probiotic product in terms of its microbial composition, concentration, stability, authenticity, and labeling.

status: Published

Abstract BACKGROUND Whereas multi‐species biofilm reactors are commonly used for the treatment of liquid and solid wastes, new strategies are progressing for the development of single species biofilm for the production of high‐value metabolites. Technically, this new concept relies on the design of bioreactors able to promote biofilm formation and on the identification of the key physico‐chemical parameters involved in biofilm formation. RESULTS An experimental setting comprising a liquid continuously recirculated on a metal structured packing has been used to promote Bacillus subtilis GA1 biofilm formation. The colonization of the packing has been visualized non‐invasively by X‐ray tomography. This analysis revealed an uneven, conical, distribution of the biofilm inside the packing. Compared with a submerged culture carried out in a stirred tank reactor, significant modification of the lipopeptide profile has been observed in the biofilm reactor with the disappearance of fengycin and iturin fractions and an increase of the surfactin fraction. In addition, considering the biofilm reactor design, no foam formation has been observed during the culture. CONCLUSIONS The configuration of this biofilm reactor set‐up allows for a higher surfactin production by comparison with a submerged culture while avoiding foam formation. Additionally, scale‐up could easily be performed by increasing the number of packing elements. © 2013 Society of Chemical Industry

Cell density and cell viability have been followed on-line by using a three-dimensional optical reflectance method (3D-ORM) probe. This method has allowed to highlight the differences between a well-mixed and a scale-down bioreactor configured in order to reproduce mixing deficiencies during a fed-batch culture of Escherichia coli. These differences have been observed both for the obscuration factor (OBF) and the coincidence probability delivered by the probe. These parameters are correlated to flow cytometry measurement based on the PI-uptake test and cell density based on optical density measurement. This first set of results has pointed out the fact that the 3D-ORM probe is sensitive to sub-lethal injuries encountered by microbial cells in process-related conditions. The effect of lethal injuries has been further investigated on the basis of additional experiments involving heat stress and a sharp increase of the OBF has been observed indicating that cells are effectively injured by the increase of temperature. However, further improvement of the probe are needed in order to give access to single-cell measurements.

Downstream processes have great influences on bacterial starter production. Different modifications occur to cellular compounds during freeze-drying process and storage of bacterial starters. Consequently, viability and culturability (multiplication capacity) undergo some changes. In this study, the effects of freeze-drying process and storage conditions were examined on cell envelope integrity, respiration and culturability of Acetobacter senegalensis. Freezing of cells protected with mannitol (20% w/w) did not affect cell multiplication and respiration considerably; however, 19% of cells showed compromised cell envelope after freezing. After drying, 1.96×1011 CFU/g were enumerated, indicating that about 34% of the cells could survive and keep their culturability. Drying of the cells induced further leakage in cell envelope and finally 81% of cells appeared as injured ones; however, 87% of the dried cells maintained their respiration capacity. Storage temperature had significant effect on cell multiplication ability; higher storage temperature (35°C) caused 8.59-log reduction in cell culturability after nine-month period of storage. Collapse of cell envelop integrity and respiration was observed at 35°C. At lower storage temperature (4°C), the culturability decreased about one-log reduction after nine months. Cell envelope integrity was subjected to minor changes during a period of nine month-storage at 4°C whereas a heterogeneous population of cells with different respiration capacity emerged at 4°C. These results indicate that a major part of cells undergone drying process and storage entered into viable but non-culturable state. In addition, usage of different culture media didn’t improve resuscitation. Besides, it seems that sub-lethal damages to cell envelope caused uptake of propidium iodide, however these kinds of injuries could not impress cell multiplications and respiration.

In this work, we present the design and characterization of Green Fluorescent Protein (GFP)-based reporter systems designed to describe cellular activity in "complex," heterogeneous bioreactors. The reporter systems consist of Escherichia coli strains carrying growth dependent promoters fused to genes expressing stable and unstable variants of GFP, respectively. The response of Escherichia coli cells to transient exposure to glucose was studied in a two-compartment scale down bioreactor (SDR) consisting of a well-stirred tank reactor (STR) connected to a plug-flow reactor (PFR). Such a SDR system is employed to mimic the situation of high glucose concentration and oxygen limitation that often encountered in large-scale, fed-batch bioreactors and the response of E. coli was simulated by continuously pumping microbial cells from STR to the PFR. We found that repeated addition of concentrated glucose pulses with varied frequency at the entrance of the PFR had consequences on strain physiological behavior. The GFP expressions were significantly marked after 10 h of cultivation in STR (control reactor) and SDR, whereas, growth rates were rather similar. Additional experiments in chemostat with programmed glucose perturbation suggested that the activities of the promoters were linked with the substrate limitation signal. Taken together with immunoblot analysis, we suppose protein leakage is responsible for the overexpression of fis and the related promoters, such as rrnB in this case study, but additional works are required in order to confirm this relationship. This investigation is useful for a better understanding of the fast dynamic phenomena occurring in heterogeneous large-scale bioreactors.

Substrate limitation responsive biosensors have been used for the development of a mini-bioreactor platform that can be used as a scale-down tool. Three green fluorescent protein (GFP) transcriptional reporters have been chosen in Escherichia coli, i.e., uspA::gfp, csiE::gfp, and yciG::gfp. Our previous studies have shown that these kinds of promoters are induced in response to substrate limitation and are significantly repressed when cultures are carried out in heterogeneous bioreactors. This sensitivity to substrate limitation has been confirmed in the case of the csiE and yciG biosensors. A mini-scale-down platform is proposed as a high throughput tool to rapidly investigate the usefulness of a given microbial biosensor. This platform is composed of shake flasks able to operate in fed-batch mode either using the slow release or the intermittent feeding principle. Local heterogeneities were reproduced at the level of these mini-bioreactors (operating under the intermittent feeding principle) and caused a decrease in GFP expression as in conventional scale-down reactors. The presence of GFP in supernatants was also noted and seems to be correlated with the substrate limitation signal for the three cultivation systems considered in this work (i.e., chemostat, conventional and mini-bioreactors) and with membrane permeability.

The storage stability of freeze-dried powders was studied by parameters such as loss of viability on the Plate Count Agar (PCA). Powder with glycerol (PG) contains 8.4x10cfu/g before storage 1.1x10cfug after 3 months at 4°C and 6.0x10cfu/g after 3 months at 20°C. The concentration of soluble proteins (mg/g) decrease during storage at 4°C from 3.77 to 0.80 after 90 days; and the ratios of unsaturated to saturated fatty acids (C18:3/C16:0 and C18:2/C16:0) decrease respectively from 0.05 to 0.04 and 0.007 to 0.004 after 3 months at 4°C. This ratio characterises the membrane fluidity. Powder without glycerol (PS) contains 1.1x10 cfu/g before storage and 1.4 x 10 cfu/g after 3 months at 4°C and 1.4 x 10 cfu/g after 3 months at 20°C. The concentration of soluble proteins (mg/g) decrease during storage at 4°C from 4.08 to 0.42 after 90 days, the glutathione concentration decrease during storage at 4°C from 2.2 to 1.4. The beneficial effect of glycerol on fatty acid composition during freezedrying is shown and the ratios of unsaturated to saturated fatty acids (C18:2/C16:0 and C18:3/C16:0) decrease respectively from 0.019 to 0.004 and 0.054 to 0.036 after 90 days storage at 4°C. Analysis by flow cytometry was used to assess the physiological state in which cells are at the end of freeze-drying. We found 13.5% live cells, 36.1% dead cells and 50.4% cells in 246 MputuKanyinda Jean-Noel et al an intermediate state for powder with glycerol (PG) after freeze-drying. These results shows that glycerol play an important role in Pseudomonas fluorescens BTP1 desiccation during freeze-drying, by maintaining a degree of viability after freeze-drying and during storage.

In order to investigate the potential occurrence of GFP leakage to the extracellular medium, the fluorescence and the total protein content of the supernatants coming from the different cultures have been investigated (Figures 4-1 and 4-2). In general, the fluorescence intensity of the supernatant increases in the case of the two biosensors and for all the operating conditions tested. However, a slightly lower extracellular fluorescence is observed in the case of the cultures operated in SDRs, compared with those operated in bioreactors without recycle loop. Consistent with this, Figure 4-2 shows that the amount of GFP protein as well as...

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A series of scale-down experiments have been carried out by our team. During these experiments, different GFP transcriptional reporters have been used in order to characterize the response of microbial cells in front of the environmental fluctuations perceived during the cultures. These transcriptional reporters have been constructed form different natural promoters involving either the general stress response, the response to nutrient limitation or a dependency to the growth rate of the strain. The experimental results are reported in the following section, but it is first useful to specify the cultivation conditions used to carry out these experiments.

The displacement of microbial cells in the environment and in engineered system such as a bioreactor can be represented roughly by convection and/or diffusion-based equations, according to the nature of the convoying medium. However, one of the biggest advantages of using whole cell biosensor is their ability to detect stress at the micrometer scale. If this kind of data as to be interpreted, the environmental fluctuations have to be described at the single cell level and the random component linked with the displacement of the cell has to be taken into account. In the case of bioreactor operations, such displacement can be described by a stochastic version of the hydrodynamic presented previously. Roughly, the same equations are kept but the algorithm used to perform the simulation is quite different since the stochastic component of the process has to be added to the problem. The basic principle is to use the same model structure as used previously for the simulation of glucose gradient by integrating a random component. The matrix M containing the exchange flow rate (m3/s) of the NOZ model will be assimilated as the generator matrix Q of the stochastic version of the model [38].

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A scale-down investigation of the impact of local dissolved oxygen limitation on lipase production by Y. lipolytica has been performed. One of the major issues encountered during this kind of process is foam formation, requiring a reduction of the overall oxygen transfer efficiency of the system in order to keep antifoam consumption to a reasonable level. A regulation strategy involving oxygen enrichment of the air flow through the reactor has allowed this issue to be partly overcome. For a second time, the scale dependency of the process operated with air enrichment has been investigated by a combination of scale-down and pilot-scale cultivation tests. The scale-down apparatus considered in this work comprised a well-mixed part connected to a plug-flow part subjected to dissolved oxygen limitation. Surprisingly, foaming intensity was greatly reduced in the case of the test performed in scale-down reactors (SDRs) while maintaining the same stirring and aeration intensities in the stirred part of the reactor. For mean residence time of 100 s in the recycle loop of the reactor, foam formation was significantly reduced while cell growth and lipase production were both unaltered. When the residence time in the recycle loop was raised to 200 s, the foam phenomena was also reduced, but the lipase yield was altered as well as lip2 gene transcription and translation as shown by real-time quantitative polymerase chain reaction (RT-qPCR) and reporter gene activity, respectively. Our results clearly show the importance of primarily taking into account cell physiology for the scaling-up procedure.

Mixing deficiencies can be potentially detected by the use of a dedicated whole cell microbial biosensor. In this work, a csiE promoter induced under carbon-limited conditions was involved in the elaboration of such biosensor. The cisE biosensor exhibited interesting response after up and down-shift of the dilution rate in chemostat mode. Glucose limitation was accompanied by green fluorescent protein (GFP) leakage to the extracellular medium. In order to test the responsiveness of microbial biosensors to substrate fluctuations in large-scale, a scale-down reactor (SDR) experiment was performed. The glucose fluctuations were characterized at the single cell level and tend to decrease the induction of GFP. Simulations run on the basis of a stochastic hydrodynamic model have shown the variability and the frequencies at which biosensors are exposed to glucose gradient in the SDR. GFP leakage was observed to a great extent in the case of a culture operated in well-mixed fed-batch mode, by comparison with those operated in SDR. GFP leakage seems to be correlated to a higher membrane permeability, confirming previous studies highlighting a better cell viability in cultures operated in a fluctuating environment. Our results suggest that GFP leakage could be used in parallel to the normal GFP biosensor function in order to assess microbial viability in process conditions.

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.

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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.

Air pollution is one of the major problems to be solved over this century. So in recent years, a strong technological base has been developed for air quality control. The driving force has been the public awareness of the need for human health and environmental protection. Consequently, environmental legislation and regulations have been initiated by governments to control air quality. Removing air pollutants from industrial gaseous emissions is achieved by different physical and chemical ways like the transfer and/or concentration of compounds in a liquid phase (condensation, absorption) or onto a solid (adsorption) or by the destruction of molecules by thermal and catalytic incineration. However, more recently new treatment alternatives have been developed, involving the biological degradation of pollutants present in the gaseous phase. Thus, these microbial systems can remove a large number of molecules, especially volatile organic compounds (VOC) and odorous molecules. Such systems present several advantages, among which is their high efficiency and low cost. Moreover, the treatment of polluted air in bioreactors allows the complete degradation or transformation of the contaminants. As a result of microbial metabolism, the pollutants are degraded into carbon dioxide and water or converted into cell biomass.

Abstract BACKGROUND: Two‐phase partitioning bioreactors (TPPBs) are considered as a new technology for xenobiotic degradation in gaseous effluents. However, there is still a need for more knowledge on how to design and scale up TPPBs. The partitioning of the two phases remains a misunderstood method of research. In particular, the impact of pollutant (isopropylbenzene), biomass and surfactant extract needs to be better evaluated. RESULTS: An adapted scale‐down apparatus has been developed in order to quantify the speed of phase partitioning (SPP) into a plug flow section. First, it was shown that isopropylbenzene (IPB) does not destabilise the system more significantly. Second, respectively 0.5 g L−1 and 0.05 g L−1 of biomass and surfactant extract, separately or in mixture, were sufficient to ensure the stability of the two‐phase system. Finally, a 100 m3 limit of scaling‐up was suggested on the basis of the circulation time comparison. CONCLUSION: The scaling up of an aqueous–silicone oil TPPB was found to be definitely conceivable when the presence of biotic compounds were considered. However, further considerations are needed to verify our assumptions, in particular by taking into account the velocity field pattern in full‐scale bioreactors and reproduce it in laboratory‐scale apparatus. Copyright © 2010 Society of Chemical Industry

peer reviewed

peer reviewed

peer reviewed

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.

Bioreactor scale-up often pose 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.

Extrait du premier article du numéro: L'École Doctorale Thématique en Génie des Procédés (EDT GEPROC) a pour objectif de promouvoir une recherche pluridisciplinaire doctorale dans les divers domaines d'application du génie des procédés. C'est donc assez logiquement que la 2e journée thématique de réflexion organisée par l'EDT-GEPROC a été consacrée au thème du Génie des procédés appliqué aux bio-industries (agroalimentaire, agrocarburants et énergies, environnement, industrie pharmaceutique). Cette manifestation s'est tenue le 16 décembre 2009 sur le site de Gembloux Agro-Bio Tech. Elle a rassemblé 80 participants provenant des 4 Institutions universitaires faisant partie de l'EDT GEPROC (ULg, UMONS, ULB et UCL). Il est important à ce niveau de noter également la participation de plusieurs représentants du monde industriel. En effet, la mise en contact des doctorants avec le monde industriel est un des objectifs visés par l'EDT GEPROC. Le programme suivant s'est étalé sur une demi-journée : 12:00-12:45 : Accueil et mise en place des posters 12:45-13:00 : Ouverture de la journée thématique : Génie des procédés appliqué aux bio-industries 13:00-13:30 : Évolutions technologiques des bioréacteurs par Didier Thébline (Sartorius-Stedim) 13:30-14:00 : Nouveaux développements dans le domaine du matériel à usage unique pour les industries biotechnologiques et pharmaceutiques par Jean-Louis Belot (Sartorius-Stedim) 14:00-14:30 : Conception d'antimousses chimiques à l'usage des bioprocédés : contraintes et performances par Jean-Pa

Yeast is a widely used microorganism at the industrial level because of its biomass and metabolite production capabilities. However, due to its sensitivity to the glucose effect, problems occur during scale-up to the industrial scale. Hydrodynamic conditions are not ideal in large-scale bioreactors, and glucose concentration gradients can arise when these bioreactors are operating in fed-batch mode. We have studied the effects of such gradients in a scale-down reactor, which consists of a mixed part linked to a non-mixed part by a recirculation pump, in order to mimic the hydrodynamic conditions encountered at the large scale. During the fermentation tests in the scale-down reactor, there was a drop in both biomass yield (ratio between the biomass produced and the glucose added) and trehalose production and an increase in both fermentation time (time between inoculation and beginning of stationary phase) and ethanol production. We have developed a stochastic model which explains these effects as the result of an induction process determined mainly by the hydrodynamic conditions. The concentration profiles experienced by the microorganisms during the scale-down tests were expressed and linked to the biomass yields of the scale-down tests.

Abstract BACKGROUND: The two‐phase partitioning bioreactor (TPPB) has become a new strategy for waste gas treatment. However, the impact of biomass and surfactants on gas/liquid (G/L) mass transfer needs to be better evaluated because the effects on the mass transfer coefficient KL and the interfacial area a, respectively, remains misunderstood. RESULTS: This study showed that, first, the surfactant extract produced by Rhodococcus erythropolis reduced the surface hydrophobicity of the biomass. Secondly, an optimal concentration appeared to exist for both components, respectively 0.5 g L−1 and 0.7 g L−1 for biomass (B) and surfactant extract (SE) when the global mass transfer coefficient (KLa) of oxygen was measured in a silicone oil/water TPPB. However, the combination of B and SE was found to induce a negative synergism. In particular, SE improved the interfacial area a by increasing the bubble diameter, while B reduced it as soon as a concentration of 1 g L−1 was exceeded. In contrast, the SE acted negatively on the KL, while B improved it overall. CONCLUSION: Better consideration is needed of the effect of biotic components in order to understand the phenomenon of G/L mass transfer in a TPPB. The behaviour of biomass growth and surfactants may strongly influence the mathematical models proposed in the literature. Copyright © 2009 Society of Chemical Industry

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.

The influence of three extracellular factors (namely, the methyl oleate dispersion in the broth, the dissolved oxygen variations, and the pH fluctuation) on the lipase production by Y. lipolytica in batch bioreactor has been investigated in different scale-down apparatus. These systems allow to reproduce the hydrodynamic phenomena encountered in large-scale equipments for the three specified factors. The effects of the extracellular factors have been observed at three distinct levels: the microbial growth, the extracellular lipase production, and the induction of the gene LIP2 encoding for the main lipase of Y. lipolytica. Among the set of environmental factors investigated, the dissolved oxygen fluctuations generated in a controlled scale-down reactor (C-SDR) have led to the more pronounced physiological effect by decreasing the LIP2 gene expression level. The other environmental factors observed in a partitioned scale-down reactor, i.e., the methyl oleate dispersion and the pH fluctuations, have led to a less severe stress traduced only by a decrease of the microbial yield and thus of the extracellular lipase specific production rate.

Abstract The dynamics of cell growth and bacteriocin production by Lactobacillus curvatus CWBI‐B28 in modified De Man/Rogosa/Sharp (mMRS) broth with various concentrations of glucose and complex nitrogen source (CNS; peptone, yeast extract and meat extract) was investigated in flask fermentations and in a laboratory fermentor using batch and fed‐batch cultivations. In fed‐batch fermentation the rate of feeding of the reactor with the substrates was either maintained constant (0.12 L h−1) or varied exponentially as a function of time. The results showed that both cell growth and bacteriocin activity were influenced by changes in the concentrations of glucose and CNS. Optimal growth and bacteriocin activity were obtained in mMRS broth containing 40 g L−1 glucose and 40 g L−1 CNS (mMRS40/40). A bacteriocin titre of 4266 AU mL−1 and a cell count of 8.7 log colony‐forming units (cfu) mL−1 were recorded when this medium was used for cultivation. In batch fermentation using the same medium, a higher cell count (9.5 log cfu mL−1) and twice as much bacteriocin as in flask fermentation were produced. The highest bacteriocin titre (8533 AU mL−1) was obtained with fed‐batch fermentation at an exponentially varying rate of feeding. Bacteriocin activity and cell dry mass did not always correlate. Copyright © 2007 Society of Chemical Industry

Xylanase is produced by Penicillium canescens 10-10c from soya oil cake in static conditions using solid-state fermentation. The impact of several parameters such as the nature and the size of inoculum, bed-loading, and aeration is evaluated during the fermentation process. Mycelial inoculum gives more production than conidial inoculum. Increasing the quantity of inoculum enhances slightly xylanase production. Forced aeration induces more sporulation of strain and reduces xylanase production. However, forced moistened air improves the production compared to production obtained with forced dry air. In addition, increasing bed-loading reduces the specific xylanase production likely due to the incapacity of the Penicillium strain to grow deeply in the fermented soya oil cake mass. Thus, the best cultivation conditions involve mycelial inoculum form, a bed loading of 1-cm height and passive aeration. The maximum xylanase activity is obtained after 7 days of fermentation and attains 10,200 U/g of soya oil cake. These levels are higher than those presented in the literature and, therefore, show all the potentialities of this stock and this technique for the production of xylanase.

A stochastic microbial growth model has been elaborated in the case of the culture of E. coli in fed-batch and scale-down reactors. This model is based on the stochastic determination of the generation time of the microbial cells. The determination of generation time is determined by choosing the appropriate value on a log-normal distribution. The appropriateness of such distribution is discussed and growth curves are obtained that show good agreement compared with the experimental results. The mean and the standard deviation of the log-normal distribution can be considered to be constant during the batch phase of the culture, but they vary when the fed-batch mode is started. It has been shown that the parameters related to the log-normal distribution are submitted to an exponential evolution. The aim of this study is to explore the bioreactor hydrodynamic effect on microbial growth. Thus, in a second time, the stochastic growth model has been reinforced by data coming from a previous stochastic bioreactor mixing model (1). The connection of these hydrodynamic data with the actual stochastic growth model has allowed us to explain the scale-down effect associated with the glucose concentration fluctuations. It is important to point out that the scale-down effect is induced differently according to the feeding strategy involved in the fed-batch experiments.

peer reviewed

Lactic acid bacteria isolated by screening method from traditional food products of African countries were tested for their ability to produce bacteriocin an antimicrobial substances acting against other bacteria. Some of these bacteriocins allow the inhibition of Listeria monocytogenes, a food-bome pathogen responsible for human listeriosis. However, food ingredients caused an inefficient action of bacteriocins produced by several lactic acid bacteria against Listeria monocytogenes achieving the phenomenon of Listeria growth rebound in bacteriocin-supplemented food models. Recently, new starter cultures of lactic acid bacteria with an industrially important functionality are being developed. The latter can contribute to inhibit the rebounding phenomenon of Listeria and offer one or more organoleptic, technological, nutritional, or health advantages.