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Flow cytometry has recently been presented as a research tool in the assessment of the viability/activity of activated sludge from municipal wastewater treatment plants, but it has not put in practice for industrial biotreatments yet. In this study, for the first time ever, the reliability and significance of the multiparameter flow cytometry applied to the biological nitrification-denitrification treatment of leachate have been evaluated. Using a double staining procedure (cFDA/PI), the viable, damaged, and dead subpopulations were determined, and the results were compared to those obtained with conventional methods, such as nitrogen and oxygen uptake rates or plate counting. Flow cytometry showed that viable cells represented approximately 47% of the total population, whereas active cells accounted for 90%. For both sludge from nitrification and denitrification processes, with less than 1% of them being also culturable in plate. Either flow cytometry or uptake rates revealed that health status of sludge remained constant throughout the biotreatment, which is consistent with the high recirculation rates. Under anaerobic starvation conditions, physiological status of sludge remained constant as well as specific oxygen and denitrification rates. Nevertheless, both the culturability in plate and the nitrification rate significantly decreased. These findings proved that multiparameter flow cytometry is a useful tool for the assessment of the viability and activity of sludge from a nitrification-denitrification biotreatment process. These results gathered all the bacterial communities in the sludge, so the decay in minority populations, such as nitrifying bacteria, requires the use of a complementary technique to evaluate specific activities.

Flow cytometry (FC) has been introduced to characterize and to assess the physiological states of microorganisms in conjunction with the classical plate-counting method. To show the applicability of the technique, in particular for the development of kinetic models, pure culture fermentation experiments were followed over time, using both prokaryotic (Lactobacillus hilgardii) and eukaryotic (Saccharomyces cerevisiae) microorganisms growing in standard culture media (MRS and YPD). The differences observed between the active and viable cells determined by FC and CFU, respectively, allowed us to determine that a large number of cells were in a viable but nonculturable (VBNC) state, which resulted in a subpopulation much larger than the damaged-cell (double-stained) subpopulation. Finally, the determination of the evolution of viable, the VBNC, and the dead cells allowed us to develop a segregated kinetic model to describe the yeast and the bacteria population dynamics and glucose consumption in batch cultures. This model, more complete than that which is traditionally used, based only on viable cell measurements, describes better the behavior and the functionality of the cultures, giving a deeper knowledge in real time about the status and the course of the bioprocesses.

The flow cytometry (FC) technique used with certain fluorescent dyes (ChemChrome V6 [CV6], DRAQ5, and PI) has proven useful to label and to detect different physiological states of yeast and malolactic bacterium starters conducting cider fermentation over time (by performing sequential inoculation of microorganisms). First, the technique was tested with pure cultures of both types of microorganisms grown in synthetic media under different induced stress conditions. Metabolically active cells detected by FC and by the standard plate-counting method for both types of microorganisms in fresh overnight pure cultures gave good correlations between the two techniques in samples taken at this stage. Otherwise, combining the results obtained by FC and plating during alcoholic and malolactic fermentation over time in the cider-making process, different subpopulations were detected, showing significant differences between the methods. A small number of studies have applied the FC technique to analyze fermentation processes and mixed cultures over time. The results were used to postulate equations explaining the different physiological states in cell populations taken from fresh, pure overnight cultures under nonstress conditions or cells subjected to stress conditions over time, either under a pure-culture fermentation process (in this work, corresponding to alcoholic fermentation) or under mixed-fermentation conditions (for the malolactic-fermentation phase), that could be useful to improve the control of the processes.

Abstract: Flow cytometry (FC) is a powerful technique for the rapid analysis of single cells in a mixture, by means of light-scattering and fluorescence measurements. In biotechnology, the power of this method lies both in the possibility of determining a wide range of cell parameters at single cell level, and in the ability to obtain information about their distribution within cell populations, providing valuable information for bioprocess design and control. The first part of this review will be devoted to describe the basic aspects of multicolour FC, the different strategies used for cell detection, and the cellular target sites and fluorescent probes which are currently used in cell assays. Next, a deep revision concerning the most recent FC applications related to cell analysis and quantification (especially both bacteria and yeast) in food, drink and pharmaceutical industries and in natural environment and water systems is presented. This review seeks to highlight the advantages of this technique in microbial fermentations monitoring and control, as well as in the development of more accurate kinetic models directed to bioprocesses optimization. Finally, an overview of some commercial FC devices is presented along with a discussion of the progress and advantages of the method for bioprocesses on-line monitoring. [Copyright &y& Elsevier]

Abstract: High-level production of lactobionic acid from whey by Pseudomonas taetrolens under fed-batch fermentation was achieved in this study. Different feeding strategies were evaluated according to the physiological status and fermentation performance of P. taetrolens. A lactobionic acid titer of 164g/L was obtained under co-feeding conditions affording specific and volumetric productivities of 1.4g/gh and 2.05g/Lh, respectively. Flow cytometry assessment revealed that P. taetrolens cells exhibited a robust physiological status, which makes them particularly well-suited for employing concentrated nutrient solutions to further prolong the growth and production phases. Such detailed knowledge of the physiological status has been revealed to be a key issue to further support the development of high-yield lactobionic acid production processes under feeding strategies. The present study has demonstrated the feasibility of P. taetrolens to achieve high-level bio-production of lactobionic acid from whey through fed-batch cultivation, suggesting its major potential for industrial-scale implementation. [Copyright &y& Elsevier]

Highlights: [•] Efficient lactic acid production by Lactobacillus casei in mixed sugar fermentation. [•] Monitorization of Lactobacillus casei physiological states through flow cytometry. [•] Lactobacillus casei cells remained metabolically active under uncontrolled-pH conditions. [•] Increase in physiological heterogeneity of Lactobacillus casei under pH-controlled conditions. [•] A segregated kinetic model derived from flow cytometry data was developed. [ABSTRACT FROM AUTHOR]