Quantitative Approach to Determining the Contribution of Viable-but-Nonculturable Subpopulations to Malolactic Fermentation Processes

Abstract

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...

Effect of acclimation medium on cell viability, membrane integrity and ability to consume malic acid in synthetic wine by oenologicalLactobacillus plantarumstrains

“…Multiparameter FC analysis represents an important tool in winemaking because it allows monitoring industrial fermentation in real time. According to our knowledge, it has been already used to monitor alcoholic fermentation by Saccharomyces cerevisiae in winemaking (Farthing et al 2007), ethanol stresses in Oenococcus oeni (da Silveira et al 2002) and MLF in cider carried out by Lactobacillus hilgardii (Quir os et al 2009). …”

Application of flow cytometry to industrial microbial bioprocesses

“…), the efficiency of different preservation and production processes and products shelf-life [4]. These cells could take an active part in bioprocesses progress and contribute actively to fermentations [8,21,29]. It has been reported that VBNC cells conducting malolactic fermentation showed a state of reduced metabolic activity and that the specific malate uptake rate of the VBNC subpopulation was approximately 50% of that found for the viable population [29].…”

“…These cells could take an active part in bioprocesses progress and contribute actively to fermentations [8,21,29]. It has been reported that VBNC cells conducting malolactic fermentation showed a state of reduced metabolic activity and that the specific malate uptake rate of the VBNC subpopulation was approximately 50% of that found for the viable population [29]. VBNC cells may also show undesirable activity or alter endproduct quality [30,31].…”

“…This model properly explained the correlation between the rate of damage and the intensity of agitation according to a firstorder equation. A segregated model involving viable, VBNC and dead cells to describe subpopulations dynamics and glucose consumption by active cells in batch yeast and bacteria pure cultures in synthetic media was also formulated [25,29] (Fig. 10).…”

Viability study of biofilm-former strains from paper industry by flow cytometry with application to kinetic models

“…These kinetic models describe the temporal evolution of the concentration of microorganisms in a system, as a function of relevant intrinsic or extrinsic variables based on different stages of viability (Herrero et al, 2006;Marks, 2008;Quiros et al, 2007;Quiros et al, 2009). …”