INTRODUCTION

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Homofermentative lactic acid bacteria (LAB) are used primarily in the dairy industry but may also prove to be advantageous hosts for production of lactate as a bulk chemical or of food additives such as bacteriocins or amino acids using recombinant DNA technology (10, 15). The metabolism of these bacteria is constrained by the requirement for a balance between NADH-producing and -consuming reactions. In the absence of external electron acceptors (e.g., oxygen), the carbon fluxes of catabolism are tightly coupled. In anaerobic culture, glucose may be redox-neutrally converted to lactate or, alternatively, to the mixed acid products formate, acetate, and ethanol in a molar ratio of 2:1:1 (12, 26). However, when oxygen is present in the growth medium, the catabolic carbon fluxes are uncoupled from the redox metabolism due to the NAD⁺-regenerating activity of NADH oxidases (NOX). This oxygen-related potential for redirecting fluxes is used industrially in connection with the formation of other metabolites such as diacetyl in fermented milk products. Even submillimolar concentrations of diacetyl have a strong impact on the taste of buttermilk and cheese (22). Hence, minute amounts of oxygen may influence an industrial process greatly. In this light, it is surprising that, to our knowledge, no investigations have been carried out on the microaerobic physiology of starter cultures. A number of studies on the effect of oxygen in fully aerated cultures have been published, but little light has been shed on the window ranging from anaerobic to fully aerobic conditions of growth (4, 7, 8, 20).

Interest in the microaerobic physiology of LAB is furthermore spurred by reports on the extreme sensitivity of pyruvate formate-lyase (PFL) to oxygen (1, 25) and the negative effect of oxygen on pfl gene expression (2, 19). Similarly, it has recently been reported that vigorous aeration reduces the transcription of the adhE gene, which encodes alcohol dehydrogenase (ADH) in Lactococcus lactis (3). However, to our knowledge, no data have been published on the effect of controlled aeration on the ADH enzyme protein level. While oxygen inactivates the PFL enzyme, the activity levels of other enzymes are stimulated under aerobic conditions. This is the case for the -acetolactate synthase (ALS), the initial step in the conversion of pyruvate to acetoin and diacetyl, and the pyruvate dehydrogenase complex (PDH) (8).

Thus, the presence of oxygen in the growth medium influences the metabolism of LAB not only by loosening the tight coupling between catabolic carbon fluxes and the redox metabolism but also by affecting the cellular content of key enzymes. The present investigation was initiated to determine the effect of different levels of controlled mild aeration on product formation and hence on in vivo activities of the enzymes downstream of the pyruvate branch point: PDH, ALS, PFL, and lactate dehydrogenase (LDH). The effect of oxygen on the synthesis of PFL and ADH proteins and on the activity of the enzymes NOX, PDH, ALS, LDH, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was quantified under different growth conditions. As such, the microaerobic physiology of L. lactis MG1363 was determined and the link to levels of enzyme protein or activity under the growth conditions studied was found. To the best of our knowledge, no study of microaerobic conditions of growth has previously been carried out for a homofermentative LAB strain.

The relevance of the present study is both academic and industrial. Diffusion of air into the growth medium brings about a microaerobic environment at the interface, which may have important implications for production of flavor-associated secondary metabolites. Academically, the influence of oxygen on the distribution of pyruvate among four competing enzymes is a challenge since it involves regulatory control at several levels (Fig. 1). Continuous cultivation was the tool of choice for investigating the effects of varying the dissolved oxygen tension (DOT), since a constant growth rate can be fixed and the in vivo activities of the product forming pathways can be precisely determined. In this way, we showed that minute amounts of oxygen have a dramatic effect on the resulting end product profile.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Metabolic network around the pyruvate node. AK, acetate kinase; PYK, pyruvate kinase. Other abbreviations are defined in the text.

	MATERIALS AND METHODS

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Strain and culture conditions. The homofermentative laboratory strain L. lactis ssp cremoris MG1363 (13) was the only organism used in this study. The organism was grown at 30°C in continuous mode in a baffled bioreactor (Applikon, Schiedam, The Netherlands) with a 1,000-ml working volume and fitted with a four-blade Rushton turbine rotating at 350 rpm. The dilution rate (D) was set at 0.1 h¹. The pH was kept constant at 6.6 by automatic addition of 5 M KOH.

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Analysis of fermentation end products and glucose. Ethanol, acetoin, acetate, formate, lactate, and pyruvate were separated by high-pressure liquid chromatography (AminoHPX-87H column; Bio-Rad, Richmond, Calif.) at 65°C using 5 mM H₂SO₄ flowing at 0.6 ml · min¹ as the eluent. Ethanol and acetoin were quantified with a Waters 410 refractive index detector (Millipore, Badford, Mass.), while acetate, formate, lactate, and pyruvate were quantified with an Waters 486 absorbance detector set at 210 nm.

Determination of bacterial cell dry weight. Nitrocellulose filters (pore size, 0.45 µm) were used to determine the cell dry weight. The filters were tared after having been dried in a microwave oven at 150 W for 10 min. The biomass from 10 ml of culture was collected on the filter and washed with 10 ml of distilled water before being dried and tared as indicated above.

Enzyme assays in vitro. Enzyme extracts were prepared essentially as described previously (12). Approximately 100 ml of culture volume was collected from the bioreactor and centrifuged (6,000 × g for 10 min at 4°C). The supernatant was discarded, and the biomass washed twice in 200 ml of 0.2% (wt/vol) KCl and subsequently resuspended in 5 ml of a pH 7.2 buffer of the following composition: 45 mM Tris, 15 mM tricarballylic acid, 20% (vol/vol) glycerol, 1 mM dithiothreitol, and 4.5 mM MgCl₂. The samples were stored at 25°C until analysis. Enzymes were extracted by sonication of cells (five cycles of 30 s alternating with 60 s of cooling on ice). The resulting extract was clarified by centrifugation (10,000 × g for 10 min at 4°C) and immediately analyzed for enzyme activity and enzyme protein levels as specified below. The protein concentration of the enzyme extract was determined by the assay of Lowry et al. (18) using bovine serum albumin as standard.

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	RESULTS

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A supply of oxygen leads to an increased yield of biomass on glucose. In preliminary experiments with a glucose feed concentration of 10 g · liter¹, it was observed that mild aeration of the bioreactor changed the situation of glucose limitation prevalent under anaerobic conditions to one of glucose excess at 5% DOT (data not shown). Since our primary interest lay in studying glucose-limited cultures, the reason for the accumulation of glucose in the bioreactor was not investigated (previous work [14] suggests an inhibitory effect of H₂O₂ under aerobic conditions and with a high sugar concentration in the feed). By reducing the concentration of glucose in the feed to 3 g · liter¹, we were able to determine five distinct steady states which were all glucose limited (Table 1). One steady state (steady state 1) was anaerobic, while three (steady states 2 to 4) were mildly aerated with increasing ratios of air to N₂ in the gas feed but with unmeasurable values of DOT. The final steady state (steady state 5) was obtained for a measured and controlled DOT value of 5%.

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A supply of oxygen strongly affects pyruvate metabolism. Based on the known dilution rate (D = 0.1 h¹) and the measured steady-state concentrations of glucose, biomass, and catabolic products (Tables 1 and 2), we estimated the specific in vivo activities through the enzymes metabolizing pyruvate and involved in NAD⁺ regeneration (Fig. 2 and 3). For all five steady states, 94% or more of the glucose carbon was recovered in biomass and end products. These data furthermore showed that in our experimental plan, several patterns of behavior ranging from anaerobic metabolism (ethanol, formate, and acetate besides lactate) to aerobic metabolism (more acetate, some CO₂, but no ethanol or formate) were covered (Table 2).

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Distribution of carbon at the pyruvate branch point as a function of aeration. As an example, the in vivo specific activity of PFL at steady state 3 was computed as follows: rPFL = concentration of formate × dilution rate/biomass concentration = 26.25 (C-mmol · liter1) × 0.1 (h1)/0.58 (g [dry weight] · liter1 = 4.77 C-mmol · g (dry weight)1 · h1. The in vivo specific activity of PDH was computed as rPDH = [(racetate kinase + rADH)/2]  rPFL in millimoles of carbon per gram (dry weight) per hour.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Synthesis of NAD+ by ADH, LDH, NOX, and PDH as function of growth conditions. As an example, the in vivo specific activity through ADH for steady state 2 was calculated as follows: rADH = (concentration of ethanol) × (moles of NAD+ generated per C-mole of ethanol generated) × (dilution rate)/(biomass concentration) = 15.92 (C-mmol · liter1) × 1 (mmol of NAD+ · C-mmol1) × 0.1 (h1)/0.60 (g [dry weight] · liter1) = 2.65 mmol of NAD+ · g (dry weight)1 · h1. The in vivo flux through NOX was calculated as rNOX = (number of moles of NAD+ generated per mole of O2 consumed) × (specific uptake rate of oxygen).

Aeration affects the enzyme levels. The in vitro levels of GAPDH and of enzymes downstream of the pyruvate branch point were determined in an attempt to obtain the pattern of regulation triggered by the presence of oxygen in the medium (Table 3). An increase in aeration had no effect on the enzyme activity levels of GAPDH, while the flux through the enzyme, quantified as the specific rate of glucose uptake, decreased with increasing aeration (Table 1). This was surprising since increasing aeration was expected to reduce the ratio of NADH to NAD⁺ (9, 23), hence relieving the inhibitory effect of an elevated ratio on GAPDH activity (11). The data therefore suggest that the enzyme was insensitive to the putative variation of the NADH/NAD⁺ ratio. This led us to believe that the ratio, even for the anaerobic steady state, was so low that it hardly had any inhibitory effect on the in vivo activity of the GAPDH enzyme. The level of LDH in vitro activity was not affected by increasing aeration.

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Regulation of in vivo activities. (i) PFL. For steady states 2 and 3, the specific in vivo activity of PFL was unaltered compared to the anaerobic level. This was the case despite the approximately two fold-lower PFL protein level observed for steady states 2 and 3 (Table 3). This shows that the PFL protein level was not limiting for formate synthesis under the anaerobic conditions studied here. It is doubtful that the limitation of PFL activity under anaerobic conditions could be attributed to the intracellular levels of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, both allosteric inhibitors of the enzyme (25), since it has been shown that at a comparable dilution rate (D = 0.12 h¹), the intracellular levels of the two metabolites are far below the values which would lead to inhibition (28).

View larger version (17K): [in this window] [in a new window]   FIG. 4.   In vivo specific activity of PFL versus the intracellular level of the enzyme (sum of active and inactive forms), expressed as a percentage of the amount found under anaerobic conditions of growth. Horizontal error bars represent the standard deviations of five duplicate determinations of the protein in the sample. Vertical error bars representing the standard deviation of three determinations of the in vivo specific activity of the enzyme are not visible since the standard deviations were low on the scale of the figure.

(ii) ADH. Ethanol synthesis decreased drastically on slight aeration (Table 2), which is ascribed partly to a reduced protein level of ADH in the cell (Table 3). Furthermore, the sigmoidal shape of the relationship linking flux through ADH to the in vitro ADH level suggests that increased aeration lowered the NADH/NAD⁺ ratio, leading to a higher degree of inhibition of ADH (Fig. 5). It appears from our data that the ADH protein level present under anaerobic conditions does not limit the rate of ethanol synthesis. The same conclusion was reached for the PFL protein level and formate synthesis.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   In vivo specific activity of ADH versus the intracellular level of the enzyme, expressed as a percentage of the amount found under anaerobic conditions of growth. Error bars are defined in the legend to Fig. 4. Steady states 4 and 5 are both close to the origin.

	DISCUSSION

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Under anaerobic conditions, pyruvate is converted to lactate by LDH and to acetyl-CoA and formate by PFL. Acetyl-CoA fuels the synthesis of lipids, of acetate through acetate kinase, and of ethanol through ADH. ATP production accompanies the synthesis of acetate, whereas ethanol production regenerates NAD⁺ consumed in the glycolysis and in the biomass synthesis. By supplying oxygen to the growth medium, NAD⁺ regeneration was taken over by NADH oxidases, whereby the organism can redirect the flux from ethanol to increased acetate synthesis, thus producing more ATP. This led to a higher yield of biomass on glucose and clearly demonstrates that ATP synthesis limits biomass formation when the organism is grown at a dilution rate of 0.1 h¹ under anaerobic conditions. NOX most probably displayed a higher affinity for NADH than did ADH, judging from the decrease in specific ethanol production as oxygen was introduced.

Oxygen, even at undetectable levels, triggered a response down regulating the capacity of the PFL-ADH pathway and increasing the in vitro activity levels of ALS, NOX, and PDH. In contrast, GAPDH and LDH in vitro activity was not affected by increasing aeration. A significant redirection of carbon at the pyruvate branch point ensued from the altered enzyme levels. Our results show that PFL and PDH can function simultaneously, although this requires fine-tuned conditions of growth.

The anaerobic product spectrum consisting of formate, acetate, ethanol, and lactate was replaced with the aerobic product pattern (acetate, CO₂, and lactate) for low values of DOT (5% DOT, corresponding to 13 µmol of O₂ · liter¹). This underlines the strong influence of low oxygen levels on a continuous cultivation operated at low dilution rate.

We ascribe the lack of acetoin synthesis to the rather low level of the glycolytic flux obtained for D = 0.1 h¹ used in our study. This prevents a large pool of intracellular pyruvate from building up; because of the low affinity of ALS for pyruvate (16, 24), a high intracellular concentration of pyruvate (e.g., by decomposition of citrate) is needed if carbon is to be directed through ALS.

The molecular mechanism of oxygen action is unknown, but it is reasonable to assume that oxygen plays a significant role on the level of gene expression and hence on enzyme levels. Increased aeration diminished the synthesis of PFL and ADH proteins while raising the activity levels of ALS, NOX, and PDH. Furthermore, oxygen is known to irreversibly inhibit PFL by inducing peptide bond cleavage. The presence of oxygen will probably also change the NADH/NAD⁺ ratio and thus the in vivo activity of enzymes involving this pair of cofactors (GAPDH, PDH, LDH, ADH, and NOX). We have tried hard to measure NADH in cellular extracts on a fluorometer by using an enzyme-linked assay described previously (12), but the presence of interferences in the extract, particularly under aerobic conditions, impeded the analysis (16). This has prevented us from confirming a correlation between the NADH/NAD⁺ ratio and the in vivo specific activities of the enzymes mentioned above.

Preliminary results from our laboratory show that the oxygen-induced regulatory cascade leading to higher ALS, NOX, and PDH activity is not only controlled by aeration but also subjected to glucose repression (data not shown).

This work demonstrates the strong impact of a minute oxygen supply on pyruvate metabolism and suggests that both altered gene expression and varied redox levels play a predominant role in regulation of this metabolism.