No abstract

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.

Analysis of microbiological samples from fermentation processes in the beverage industry (beer, wine, and cider) by traditional indirect, culture-based standard methods is timeconsuming, and the methods do not produce direct information about the physiological state of the microorganisms. Moreover, the plate-counting method detects only cells able to form colonies under the conditions of the medium that is used, ignoring the presence of cells that do not form colonies but are nevertheless metabolically active (6). As is known, standard laboratory culture media rarely resemble natural environmental conditions (32). The application of flow cytometry (FC) with fluorescent dyes is faster and more direct and has made it possible to distinguish stages beyond the classical definition of viability generally demonstrated by culturing (24). FC can be used to quantitatively measure the optical characteristics of cells as they pass, in single file, into a focused beam of light (for a review, see reference 33). As particles pass through the beam, three parameters are measured: forward light scatter, side angle light scatter, and fluorescence at selected wavelengths. Light scatter is related to cell mass, structure, surface properties, and the optical density of the internal medium. A variety of fluorescent dyes may be used to detect structural or functional cellular properties. In this way, different cellular functions, such as reproductive growth, metabolic activity, and membrane integrity, can be detected, allowing a definition of the term viable-but-not-culturable cells (VBNC) (24) (other authors have called them active but nonculturable). Previous studies have reported that there are two major adaptations that cells undergo during the formation of VBNC states: cell wall toughening and DNA condensation (29). The detection of metabolic activity provides presumptive evidence of reproductive growth by the demonstration of enzyme activity, such as esterases, along with membrane integrity. In the absence of metabolic activity, it is still possible to determine membrane integrity by either dye retention or dye exclusion, the latter using propidium iodide (PI). Cells without an intact membrane cannot maintain or generate the electrochemical gradient that generates the membrane potential and can be considered dead cells (24). The use of FC techniques was reported for the "at-line" study of Escherichia coli fermentations (14), which detected a considerable drop in cell viability (about 20%) during the latter stages of small-scale (5-liter), well-mixed fedbatch fermentations.Flow-cytometric applications using different dyes have been compared to standard methods to assess yeast cultures in baking, wine making, cider making, and brewing (1,5,7,8,10,11,17,19). The technique has also been used to study the membrane integrity of ethanol-stressed Oenococcus oeni cells (9). Most studies of FC applications have focused mainly on microorganims as pure cultures, and only a small number have analyzed mixed populations over time (30).The purp...

Different sizes of viable-but-nonculturable cell subpopulations of a lactic acid bacterium strain were induced by adding increasing amounts of SO 2 . The experimental data obtained here were fitted to a segregated kinetic model developed previously. This procedure allowed us to determine in quantitative terms the contribution of this physiological state to malolactic fermentation.The persistence of stressed, damaged, or viable-but-nonculturable (VBNC) cells during microbial fermentation underlines the requirement of alternative methods for detecting and characterizing these emergent states not otherwise detectable by traditional culture-based methods (13). Flow cytometry (FC) has evolved as an outstanding tool in bioprocesses due to its usefulness in cell physiology monitoring (5, 12). The persistence of nonculturable cells during microbial fermentation has been attributed to changes in water activity, acidity, redox potential, nutrient availability, and starvation (14,17,18,24,25) or to the use of preserved starter cultures (20). Additionally, the quantification of catalytic activity is critical to bioprocess optimization, as it measures the individual contributions of different cell subpopulations to the global process (2, 13). Despite the loss of culturability under standard conditions, it is strongly suspected that VBNC cells remain alive, maintain the transport system and biosynthesis, and are able to metabolize substrates (16,26). Gene expression has also been demonstrated previously (9, 23). However, although the physiology, biochemistry, and genetics of the VBNC state have been studied over the years, its functionality and biological implication are still issues under intense debate (1,21,22).In this work, cider malolactic fermentation (MLF) was selected as a model system to clarify the role played by VBNC cells in bioprocesses. MLF was carried out under different SO 2 concentrations (0, 30, and 60 ppm total) for inducing VBNC states. The fermentation medium was sterile apple must or "green" cider (obtained just after alcoholic fermentation and containing 5.6% [vol/vol] ethanol), obtained as previously described (11). Sodium bisulfite was used for SO 2 treatments. MLF was carried out in duplicate at 22°C statically in 250-ml bottles. An indigenous strain of Lactobacillus hilgardii was inoculated at an optical density at 600 nm of ϳ0.1 to start MLF. Flasks were shaken just before sampling in order to homogenize the biomass content. Samples were taken aseptically at time intervals until malic acid was consumed (Յ0.5 g liter Ϫ1 ), and cells were collected and processed for further analysis as described previously (20). Supernatants were filtered (0.45 m pore size) and frozen (Ϫ20°C) until chemical analysis. The amount of malic acid was determined by highpressure liquid chromatography (Alliance 2690; Waters) with a photodiode array detector (Waters 996), as reported previously (19).Evolution of bacterial subpopulations during MLF. Viable cells (measured as CFU ml Ϫ1 ) were monitored by a plate counting metho...

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