Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nordkvist, M. Articles by Villadsen, J. Search for Related Content PubMed PubMed Citation Articles by Nordkvist, M. Articles by Villadsen, J. Agricola Articles by Nordkvist, M. Articles by Villadsen, J.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, June 2003, p. 3462-3468, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3462-3468.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Glucose Metabolism in Lactococcus lactis MG1363 under Different Aeration Conditions: Requirement of Acetate To Sustain Growth under Microaerobic Conditions

Mikkel Nordkvist, Niels Bang Siemsen Jensen, and John Villadsen^*

Center for Process Biotechnology, BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark

Received 10 September 2002/ Accepted 18 March 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with glucose as the energy source under different aeration conditions, namely, anaerobic conditions, aerobic conditions, and microaerobic conditions with a dissolved oxygen tension of 5% (when saturation with air was used as the reference). The maximum specific growth rate was high (0.78 to 0.91 h^-1) under all aeration conditions but decreased with increasing aeration, and more than 90% of the glucose was converted to lactate. However, a shift in by-product formation was observed. Increasing aeration resulted in acetate, CO₂, and acetoin replacing formate and ethanol as end products. Under microaerobic conditions, growth came to a gradual halt, although more than 60% of the glucose was still left. A decline in growth was not observed during microaerobic cultivation when acetate was added to the medium. We hypothesize that the decline in growth was due to a lack of acetyl coenzyme A (acetyl-CoA) needed for fatty acid synthesis since acetyl-CoA can be synthesized from acetate by means of acetate kinase and phosphotransacetylase activities.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The homofermentative lactic acid bacterium Lactococcus lactis is used primarily in the dairy industry to manufacture fermented milk products and cheese. The members of this species are facultative anaerobes and have limited biosynthetic capacity (7, 18, 30). Consequently, the main purpose of sugar metabolism is to produce ATP for biosynthesis, and more than 95% of the sugar metabolized ends up in fermentation products (24). If the small amount of NADH gained in anabolism is neglected, catabolism is constrained by a balance between NADH-producing and -consuming steps. Under anaerobic conditions this constraint results in conversion of glucose into lactate via lactate dehydrogenase (LDH) or into the mixed acid products formate, ethanol, and acetate at a C molar ratio of 1:1:1 via pyruvate formate lyase (PFL) depending on whether the specific sugar uptake rate is high or low (6, 12, 21). When oxygen is present in the medium, the tight coupling of catabolic carbon fluxes that is needed to satisfy the redox balance is alleviated, since NAD⁺ can be regenerated by the activity of NADH oxidases (NOX).

Not only does the presence of oxygen in the medium influence metabolism by altering the NADH/NAD⁺ ratio, which has been proposed to play a key role in regulation of sugar metabolism (12, 13, 16, 17, 23), but the cellular content of key enzymes also changes with aeration. The negative effect of oxygen on expression of the pfl gene is well known (1, 22), and PFL is known to be very sensitive to oxygen (10, 22, 29). Furthermore, expression of the adhE gene, which encodes the alcohol dehydrogenase enzyme, is known to be reduced by aeration (2). In contrast, the in vitro specific activities of -acetolactate synthase (ALS) and the pyruvate dehydrogenase (PDH) complex have been reported to increase with aeration (8, 17).

For the most part, L. lactis has been studied under totally anaerobic conditions or, in some cases, under totally aerobic conditions (8, 10, 19, 26, 31). To the best of our knowledge, a recent study performed by Jensen et al. (17) was the first study to look into the behavior of L. lactis under microaerobic conditions (i.e., with small amounts of oxygen dissolved in the cultures). This study was performed in a chemostat operating at a low dilution rate (0.1 h^-1), which led to a product pattern in which more than 80% of the glucose carbon ended up in products other than lactate.

To extend our knowledge of the metabolic behavior of L. lactis at intermediate oxygen concentrations, we performed batch cultivation experiments with L. lactis growing on glucose under different aeration conditions. Under microaerobic conditions (5% dissolved oxygen tension [DOT] relative to saturation with air) we observed that growth came to a gradual halt, although more than 60% of the glucose was still left. This phenomenon was investigated closely. We found that the decline in growth was not observed when acetate was added to the medium. We obtained some evidence to explain why acetate was needed to sustain growth.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Strain and growth conditions.
The bacterium used throughout this study was the homofermentative laboratory strain L. lactis subsp. lactis MG1363 (14). This microorganism was grown in in-house bioreactors with a working volume of 1.0 liter under anaerobic and aerobic conditions or in an MBR bioreactor (MBR Bio Reactor AG, Wetzikon, Switzerland) with a working volume of 1.5 liters under microaerobic conditions. All cultures were incubated at 30°C, and the baffled bioreactors were fitted with four-blade Rushton turbines rotating at 350 rpm. A constant pH of 6.6 was maintained by automatic addition of 10 M KOH.

Unless stated otherwise, the cells were grown in defined MS10 medium (7) supplemented with the following components to allow growth under aerobic conditions: MnSO₄ (1.25 x 10^-5 g · liter^-1), thiamine (1 mg · liter^-1), and DL-6,8-thioctic acid (2.5 mg · liter^-1). The glucose concentration was 10 g · liter^-1. The bioreactors were inoculated with cells from precultures grown at 30°C in shake flasks on the medium described above buffered with threefold-higher concentrations of K₂HPO₄ and KH₂PO₄.

Anaerobic conditions were ensured by flushing the medium with N₂ (99.998% pure) prior to inoculation and by maintaining a constant flow of 50 ml of N₂ min^-1 through the headspace of the bioreactor during cultivation. The bioreactors used for microaerobic and aerobic cultivation were equipped with polarographic oxygen sensors (Mettler Toledo, Urdorf, Switzerland). The oxygen electrodes were calibrated by sparging the medium with air (DOT, 100%) and N₂ (DOT, 0%); the 100% saturation value was based on air. When defined in this way, a DOT of 100% corresponded to a saturation oxygen concentration of 2.4 x 10^-4 M (in pure water at 30°C). Aerobic conditions were obtained by sparging the bioreactor with air at a rate of 1 liter of gas · liter of reactor volume^-1 · min^-1 to ensure that the DOT was more than 80%. During microaerobic experiments the DOT was kept at 5% by sparging the reactor with 250 ml of gas composed of a mixture of N₂ and atmospheric air per min. The ratio of N₂ to air was adjusted by using two mass flow controllers (Bronkhorst, Ruurlu, Holland), and the DOT was kept constant by feedback regulation of the ratio (17).

Analytical methods. (i) Biomass.
The biomass concentration was monitored spectrophotometrically by measuring the optical density at 580 nm and correlating the optical density with cell dry weight measurements. Cell dry weight measurements were obtained as described previously (17). One unit of optical density at 580 nm was shown to be equivalent to 0.25 g (dry weight) of cells · liter^-1.

(ii) Glucose and end products.
To determine the extracellular metabolite contents, samples were filtered through a 0.22-µm-pore-size filter. Glucose, lactate, formate, acetate, ethanol, pyruvate, and acetoin were separated by high-pressure liquid chromatography (Aminex HPX-87H column [Bio-Rad, Hercules, Calif.]) at 65°C by using 5 mM H₂SO₄ at a flow rate of 0.6 ml · min^-1 as the mobile phase. Glucose, ethanol, and acetoin contents were measured refractometrically with a Waters 410 differential refractometer detector (Millipore, Milford, Mass.). Lactate, formate, acetate, and pyruvate were quantified by using a Waters 486 tunable absorbance detector set at 210 nm.

(iii) Amino acids.
For one of the microaerobic cultures, the concentrations of amino acids in the growth medium at the end of cultivation were measured by the method of Barkholt and Jensen (3) to make sure that none of the amino acids were depleted.

(iv) Hydrogen peroxide.
The hydrogen peroxide concentrations in filtered samples taken from a microaerobic culture were determined spectrophotometrically with 2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) at 433 nm essentially as described previously (31). To 3 ml of sample, 75 µl of an ABTS solution (28 g liter^-1) and 15 µl of horseradish peroxidase (500 U ml^-1) were added, and the absorbance was measured immediately.

In vitro enzyme assays.
Enzyme extracts were prepared essentially as described by Garrigues et al. (12). Approximately 200 ml of a cell culture was harvested from a bioreactor during the exponential phase and centrifuged (6,000 x g, 4°C, 10 min) to obtain a biomass pellet, which was washed twice with 200 ml of 0.2% (wt/vol) KCl and resuspended in 5 ml of protein extraction buffer (pH 7.2) containing 45 mM Tris, 15 mM tricarballylic acid, 20% (vol/vol) glycerol, 1.0 mM dithiothreitol, and 4.5 mM MgCl₂. The samples were stored at -20°C until analysis. Enzyme extraction was accomplished by sonication (five 30-s cycles separated by 1-min cooling periods on ice), and cell debris was removed by centrifugation (10,000 x g, 4°C, 10 min) to obtain a protein extract, which was used immediately in all enzyme assays. The protein concentrations of the extracts were quantified by the method of Lowry et al. (20) by using bovine serum albumin as the standard.

In vitro LDH, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), PDH, and NOX enzyme activities were determined at 30°C and pH 7.2 by monitoring the concentration of NADH spectrophotometrically at 340 nm ( = 6.22 x 10³ M^-1 · cm^-1) or, in the case of PDH, by monitoring the synthesis of the electron acceptor 2-(p-idophenyl)-3-p-nitrophenyltetrazolium chloride at 500 nm ( = 12.4 x 10³ M^-1 · cm^-1).

LDH, GAPDH, and PDH activities were assayed in reaction mixtures having the exact compositions described previously (12). The enzyme activity assay for NOX was performed with the reaction mixture described previously by Jensen et al. (17).

ALS enzyme activity was measured essentially as described by Boumerdassi et al. (5) by measuring acetoin production from pyruvate in a 1-ml reaction mixture containing 100 mM phosphate buffer (pH 6.5), 0.21 mM cocarboxylase, and 80 mM sodium pyruvate, which initiated the reaction. After incubation at 30°C for 15 min, 200 µl of 0.5 M HCl was added to stop the reaction and convert -acetolactate into acetoin. After incubation at 45°C for 30 min, the resulting solution was used for quantification of acetoin by the colorimetric method of Westerfeld (32).

All in vitro enzyme activities were determined relative to protein content. However, by assuming that the protein pool constitutes 45% of the total cell dry weight, as reported by Novák and Loubiere (24), the activities were calculated relative to cell dry weight.

Measurement of cellular coenzyme concentrations.
Cellular coenzyme concentrations were measured by using in vitro procedures based on rapid inactivation of the metabolism of exponentially growing cells, followed by metabolite extraction. Cell samples (10 ml) were transferred from a bioreactor to tubes, which were precooled in liquid nitrogen, and the metabolic activity was quenched by immersion of the tubes in liquid nitrogen. The samples were stored at -20°C until further analysis.

Acidic metabolite extraction and analysis of NAD⁺ were performed essentially as described previously (12). HCl (37%) was added during thawing of samples in a water bath at 50°C to give a final pH of 1.2. Extraction was completed by incubation at 50°C for 8 min, followed by neutralization with 10 M KOH to obtain a pH of 7. After centrifugation (6,000 x g, 4°C, 10 min) the supernatant was immediately used for measuring the metabolite concentration by coupling an enzyme assay with fluorimetric determination of the coenzyme NADH. Emission was measured at 460 nm after excitation at 340 nm with a Perkin-Elmer LS 50 B luminescence spectrometer (Perkin-Elmer, Beaconsfield, England). The reaction mixture used in the assay was exactly the reaction mixture described by Garrigues et al. (12).

The cellular NADH concentration was assessed by a method that relied on alkaline extraction of NADH with concomitant destruction of NAD⁺, followed by quantification of the NADH by a cycling assay. This method gives results with a much lower standard deviations than measurement with a fluorimetric assay gives. NADH was extracted by adding 10 M KOH to cell samples during thawing in a water bath at 56°C to obtain a final pH of 12.5. Samples were taken directly from these extraction solutions and incubated at 56°C for 10 min. After centrifugation (10,000 x g, 4°C, 5 min) the samples were neutralized to obtain a final pH of 7.5. The prepared extracts were used immediately in a microcycling assay mixture, which was prepared essentially as described by Bernofsky and Swan (4) by mixing 500 µl of neutralized extract, 400 µl of water, 50 µl of 3-(4,5-dimethyl-thiazoyl-2)-2,5-diphenyltetrazolium bromide (MTT) (8.4 mM), 50 µl of phenazine ethosulfate (33.2 mM), and 10 µl of ethanol (96%). After incubation for 5 min at 35°C, 20 µl of alcohol dehydrogenase (900 U/ml) was added to initiate the cycling reaction, and the rate of MTT reduction was monitored spectrophotometrically at 570 nm. Calibration was performed by adding 0, 0.25, 0.50, 0.75, and 1.00 µM NADH during the extraction procedure.

All cofactor concentrations were determined relative to cell dry weight by the experimental procedures described above. By using the intracellular volume reported by Sjöberg and Hahn-Hägerdal (25) (1.7 ml · g [dry weight] of cells^-1), the aqueous molar volumes were calculated.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Analysis of growth and product formation.
Growth of L. lactis subsp. lactis MG1363 on glucose was investigated in pH-regulated batch cultures under the following aeration conditions: anaerobic, microaerobic (DOT, 5%), and aerobic (DOT, >80%). Under all aeration conditions the maximum specific growth rates were high (range, 0.78 to 0.91 h^-1) (Table 1), and the specific growth rate decreased with increasing aeration. Under anaerobic and aerobic conditions, growth was exponential after a variable lag phase until a very short transient of less than 1 h, during which the specific growth rate gradually declined. The transient was followed by stationary phase. However, under microaerobic conditions, the specific growth rate declined when more than 60% of the glucose in the medium was not converted (Fig. 1A). This was shown to be due to a declining biomass yield on glucose (Fig. 2) and not to a lower specific sugar uptake rate.

View this table: [in this window] [in a new window]

TABLE 1. Maximum specific growth rate and yield coefficients as a function of the aeration conditionsa

View larger version (29K): [in this window] [in a new window]

FIG. 1. Biomass and end product time courses under microaerobic conditions (A), under microaerobic conditions with acetate addition during cultivation (0.3 g of sodium acetate liter-1 was added after 7 h 38 min) (B), and under microaerobic conditions on a modified medium without DL-6,8-thioctic acid but with 0.82 g of sodium acetate liter-1 (C). Symbols: , biomass; , glucose; , lactate; •, acetate; x, ethanol; , pyruvate; , acetoin.

View larger version (17K): [in this window] [in a new window]

FIG. 2. Biomass concentration under microaerobic conditions () and during microaerobic cultivation with acetate addition during cultivation () as a function of the glucose taken up since the start of the batch cultivation experiment. The slope is equal to the yield coefficient on glucose.

The glucose carbon recovery data were similar for all three aeration conditions. The end products analyzed accounted for 98% of the converted glucose carbon, and there was a clear predominance of lactate (>90%) (Table 1). However, a shift in by-product formation from formate, ethanol, and acetate under anaerobic conditions to acetate, acetoin, pyruvate, and CO₂ under aerobic conditions was observed. Under anaerobic conditions the by-products formate, acetate, and ethanol were produced at a C molar ratio of approximately 1:1:1, indicating that the PFL pathway was the only active pathway of pyruvate metabolism besides LDH; i.e., there was no flux through the PDH enzyme complex. Furthermore, the redox constraint under anaerobic conditions was fulfilled since equimolar quantities of acetate and ethanol were produced, resulting in redox neutral conversion of glucose. Under microaerobic and aerobic conditions this tight constraint on the by-product fluxes was alleviated due to NOX activity. Under aerobic conditions, the in vivo fluxes through PFL and alcohol dehydrogenase were zero since neither formate nor ethanol was formed, and thus the PDH enzyme complex was responsible for the first step in the conversion of pyruvate to acetate. Only a small amount of in vivo ALS activity was observed under aerobic conditions, which confirms that the amount of acetoin produced in L. lactis is small, unless the intracellular pyruvate concentration is high (11). Under microaerobic conditions product formation was extremely homolactic, and only 2% of the glucose carbon ended up in products other than lactate. The PFL enzyme was inactive since no formate was formed. Consequently, the very small amounts of ethanol and acetate observed in the medium resulted from activity of the PDH complex.

The biomass yield during the exponential phase correlated with the acetate yield (Table 1). According to Jensen et al. (17) 43% of the biomass carbon comes from glucose, but still the main portion of the glucose metabolized goes into production of ATP and catabolic end products. For each acetate molecule formed from pyruvate an extra ATP is formed (Fig. 3). Thus, there is a correlation between specific acetate production and specific production of ATP in catabolism, and as a consequence there is a correlation between biomass yield and acetate yield. This indicates that biomass synthesis is energy limited except when the decline in growth occurs under microaerobic conditions.

View larger version (19K): [in this window] [in a new window]

FIG. 3. Metabolic network around the pyruvate node. PYK, pyruvate kinase; AK, acetate kinase. For other enzyme abbreviations see the text.

Analysis of the decline in growth under microaerobic conditions.
A number of experiments were carried out to explain why the specific growth rate declined gradually under microaerobic conditions although more than 60% of the glucose was still left in the medium. We investigated whether this was due to depletion of an amino acid in the growth medium, but this was not the case. Grufferty and Condon (15) and van Niel et al. (31) have shown how a decline in growth can be a consequence of accumulation of autoinhibitory levels of hydrogen peroxide. Consequently, we tested whether hydrogen peroxide was present in the growth medium, but this compound could not be detected.

We then hypothesized that the decline in growth could be due to a lack of acetyl coenzyme A (acetyl-CoA), a precursor for fatty acid synthesis, which can be synthesized by PFL or PDH (Fig. 3). Additionally, acetyl-CoA may be synthesized from acetate by the acetate kinase and phosphotransacetylase enzymes (9, 11). PFL was clearly inactive under microaerobic conditions (Table 1), and the low yields of acetate and ethanol indicate that there was low PDH activity. If the decline in growth was due to a lack of acetyl-CoA, the decline might have been reversed by addition of a sufficient amount of acetate to the medium. Actually, acetate is part of some media (18) which do not contain 6,8-thioctic acid, which functions as a cofactor of the PDH complex (9, 27, 30).

Indeed, addition of acetate during microaerobic cultivation prevented a decline in growth (Fig. 1B), and it was found that the biomass yield did not decrease when acetate was added (Fig. 2). The product yields did not change when acetate was added during microaerobic cultivation compared to the product yields during cultivation without acetate addition (Tables 1 and 2). Furthermore, an experiment was conducted in which the decline in growth was allowed to occur before acetate was added. Figure 4 shows that addition of acetate immediately eliminated the decline in growth.

View this table: [in this window] [in a new window]

TABLE 2. Maximum specific growth rate and yield coefficients for microaerobic cultivation with addition of sodium acetate during cultivation (0.3 g of sodium acetate liter-1 added after 7 h 38 min) and for microaerobic cultivation on a modified medium without DL-6,8-thioctic acid but with 0.82 g of sodium acetate liter-1

View larger version (16K): [in this window] [in a new window]

FIG. 4. Time course of biomass concentrations during microaerobic cultivation when acetate was added after a decline in the specific growth rate was observed. The addition eliminated the decline in growth.

To investigate the influence of acetate on the metabolism of L. lactis subsp. lactis MG1363 further, we performed a microaerobic cultivation in which 6,8-thioctic acid, which is essential for a functional PDH complex (28), was replaced by 0.82 g of sodium acetate liter^-1, the amount used in SA medium (18). One C-mole of acetate (i.e., 1 mol of acetate carbon) was taken up per 100 C-mol of glucose during this cultivation (Fig. 1C and Table 2), which corresponded to an acetate yield of 2.27 C-mmol of acetate g (dry weight) of cells^-1. The biomass yield was lower than the yield observed under microaerobic conditions when acetate was added during cultivation on a medium containing 6,8-thioctic acid. The lactate yield with glucose was comparable to the yields in the other microaerobic experiments (Table 2). However, a larger amount of pyruvate was detected and acetoin was produced, while no ethanol was formed.

Intracellular cofactor concentrations.
A high NADH/NAD⁺ redox ratio could have been responsible for the low PDH activity under microaerobic conditions. Consequently, intracellular cofactor concentrations were measured in exponentially growing cells. The NAD⁺ concentration increased twofold from anaerobic to aerobic conditions, while the NADH concentration decreased by 36% (Table 3). Unfortunately, we were unable to determine the NADH concentration under microaerobic conditions. It is difficult to measure low cellular NADH concentrations in L. lactis, and with the method used it was extremely difficult to determine the concentration when the sample contained less than 1 g of biomass liter^-1. Under microaerobic conditions the biomass concentration during the exponential phase (before the decline in growth) was below this limit.

View this table: [in this window] [in a new window]

TABLE 3. Cellular concentrations of NADH and NAD+ and NADH/NAD+ redox ratio as a function of the aeration conditionsa

Enzyme activities and kinetic analysis of PDH.
In vitro specific activities of the key enzymes GAPDH, LDH, PDH, ALS, and NOX in exponentially growing cells (Table 4) were assayed in order to determine the pattern of regulation mediated by the increasing level of oxygen present in the medium. Considering the standard deviations, the in vitro GAPDH and LDH specific activities were not affected by aeration. This finding is in accordance with a recent study of Jensen et al. (17). Surprisingly, the in vitro PDH specific activity did not increase with aeration, and the in vitro activity was not zero even under anaerobic conditions, under which the in vivo PDH activity was zero. Both the ALS and NOX in vitro specific activities increased with aeration.

View this table: [in this window] [in a new window]

TABLE 4. In vitro specific activities of key enzymes as a function of the aeration conditionsa

We speculated that the decline in growth observed under microaerobic conditions resulted from a low availability of acetyl-CoA due to low PDH in vivo activity. Since the in vitro specific activity of the enzyme did not change with aeration, we examined how the PDH enzyme complex responds to biochemical modulation by the redox ratio. At an NADH/NAD⁺ ratio equivalent to 0.019 (the ratio measured under aerobic conditions) the specific activity was found to be less than 5% of the full in vitro activity (Fig. 5), and at the ratio found under anaerobic conditions the specific activity was less than 0.6%.

View larger version (33K): [in this window] [in a new window]

FIG. 5. Effect of the NADH/NAD+ ratio on the in vitro specific activity of PDH. The NAD+ concentration was 5 mM for all measurements. The activity was assessed in an anaerobic glove box to prevent parasitic NOX activity.

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study showed that acetate is needed to sustain growth of strain MG1363 under microaerobic conditions. This was the case even though small amounts of acetate were produced from glucose with a constant yield under microaerobic conditions, both when acetate was supplied during cultivation and when it was not supplied. We hypothesize that the flux through the PDH enzyme was too low to maintain a sufficient acetyl-CoA pool size under microaerobic conditions and that addition of extracellular acetate increased the sizes of the pools of intracellular acetyl-phosphate and acetyl-CoA at which fatty acids could be synthesized. The results of in vitro characterization of PDH and measurement of cofactors, even though NADH could not be measured under microaerobic conditions, suggest that the small flux through PDH could be caused by a high NADH/NAD⁺ ratio.

The effect on glucose metabolism under microaerobic conditions when 6,8-thioctic acid was replaced with acetate is consistent with the results of a study performed by Curic et al. (11), in which L. lactis subsp. lactis biovar diacetylactis DB0410 and the derived -acetolactate decarboxylase-deficient strain MC010 were grown in aerobic batch cultures in media containing either 6,8-thioctic acid or acetate. Replacement of 6,8-thioctic acid with acetate led to redirection of the pyruvate catabolism from acetate towards -acetolactate, diacetyl, and acetoin. The amount of acetate taken up was 1 C-mol per 100 C-mol of glucose consumed, the same as in this study. Collins and Bruhn (9) measured incorporation of [2-¹⁴C]acetate into lipids in L. lactis subsp. lactis biovar diacetylactis grown under aerobic conditions in a complex medium lacking 6,8-thioctic acid. They found that 3.56 C-mmol of acetate was incorporated per g (dry weight) of cells, a value which is 57% higher than the acetate yield for biomass obtained in this study. The resolution of the method used by Collins and Bruhn (9) was greater than the resolution that can be achieved by high-pressure liquid chromatography measurement of extracellular metabolites.

In the recent study performed by Jensen et al. (17), in which L. lactis subsp. lactis MG1363 was cultivated in a chemostat operated at a low dilution rate (0.1 h^-1) and under glucose-limited conditions, a shift in product formation from formate and ethanol to acetate and CO₂ was observed when aeration was increased. The same shift in by-product formation was observed in this study, but whereas the product formation pattern was homolactic in this study, less than 20% of the glucose carbon ended up in lactate in the study of Jensen et al. This shift away from homolactic product formation with a decreasing specific sugar uptake rate is analogous to what is observed under anaerobic conditions (6, 12).

In this study the in vitro specific activity of PDH was low irrespective of the aeration conditions, which contrasts with the results obtained by Jensen et al. (17), who observed that the specific activity of PDH increased with increasing aeration from 1.89 to 21.1 mmol of product · h^-1 · g (dry weight) of cells^-1 under anaerobic and microaerobic (DOT, 5%) conditions, respectively. Even though the presence of oxygen clearly induced expression of the nox genes, the in vitro specific activity of NOX was 11 times higher in the study of Jensen et al. (17) at a DOT of 5% (17.0 mmol of product · h^-1 · g [dry weight] of cells^-1) than under the microaerobic conditions used in this study (1.49 mmol of product · h^-1 · g [dry weight] of cells^-1). Furthermore, the in vitro ALS specific activity was six times higher at a DOT of 5% and under glucose-limited conditions (24.1 mmol of product · h^-1 · g [dry weight] of cells^-1) than under microaerobic batch cultivation conditions (4.17 mmol of product · h^-1 · g [dry weight] of cells^-1). This could suggest that the regulatory systems leading to higher PDH, NOX, and ALS specific activities are subject to glucose repression. Currently, we are investigating this possibility more closely.

One conclusion of both academic and industrial interest from this study is that inclusion of acetate in the medium is recommended. Furthermore, our results show how oxygen can be used to control by-product formation.

 
* Corresponding author. Mailing address: Center for Process Biotechnology, BioCentrum-DTU, Technical University of Denmark, Building 223, DK-2800 Lyngby, Denmark. Phone: (45) 45252668. Fax: (45) 45884148. E-mail: jv{at}biocentrum.dtu.dk.

Present address: Chr. Hansen A/S, DK-2970 Hørsholm, Denmark.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Arnau, J., F. Jørgensen, S. M. Madsen, A. Vrang, and H. Israelsen. 1997. Cloning, expression, and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase. J. Bacteriol. 179:5884-5891.[Abstract/Free Full Text]
2
2. Arnau, J., F. Jørgensen, S. M. Madsen, A. Vrang, and H. Israelsen. 1998. Cloning of the Lactococcus lactis adhE gene, encoding a multifunctional alcohol dehydrogenase, by complementation of a fermentative mutant of Escherichia coli. J. Bacteriol. 180:3049-3055.[Abstract/Free Full Text]
3
3. Barkholt, V., and A. L. Jensen. 1989. Amino acid analysis: determination of cysteine plus half-cystine in proteins after hydrochloric acid hydrolysis with a disulfide compound as additive. Anal. Biochem. 177:318-322.[CrossRef][Medline]
4
4. Bernofsky, C., and M. Swan. 1973. An improved cycling assay for nicotinamide adenine dinucleotide. Anal. Biochem. 53:452-458.[CrossRef][Medline]
5
5. Boumerdassi, H., C. Monnet, M. Desmazeaud, and G. Corrieu. 1997. Isolation and properties of Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 mutants producing diacetyl and acetoin from glucose. Appl. Environ. Microbiol. 63:2293-2299.[Abstract/Free Full Text]
6
6. Cocaign-Bousquet, M., C. Garrigues, P. Loubiere, and N. D. Lindley. 1996. Physiology of pyruvate metabolism in Lactococcus lactis. Antonie Leeuwenhoek 70:253-267.[CrossRef][Medline]
7
7. Cocaign-Bousquet, M., C. Garrigues, L. Novak, N. D. Lindley, and P. Loubiere. 1995. Rational development of a simple synthetic medium for the sustained growth of Lactococcus lactis. J. Appl. Bacteriol. 79:108-116.
8
8. Cogan, J. F., D. Walsh, and S. Condon. 1989. Impact of aeration on the metabolic end products formed from glucose and galactose by Streptococcus lactis. J. Appl. Bacteriol. 66:77-84.
9
9. Collins, E. B., and J. C. Bruhn. 1970. Roles of acetate and pyruvate in the metabolism of Streptococcus diacetilactis. J. Bacteriol. 103:541-546.[Abstract/Free Full Text]
10
10. Condon, S. 1987. Responses of lactic acid bacteria to oxygen. FEMS Microbiol. Rev. 46:269-280.[CrossRef]
11
11. Curic, M., M. de Richelieu, C. M. Henriksen, K. V. Jochumsen, J. Villadsen, and D. Nilsson. 1999. Glucose/citrate cometabolism in Lactococcus lactis subsp. lactis biovar diacetylactis with impaired -acetolactate decarboxylase. Metab. Eng. 1:291-298.[CrossRef][Medline]
12
12. Garrigues, C., P. Loubiere, N. D. Lindley, and M. Cocaign-Bousquet. 1997. Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD⁺ ratio. J. Bacteriol. 179:5282-5287.[Abstract/Free Full Text]
13
13. Garrigues, C., M. Mercade, M. Cocaign-Bousquet, N. D. Lindley, and P. Loubiere. 2001. Regulation of pyruvate metabolism in Lactococcus lactis depends on the imbalance between catabolism and anabolism. Biotechnol. Bioeng. 74:108-115.[CrossRef][Medline]
14
14. Gasson, M. J. 1983. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J. Bacteriol. 154:1-9.[Abstract/Free Full Text]
15
15. Grufferty, R. C., and S. Condon. 1983. Effects of fermentation sugar on hydrogen peroxide accumulation by Streptococcus lactis C10. J. Dairy Res. 50:481-489.
16
16. Hols, P., A. Ramos, J. Hugenholtz, J. Delcour, W. M. de Vos, H. Santos, and M. Kleerebezem. 1999. Acetate utilization in Lactococcus lactis deficient in lactate dehydrogenase: a rescue pathway for maintaining redox balance. J. Bacteriol. 181:5521-5526.[Abstract/Free Full Text]
17
17. Jensen, N. B. S., C. R. Melchiorsen, K. V. Jokumsen, and J. Villadsen. 2001. Metabolic behavior of Lactococcus lactis MG1363 in microaerobic continuous cultivation at a low dilution rate. Appl. Environ. Microbiol. 67:2677-2682.[Abstract/Free Full Text]
18
18. Jensen, P. R., and K. Hammer. 1993. Minimal requirements for exponential growth of Lactococcus lactis. Appl. Environ. Microbiol. 59:4363-4366.[Abstract/Free Full Text]
19
19. Lopez de Felipe, F., M. Kleerebezem, W. M. de Vos, and J. Hugenholtz. 1998. Cofactor engineering: a novel approach to metabolic engineering in Lactococcus lactis by controlled expression of NADH oxidase. J. Bacteriol. 180:3804-3808.[Abstract/Free Full Text]
20
20. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.[Free Full Text]
21
21. Melchiorsen, C. R., N. B. S. Jensen, B. Christensen, K. V. Jokumsen, and J. Villadsen. 2001. Dynamics of pyruvate metabolism in Lactococcus lactis. Biotechnol. Bioeng. 74:271-279.[CrossRef][Medline]
22
22. Melchiorsen, C. R., K. V. Jokumsen, J. Villadsen, M. G. Johnsen, H. Israelsen, and J. Arnau. 2000. Synthesis and posttranslational regulation of pyruvate formate-lyase in Lactococcus lactis. J. Bacteriol. 182:4783-4788.[Abstract/Free Full Text]
23
23. Neves, A. R., R. Ventura, N. Mansour, C. Shearman, M. J. Gasson, C. Maycock, A. Ramos, and H. Santos. 2002. Is the glycolytic flux in Lactococcus lactis primarily controlled by the redox charge? Kinetics of NAD⁺ and NADH pools determined in vivo by ¹³C NMR. J. Biol. Chem. 277:28088-28098.[Abstract/Free Full Text]
24
24. Novák, L., and P. Loubiere. 2000. The metabolic network of Lactococcus lactis: distribution of ¹⁴C-labeled substrates between catabolic and anabolic pathways. J. Bacteriol. 182:1136-1143.[Abstract/Free Full Text]
25
25. Sjöberg, A., and B. Hahn-Hägerdal. 1989. ß-Glucose-1-phosphate, a possible mediator for polysaccharide formation in maltose-assimilating Lactococcus lactis. Appl. Environ. Microbiol. 55:1549-1554.[Abstract/Free Full Text]
26
26. Snoep, J. L., M. R. de Graef, M. J. Teixeira de Mattos, and O. M. Neijssel. 1994. Effect of culture conditions on the NADH/NAD ratio and total amounts of NAD(H) in chemostat cultures of Enterococcus faecalis NCTC 775. FEMS Microbiol. Lett. 116:263-268.[CrossRef][Medline]
27
27. Snoep, J. L., M. J. Teixeira de Mattos, M. J. Starrenburg, and J. Hugenholtz. 1992. Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and -acetolactate synthase of Lactococcus lactis subsp. lactis biovar diacetylactis. J. Bacteriol. 174:4838-4841.[Abstract/Free Full Text]
28
28. Snoep, J. L., M. van Bommel, F. Lubbers, M. J. Teixeira de Mattos, and O. M. Neijssel. 1993. The role of lipoic acid in product formation by Enterococcus faecalis NCTC 775 and reconstitution in vivo and in vitro of the pyruvate dehydrogenase complex. J. Gen. Microbiol. 139:1325-1329.[Abstract/Free Full Text]
29
29. Takahashi, S., K. Abbe, and T. Yamada. 1982. Purification of pyruvate formate-lyase from Streptococcus mutans and its regulatory properties. J. Bacteriol. 149:1034-1040.[Abstract/Free Full Text]
30
30. van Niel, E. W. J., and B. Hahn-Hägerdal. 1999. Nutrient requirements of lactococci in defined growth media. Appl. Microbiol. Biotechnol. 52:617-627.[CrossRef]
31
31. van Niel, E. W. J., K. Hofvendahl, and B. Hahn-Hägerdal. 2002. Formation and conversion of oxygen metabolites by Lactococcus lactis subsp. lactis ATCC 19435 under different growth conditions. Appl. Environ. Microbiol. 68:4350-4356.[Abstract/Free Full Text]
32
32. Westerfeld, W. W. 1945. A colorimetric determination of blood acetoin. J. Biol. Chem. 161:495-502.[Free Full Text]

---

Applied and Environmental Microbiology, June 2003, p. 3462-3468, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3462-3468.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Hernandez, I., Molenaar, D., Beekwilder, J., Bouwmeester, H., van Hylckama Vlieg, J. E. T. (2007). Expression of Plant Flavor Genes in Lactococcus lactis. Appl. Environ. Microbiol. 73: 1544-1552 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nordkvist, M. Articles by Villadsen, J. Search for Related Content PubMed PubMed Citation Articles by Nordkvist, M. Articles by Villadsen, J. Agricola Articles by Nordkvist, M. Articles by Villadsen, J.