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

Different sizes of viable-but-nonculturable cell subpopulationsof a lactic acid bacterium strain were induced by adding increasingamounts of SO₂. The experimental data obtained here were fittedto a segregated kinetic model developed previously. This procedureallowed us to determine in quantitative terms the contributionof this physiological state to malolactic fermentation.

INTRODUCTION

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INTRODUCTION

The persistence of stressed, damaged, or viable-but-nonculturable(VBNC) cells during microbial fermentation underlines the requirementof alternative methods for detecting and characterizing theseemergent states not otherwise detectable by traditional culture-basedmethods (13). Flow cytometry (FC) has evolved as an outstandingtool in bioprocesses due to its usefulness in cell physiologymonitoring (5, 12). The persistence of nonculturable cells duringmicrobial fermentation has been attributed to changes in wateractivity, acidity, redox potential, nutrient availability, andstarvation (14, 17, 18, 24, 25) or to the use of preserved startercultures (20). Additionally, the quantification of catalyticactivity is critical to bioprocess optimization, as it measuresthe individual contributions of different cell subpopulationsto the global process (2, 13). Despite the loss of culturabilityunder standard conditions, it is strongly suspected that VBNCcells remain alive, maintain the transport system and biosynthesis,and are able to metabolize substrates (16, 26). Gene expressionhas also been demonstrated previously (9, 23). However, althoughthe physiology, biochemistry, and genetics of the VBNC statehave been studied over the years, its functionality and biologicalimplication are still issues under intense debate (1, 21, 22).

In this work, cider malolactic fermentation (MLF) was selectedas a model system to clarify the role played by VBNC cells inbioprocesses. MLF was carried out under different SO₂ concentrations(0, 30, and 60 ppm total) for inducing VBNC states. The fermentationmedium was sterile apple must or "green" cider (obtained justafter alcoholic fermentation and containing 5.6% [vol/vol] ethanol),obtained as previously described (11). Sodium bisulfite wasused for SO₂ treatments. MLF was carried out in duplicate at22°C statically in 250-ml bottles. An indigenous strainof Lactobacillus hilgardii was inoculated at an optical densityat 600 nm of $~$ 0.1 to start MLF. Flasks were shaken just beforesampling in order to homogenize the biomass content. Sampleswere taken aseptically at time intervals until malic acid wasconsumed ( $≤$ 0.5 g liter^–1), and cells were collected andprocessed for further analysis as described previously (20).Supernatants were filtered (0.45 µm pore size) and frozen(–20°C) until chemical analysis. The amount of malicacid was determined by high-pressure liquid chromatography (Alliance2690; Waters) with a photodiode array detector (Waters 996),as reported previously (19).

Evolution of bacterial subpopulations during MLF.

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Evolution of bacterial...

Viable cells (measured as CFU ml^–1) were monitored bya plate counting method on MRS (in triplicate), as reportedpreviously (20). Total cell counts were determined by DRAQ5single staining, and metabolically active and dead cells weremonitored by a dual-staining (Chem Chrome 6 [CV6] and propidiumiodide [PI]) FC protocol carried out using a Cytomics FC 500instrument (Beckman Coulter), as described previously (20).This dual-staining protocol was based on the detection of membraneintegrity (PI) and intracellular esterase activity (CV6) asthe metabolic probe. The evolution of total, viable, VBNC, anddead cells during MLF is shown in Fig. 1. A subpopulation ofVBNC cells (calculated as the difference between metabolicallyactive and viable cells) was found in both controls (Fig. 1a and d),as was detected previously under stress conditions during ciderMLF (11). The use of sodium bisulfite caused a drop in cellviability even to the extent of a total absence (Fig. 1b, c, e, and f).Malic acid was consumed in all assays, showing the metabolicactivity associated with the VBNC state, as previously observed(26). The addition of increasing SO₂ amounts did not reallycorrespond to an increase in cell death rate but acceleratedthe transition to VBNC states. The effect was more drastic withgreen cider fermentation, due to a synergistic effect of SO₂and other inhibitors, such as ethanol. Actually, SO₂ has beenreported to lead wine yeast and bacteria to adopt VBNC statesrather than to undergo cell death (6, 7, 15).

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FIG. 1. Evolutions of different L. hilgardii subpopulations during MLF in apple must (A), apple must plus 30 ppm SO₂ (B), apple must plus 60 ppm SO₂ (C), green cider (D), green cider plus 30 ppm SO₂ (E), and green cider plus 60 ppm SO₂ (F). Subpopulations of VBNC cells, viable cells (CFU), and dead cells are plotted as percentages of the total cell counts on the principal y axis. Total ( ${blacklozenge}$ ), VBNC ( ${triangleup}$ ), viable (CFU) (•), and dead ( ${square}$ ) cells are expressed as cells ml^–1 on the secondary y axis. Note the different values along the x axis in the different graphs.

Determination of malolactic activities of viable and VBNC subpopulations.

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Determination of malolactic...

In order to quantify the malolactic activity of VBNC cells,experimental data for cell subpopulations and malate uptakewere fitted (MicroMath Scientist version 2.0) to a segregatedkinetic model developed previously (20). For kinetic modeling,the cell concentrations of bacterial subpopulations were expressedin g liter^–1, using corresponding calibration curves determinedpreviously (20). This model includes different cell physiologicalstates and levels of malate consumption by metabolically activecells (Fig. 2). Enzymatic assays carried out as reported previously(10), using CV6-negative, PI-positive whole cells (referredto as dead cells in this work), showed a lack of malolacticactivity, as expected. Thus, the total biomass involved in malateconversion (C_X) was considered to be formed by VBNC (C_N) andviable (C_V) subpopulations. Since malate metabolism is not directlylinked to cell growth or energy obtainment (4), the initialequation for substrate uptake (equation 6 in Fig. 2) is simplifiedas follows: –(dS/dT) = m_VC_V + m_NC_N, where m_V and m_N arecell maintenance coefficients of viable and VBNC subpopulations,respectively. These parameters can be considered a measure ofthe individual contribution of each subpopulation to the globalprocess. This equation was finally expressed as follows: –(dS/dT)= q_VC_V + q_NC_N, where q_V and q_N are specific uptake rates ofviable and VBNC subpopulations, respectively.

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FIG. 2. Scheme and equations of the segregated kinetic model (adapted from reference 20).

The experimental data and predicted values of different cellsubpopulations during MLF are shown in Fig. 3. The values ofthe kinetic parameters are given in Table 1. The values obtainedfor k₁ and k₅ (growth kinetic constants) indicated that VBNCcells divided in apple must at a rate higher than that of viablecells (k₅ > k₁). The values of death constants, k₃ and k₄,showed slightly higher cell death rates as SO₂ content increased,with k₃ > k₄ in all cases (higher VBNC death net rate).

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FIG. 3. Experimental data (symbols) and calculated values (lines) for L. hilgardii VBNC ( ${circ}$ ), viable (CFU) (•), and dead ( ${square}$ ) cells and L-malic acid consumption ( ${blacktriangleup}$ ) during MLF in apple must (a), apple must plus 30 ppm SO₂ (b), apple must plus 60 ppm SO₂ (c), green cider (d), green cider plus 30 ppm SO₂ (e), and green cider plus 60 ppm SO₂ (f).

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TABLE 1. Kinetic parameters^a determined during MLF in apple must and green cider in the absence and presence of SO₂

Finally, malic uptake experimental data were also fitted tothe equation –(dS/dT) = q_VC_V + q_NC_N, and parameters werecalculated in each case, assuming a constant value during fermentation(Fig. 3; Table 2). In all cases, the q_V/q_N ratio was close to2.0 during fermentation experiments. It was stated previouslythat the catalytic capacity of damaged cells is generally lowerthan that of viable cells (3). Entrance into the VBNC statecan be accompanied by a reduction in substrate transport andmetabolic activity levels in order to minimize cellular energeticrequirements (16), as has been observed with nonculturable lactococciin response to sugar starvation (8).

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TABLE 2. Specific uptake rates of viable and VBNC subpopulations during MLF in apple must and green cider at concentrations of 0, 30, and 60 ppm SO₂

Conclusions.

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Conclusions.

VBNC cells conducting MLF showed a state of reduced metabolicactivity. The specific malate uptake rate of the VBNC subpopulationwas approximately 50% of that found for the viable populationin all cases tested, irrespective of medium composition andSO₂ concentration. VBNC cells became the majority of the totalpopulation in all cases (70 to 90%). These results may helpto clarify, in quantitative terms, the real contribution ofthe VBNC subpopulation to fermentation processes. This outcomecould be useful for bioprocess control and optimization on anindustrial scale.

FOOTNOTES

* Corresponding author. Mailing address: Department of Chemical Engineering and Environmental Technology, Faculty of Chemistry, University of Oviedo, C/ Julián Clavería s/n., 33071 Oviedo, Asturias, Spain. Phone: 34 985 103439. Fax: 34 985 103434. E-mail: mariodiaz{at}uniovi.es

Published ahead of print on 6 March 2009.

REFERENCES

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