Departament de Microbiologia i Ecologia, Facultat de Biologia, Universitat de València, C/Dr Moliner, 50, Burjassot, València, Spain 46100-E1
Author for correspondence: Isabel Pardo. Tel: +34 6 3864390. Fax: +34 6 3864372. e-mail: Isabel.Pardo{at}uv.es
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ABSTRACT |
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Keywords: aerobic glucose catabolism, NADH, ethanol-forming pathway, Leuconostoc oenos
Abbreviations: ADH, alcohol dehydrogenase; EFP, ethanol-forming pathway; LAB, lactic acid bacteria; MBB, MLO basal broth; MBBG, MLO basal broth plus glucose (55mM); Xmax, maximum biomass
a Present address: Facultad de Ciencias Experimentales y de la Salud, Universidad Cardenal HerreraCEU, Edifici Seminari S/U, 46113, Montcada, Spain.
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INTRODUCTION |
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METHODS |
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Media and cultivation conditions
Effect of anaerobic and aerobic conditions on catabolism of sugar and growth.
O. oeni M42 was grown in 200 ml MLO basal broth (MBB), i.e. MLO without sugars, citrate, tomato juice or cysteine, in a 350 ml Biostat-Q multiple bioreactor (B. Braun). Sugars (glucose or fructose) were supplied to the medium before sterilization at a final concentration of 55 mM. MBB plus 55 mM glucose (MBBG) was used to test the effect of adding external electron acceptors. The compounds added were pyruvate (20 mM), fructose (20 mM), or L-cysteine.HCl (1·5, 7·5, 15 or 30 mmol l-1; Sigma). In each case the pH was adjusted to 4·8 with 1 M KOH. After autoclaving, media were supplemented with 0·01 g l-1 filter-sterilized D-pantothenic acid. The bioreactors were inoculated with an initial biomass concentration of 2 mg l-1 from a pre-culture grown in flasks at 28 °C for 48 h, without stirring. The cultures in the bioreactors were grown at 28 °C with constant stirring (200 r.p.m.). The aerobic or anaerobic conditions were obtained by a continuous flush (0·50 l min-1) of sterile air or N2, respectively. O2 partial pressure was continuously controlled and registered in bioreactors to ensure aeration conditions (anaerobic, pO2<5%, or aerobic, pO2>95%, as required). Microbial growth was recorded by periodic sampling of the culture broth. Optical densities of samples were measured at 600 nm. A calibration curve was constructed to correlate OD600 and dry weight (g l-1). Specific growth rates were calculated from the cell mass profiles as described by Salou et al. (1994) . The changes in pH of the media were recorded. The concentrations of residual sugars and the final products of fermentation were quantified in the broth at the end of the experiments, by HPLC (see Analysis of chemical compounds).
Effect of anaerobic and aerobic conditions on the internal pools of NADH and NADPH.
A separate experiment was designed to determine the effect of aeration conditions on the pool concentrations of the cofactors and on the activity of cell enzymes related to their reoxidation. Cells were grown in 1 l MBBG under N2 flushing, in a 2 l Biostat-B bioreactor (B. Braun). When the culture reached a level of 0·08 g cell dry matter per litre, 200 ml was aseptically transferred to another bioreactor and then continuously aerated, whereas the rest of the culture continued growing anaerobically. The experiments lasted 5 d in both reactors, sparged with either N2 or air. Samples were collected at the end of this period to measure the ethanol forming pathway (EFP) enzyme activities. Duplicate experiments were performed for each culture condition.
Analysis of chemical compounds.
Samples were withdrawn directly from the bioreactors through the sampling ports and subsequently stored at -20 °C. For chemical analysis, supernatants from centrifuged samples (16000 r.p.m., 30 min) were filtered through a C-18 cartridge (Sep-Pak, Waters) and then through a 0·22 µm membrane filter. The concentrations of sugars, organic acids and ethanol in the fermentation broth were quantified by HPLC using an HPX-87H Aminex ion-exclusion column as previously described (Maicas et al., 1999 ). ATP yields were determined from experimental lactate and acetate concentrations as described by Firme et al. (1994)
.
Preparation of cell-free extracts.
Culture broth was withdrawn from the bioreactor into an ice-cooled bottle, centrifuged and washed twice with 10 mM potassium phosphate buffer (pH 7·0, 4 °C) and once with 3 mM Tris/HCl (pH 7·0, 4 °C). Cells were then resuspended in 2 ml 6·4 mM Tris/HCl (pH 8·4, 4 °C) and disrupted by breaking them in a vortex with 1 vol. of 0·1 mm glass beads for 10 min. Cells and cell extracts from the anaerobic cultures were maintained under N2 flushing to prevent exogenous oxidation. The suspension was centrifuged (16000 r.p.m., 30 min, 2 °C) and the cell-free supernatant was pooled in a pre-cooled Eppendorf tube and stored at -20 °C for further analyses.
Protein quantification in the cell-free extracts.
The Micro BCA Protein Assay Reagent (Pierce) was used to measure the protein concentration in the cell-free extracts, according to the manufacturers instructions, using BSA as a standard.
Analysis of in vitro enzyme activities.
Enzyme assays were performed at 30 °C in quartz cuvettes with a 1 cm light path using a Beckman model DU-7 spectrophotometer. The activity of alcohol dehydrogenase (ADH; EC 1.1.1.1) was measured in 3 ml of a reaction mixture containing 0·2 M glycine/NaOH buffer (pH 9·0), 3·3% (v/v) ethanol, 7·5 mM ß-NAD+ and 150 µg protein extract (Klingenberg, 1985 ). Enzyme activity was calculated using the increase in A340 resulting from the coenzyme reduction. The reaction for ADH (NADP+) (EC 1.1.1.2) activity determination was identical to that described above, except that ß-NAD+ was replaced by ß-NADP+. For acetaldehyde dehydrogenase (acetylating) (EC 1.2.1.10) determination, the coenzyme reduction was measured in a final volume of 3 ml containing: 0·2 M K2HPO4/KH2PO4 buffer pH 7·4; 10 mM 2-mercaptoethanol; 5 mM acetaldehyde; 50 mM ß-NAD+ (or ß-NADP+); and, 150 µg protein extract. Phosphate acetyltransferase (EC 2.3.1.8) activity was determined by measuring acetyl phosphate decomposition in the presence of coenzyme A and arsenate according to the method of Kelly & Patchett (1996)
. NAD(P)H oxidase activities were assayed in 50 mM potassium phosphate buffer (pH 7·0) containing 1 mM EDTA and 25 µm ß-NADH (or ß-NADPH) (Anders et al., 1970
). The reactions were started by adding the extract; the decreases in A340 were monitored. One unit of enzyme activity was defined as the amount of enzyme reducing 1 µmol coenzyme min-1; the specific activity was expressed as mmol (g protein)-1 min-1. The functionality of the assays was verified with enzymes purchased from Sigma.
Enzyme inactivation in vitro.
Protein extracts were obtained from cells grown on MBBG under anaerobic conditions. Cells were recovered from culture medium at late-exponential phase and extracts were made as described above. Extracts were air-flushed in 0·2 M glycine/NaOH buffer for 6 h (with or without 3 mM cysteine). Samples were periodically withdrawn and ADH activity was measured as described. The enzyme activity of unflushed extracts was recorded and used as a control.
Quantification of cofactors.
Assays were performed in cuvettes equilibrated at 30 °C using a Perkin-Elmer model LS-50 Luminescence spectrometer with excitation at 340 nm and emission peak at 460 nm. Buffers and stock solutions were prepared with ultrapure water and were filtered through 0·1 µm filters. Each enzyme was prepared at the highest possible concentration, and fresh dilutions were made each day. Standard solutions of pyrimidine nucleotides were prepared each day and stored on ice. Standard substrate solutions were prepared daily and were assayed spectrophotometrically on the day of use.
ß-NAD+ quantification was performed in 1·5 ml 0·1 M Tris/HCl containing 0·4 M hydrazine hydrate (pH 8·5). The reaction was started by adding 0·5% (v/v) ethanol. When temperature equilibration was completed (12 min), 7·5 µl ADH (EC 1.1.1.1) (566 U ml-1) was added. After 23 min, 7·5 µl standard solution (0·1 mM ß-NAD+) or cell-free extracts were added to the reaction solution. ß-NADH production was measured by reading the increase of fluorescence. ß-NADP+ quantification was performed in 1·5 ml 0·05 M triethanolamine/HCl, 10 mM MgCl2, 5 mM EDTA (pH 7·4) as described for ß-NAD+ quantification, but using 0·1 M glucose 6-phosphate as substrate and 50 µM ß-NADP+ as cofactor. The reaction was catalysed by glucose-6-phosphate dehydrogenase:NADP+ oxidoreductase (EC 1.1.1.49) (27·8 U ml-1). NAD(P)H quantification was carried out by luminescence as described by Klingenberg (1985) . Anaerobic conditions were maintained by N2-flushing to prevent indirect effects, when required.
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RESULTS AND DISCUSSION |
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NADH- and NADPH-oxidase activities were recorded under aerobic conditions for O. oeni (Table 2), although the reported levels were probably not sufficient to efficiently reoxidize the cofactors given that growth was negligible under aerobic conditions. On the other hand, cells grown under anaerobic conditions produced up to 0·30 g cell dry matter per litre of medium. The lack of significant activity for NAD(P)H oxidases under aerobic conditions has been previously reported in other LAB (Nuraida et al., 1992
; Warriner & Morris, 1995
). Although NADH oxidases appear to be widely distributed among LAB, some strains lack them, whereas NADPH oxidases have only rarely been reported in LAB (Warriner & Morris, 1995
). Warriner & Morris (1995)
described the inability of Lactobacillus hilgardii to benefit from the presence of O2, which was explained by the lack of a NAD(P)H oxidase system. However, the absence of NAD(P) oxidases is not enough to explain low cell growth under aerobic conditions, because Lucey & Condon (1986)
reported that the aerobic growth of a mutant of Leuconostoc mesenteroides X2 lacking NADH oxidase was similar to its growth under anaerobic conditions, with the relative proportions of lactate and ethanol being the same under aerobic and anaerobic conditions. The low glucose consumption under aerobic conditions, and the low quantity of ethanol produced, suggested that the EFP was very inefficient. Ito et al. (1974
, 1983
) suggested that the effect of aeration on glycolysis in Leuconostoc mesenteroides was the consequence of the inability of cellular activities to reoxidize NAD(P)H via the EFP. The results of our enzyme assays to check EFP activity in O. oeni showed that both alcohol and acetaldehyde dehydrogenase (NAD+ and NADP+ dependent) activities were high enough to support cell growth in glucose-containing anaerobic cultures (Table 2
); however, these activities could not be detected in cell extracts transferred to aerobic conditions. This suggests that the EFP enzymes were inhibited by O2, thus stopping the normal way in which cofactors are reoxidized during the heterofermentative catabolism of glucose (Fig. 1
, Table 2
). Although it could be possible that O2 represses the synthesis of EFP enzymes instead of inhibiting the enzymes, it is reasonable to assume that the enzymes are constitutive, as has been reported for Leuconostoc mesenteroides (Sakamoto & Komagata, 1996
). This route is essential for reoxidation of the cofactors produced in the first steps of heterolactic sugar catabolism, and inhibiting this pathway would seriously compromise cellular development. There is evidence that O2 inhibits enzyme activity of O. oeni in vivo: transfer of an aliquot of a bioreactor culture grown on glucose from anaerobic to aerobic conditions resulted in loss of activity of EFP enzymes after 1 h. In addition, we found that normal activity in anaerobic extracts dramatically decreased when they were flushed with air.
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Aerobic growth in a glucose medium supplied with reducing substrates
The previous results suggest that adding reducing agents to MBBG under aerobic conditions could improve the regeneration of cofactors to some extent. Given that ADH and acetaldehyde dehydrogenase were inhibited by O2 in O. oeni M42, pyruvic acid, fructose or cysteine were added to MBBG in order to test the ability of these products to reoxidize NAD(P)H and, hence, to improve growth. Growth on glucose alone was negligible (Fig. 3). When pyruvic acid was supplied it was completely metabolized to lactic acid and acetic acid, reoxidizing NADH and leading to higher biomass production (Fig. 3
). The stoichiometrical balance showed a twothreefold greater yield of acetic acid than when O. oeni was grown on glucose alone (Table 4
), whilst higher levels of ATP [YATP=0·11 mol ATP (mol sugar)-1] were produced. The addition of pyruvic acid resulted in 20-fold higher levels of biomass production (Xmax=0·09 g l-1) than those seen in its absence (Fig. 3
). Nevertheless, as lactate dehydrogenase can reoxidize NADH, but not NADPH, addition of pyruvic acid to MBBG does not allow complete reoxidation of NADPH generated in the first reactions of the carbohydrate pathway and, therefore, glucose could not have been exhausted. Ito et al. (1983)
reported a beneficial effect of pyruvic acid on Leuconostoc mesenteroides growth when it was used as an external electron acceptor.
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The data presented in Table 5 indicate that the addition of quantities of cysteine, ranging from 030 mM, have a beneficial effect on glucose consumption and bacterial growth. The higher the quantity of supplied cysteine, the higher the degradation of glucose and the greater final biomass reached. When 30 mM cysteine was added, biomass production (Xmax=0·28 g l-1) and the consumption of glucose mirrored that in an O2-free atmosphere. Lactic acid and ethanol production from cultures grown on MBBG plus cysteine were shown to be equimolar, by analysis of the end-products of metabolism. The other end-product found in the culture broth was acetic acid, the yield of which varied from 0·20·6 (where yield is mmol acetic acid produced l-1 versus mmol glucose consumed l-1), depending on the levels of cysteine supplied (Table 5
). Part of this acetate would come from cysteine that was metabolized via pyruvate to yield extra ATP, as has been described in other LAB (Bruinenberg et al., 1997
). Additionally, two extra NADH molecules were reoxidized.
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In conclusion, we have proved that O. oeni M42 does not benefit from the presence of O2 in the atmosphere, as has already been observed for other LAB (Lucey & Condon, 1986 ). The absence of active NAD(P)H oxidases and the inhibition of EFP enzymes by O2 resulted in a low NAD(P)+:NADP(H) ratio and, as a consequence, growth of O. oeni M42 on MBBG was negligible (Fig. 3
). Growth can be improved by adding other substrates that act as electron acceptors, such as fructose (Nuraida et al., 1992
) or cysteine, to glucose cultures. This is of great interest when the objective is to obtain the highest biomass possible, as in the manufacture of commercial starters for malolactic fermentation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 March 2001;
revised 12 September 2001;
accepted 17 September 2001.
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