This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Van der Meulen, R.
Right arrow Articles by De Vuyst, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Van der Meulen, R.
Right arrow Articles by De Vuyst, L.
Agricola
Right arrow Articles by Van der Meulen, R.
Right arrow Articles by De Vuyst, L.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, August 2006, p. 5204-5210, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00146-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Kinetic Analysis of Bifidobacterial Metabolism Reveals a Minor Role for Succinic Acid in the Regeneration of NAD+ through Its Growth-Associated Production

Roel Van der Meulen, Tom Adriany, Kristof Verbrugghe, and Luc De Vuyst*

Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Department of Applied Biological Sciences and Engineering, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium

Received 19 January 2006/ Accepted 21 May 2006


arrow
ABSTRACT
 
Several strains belonging to the genus Bifidobacterium were tested to determine their abilities to produce succinic acid. Bifidobacterium longum strain BB536 and Bifidobacterium animalis subsp. lactis strain Bb 12 were kinetically analyzed in detail using in vitro fermentations to obtain more insight into the metabolism and production of succinic acid by bifidobacteria. Changes in end product formation in strains of Bifidobacterium could be related to the specific rate of sugar consumption. When the specific sugar consumption rate increased, relatively more lactic acid and less acetic acid, formic acid, and ethanol were produced, and vice versa. All Bifidobacterium strains tested produced small amounts of succinic acid; the concentrations were not more than a few millimolar. Succinic acid production was found to be associated with growth and stopped when the energy source was depleted. The production of succinic acid contributed to regeneration of a small part of the NAD+, in addition to the regeneration through the production of lactic acid and ethanol.


arrow
INTRODUCTION
 
Bifidobacteria have been in the spotlight of scientific research since the 1990s. This attention has to do with the health-promoting effects of these organisms in humans; because of these effects bifidobacteria are often used in probiotic preparations or as target organisms for prebiotic substrates (9, 20, 41).

Bifidobacteria are gram-positive, nonsporulating, nonmotile, saccharoclastic, and usually catalase-negative rods with various shapes (35). They can dominate the intestinal microbiota of babies, but the amounts decrease over time and the bifidobacteria usually account for approximately 3% of the human colon microbiota of adults (10, 14, 18, 22). Bifidobacteria are considered key commensals in human-microbe interactions and contribute to the degradation of undigested polysaccharides in the human colon (1, 37). Because of their ability to break down and metabolize a variety of substrates, different polysaccharides can be used as prebiotics to specifically stimulate bifidobacteria in the human colon; this is the so-called bifidogenic effect (12). Of all the candidate prebiotics tested to date, inulin-type fructans are the most promising, and consequently, the consumption of fructans by bifidobacteria has been studied in detail both in vitro and in vivo (13, 15, 24, 31, 33, 39-41).

Bifidobacteria are nutritionally less fastidious than lactobacilli (32, 38). Carbohydrates are degraded exclusively and characteristically by the fructose-6-phosphate shunt, and acetic acid and lactic acid are major metabolites (6, 36, 44). Some researchers have proposed a theoretical molar ratio of acetic acid to lactic acid of 1.5, although other workers have proven that this ratio is not always obtained (1, 5, 19, 26, 29, 31, 34, 39). The variation is explained by the production of other sugar metabolites, such as formic acid and ethanol (5). This limits the production of lactic acid, which results in an increase in the theoretical ratio of acetic acid to lactic acid. Moreover, degradation of inulin-type fructans increases acetic acid production at the cost of lactic acid (39, 40). Detailed knowledge about the metabolism of bifidobacteria is very important because of the widespread use of these microorganisms as probiotics or as target organisms for prebiotic substrates.

Bifidobacteria are also able to produce small amounts of succinic acid, although this has never been studied in detail (35). Lauer and Kandler (19) have shown that small amounts of succinic acid are probably produced through a CO2 fixation reaction. However, Chiappini (4) showed that although external CO2 can be fixed by bifidobacteria and seems to stimulate growth, it is not incorporated during the conversion of phosphoenolpyruvate (PEP) to oxaloacetate. An examination of the genomes of Bifidobacterium longum NCC2705 (16, 37) (GenBank accession no. NC_004307) and B. longum DJO10A (GenBank accession no. NZ_AABM00000000) revealed that several genes that encode enzymes that may be involved in the production of succinic acid from carbohydrates, such as PEP carboxylase and fumarate reductase, are present. Although this metabolic trait has been known for a long time, qualitative or quantitative data are scarce (19, 44).

The aim of this study was to obtain detailed information about the production of succinic acid by bifidobacteria. An effort was made to quantify the succinic acid production by different species of Bifidobacterium. Moreover, the effects of different energy sources on the metabolism and production of succinic acid were examined with two strains in an effort to elucidate the function of succinic acid production in the general sugar metabolism of bifidobacteria.


arrow
MATERIALS AND METHODS
 
Microorganisms and media.
Bifidobacterium adolescentis LMG 10502, Bifidobacterium bifidum LMG 11583, Bifidobacterium breve LMG 11084, Bifidobacterium pseudolongum subsp. globosum LMG 11614, Bifidobacterium infantis LMG 11570, Bifidobacterium angulatum LMG 11568, Bifidobacterium pseudocatenulatum LMG 10505, and Bifidobacterium dentium LMG 10507 were obtained from the BCCM/LMG Bacteria Collection (Ghent, Belgium). B. longum strain BB536 was obtained from Morinaga Industry Co. Ltd. (Tokyo, Japan). Bifidobacterium animalis subsp. lactis strain Bb 12 was obtained from Christian Hansen (Hørsholm, Denmark). All strains were stored at –80°C in de Man-Rogosa-Sharpe medium (Oxoid Ltd., Basingstoke, United Kingdom) supplemented with 25% (vol/vol) glycerol.

Fermentations were performed in MCB (medium for colon bacteria), which supported the growth of different members of the human colon microbiota (40) and contained (per liter) 6.5 g bacteriological peptone (Oxoid), 5.0 g soy peptone (Oxoid), 2.5 g tryptone (Oxoid), 3.0 g yeast extract (VWR International, Darmstadt, Germany), 2.0 g KCl, 0.2 g NaHCO3, 4.5 g NaCl, 0.5 g MgSO4 · 7H2O, 0.45 g CaCl2 · 2H2O, 0.2 g MnSO4 · H2O, 0.005 g FeSO4 · 7H2O, 0.005 g ZnSO4 · 7H2O, 0.4 g cysteine-HCl, 0.005 g hemin, and 0.005 g menadione. The medium also contained 0.5 ml liter–1 H3PO4 and 2 ml liter–1 Tween 80. For small-scale fermentations (100 ml), glucose was used as the sole added energy source at a concentration of 15 g liter–1, and the pH of the medium was adjusted to 6.50 before sterilization (210 kPa, 121°C, 20 min). For large-scale fermentations (1.5 liters), glucose, fructose, lactose, and oligofructose were used as the sole added energy sources at a concentration of 15 g liter–1. Glucose, fructose, and lactose were sterilized (210 kPa, 121°C, 20 min) separately and added aseptically to the growth medium.

Oligofructose (RaftiloseP95) was kindly provided by ORAFTI N.V. (Tienen, Belgium). RaftiloseP95 is a commercial powder produced by enzymatic hydrolysis of chicory inulin. This powder contains oligofructose (≥93.2% [wt/wt]) and small amounts of glucose, fructose, and sucrose. The degree of polymerization of the oligofructose chains varies from 2 to 8, and the average degree of polymerization is 4. Oligofructose was filter sterilized using Sartolab P-20 filters (Sartorius AG, Goettingen, Germany) and was added aseptically to the growth medium.

Solid media were prepared by adding 1.5% (wt/vol) agar (Oxoid) to MCB with the appropriate energy source (MCB agar).

Fermentation experiments.
The metabolism of all 10 Bifidobacterium strains was first examined using small-scale fermentations (100 ml) in MCB with glucose as the sole added energy source and without pH control. Initially, the strains were transferred from storage at–80°C to de Man-Rogosa-Sharpe medium and incubated anaerobically at 37°C in a modular atmosphere-controlled system (MG anaerobic work station; Don Withley Scientific, West Yorkshire, United Kingdom) that was continuously sparged with a mixture of 80% N2, 10% CO2, and 10% H2 (Air Liquide, Paris, France). After this, the strains were propagated twice in MCB with glucose as the sole added energy source and finally added (5% [vol/vol]) to 100 ml of MCB with glucose as the sole added energy source. After 24 h of anaerobic incubation at 37°C, colony counts were obtained by plating the strains on MCB agar with glucose, and the final pH of the medium was determined. Samples were also removed for further analysis.

B. longum BB536 and B. animalis subsp. lactis Bb 12 were used for a detailed kinetic analysis. This analysis was performed by using 1.5-liter fermentations in a Biostat B-DCU fermentor (Sartorius AG). The inoculum build-up was the same as that described above (transfer volume, 5% [vol/vol]), using the same added energy source that was used later during the fermentation (glucose, fructose, lactose, or oligofructose). All fermentations were carried out anaerobically by sparging the medium with a mixture of 90% N2 and 10% CO2 (Air Liquide) at 37°C for 48 h. During fermentation, a linear pH profile, starting at pH 5.80 and ending at pH 6.80 after 48 h, was used to simulate the pH change during transit through the colon. The pH was controlled by automatic addition of 1.5 M NaOH. Gentle stirring (100 rpm) was used to keep the medium homogeneous. The temperature, pH, and agitation speed were controlled online (MFCS/win 2.1; Sartorius AG). Samples were removed at regular times for analysis.

All fermentations were carried out in duplicate. The results presented below are representative of both fermentations.

Analysis of growth.
Colony counts (CFU per milliliter) were obtained by plating preparations on MCB agar with the appropriate energy source. The plates were incubated anaerobically (modular atmosphere-controlled system) at 37°C for 48 h. The biomass, expressed as cell dry mass (CDM), was determined by membrane filtration of a fixed volume of sample. The filters (0.45-µm cellulose nitrate filters; Sartorius AG) were dried at 105°C for 24 h and weighed.

Analysis of metabolites.
The amounts of glucose, fructose, lactose, lactic acid, acetic acid, ethanol, and formic acid were determined by high-performance liquid chromatography with a Waters chromatograph (Waters Corp., Milford, MA) equipped with a 2414 differential refractometer, a 600S controller, a column oven, and a 717plus autosampler. An ICSep ICE ORH-801 column (Interchim, Montluçon, France) was used with 10 mN H2SO4 as the mobile phase at a flow rate of 0.4 ml min–1. The column temperature was kept at 35°C. Samples were centrifuged (16,060 x g for 15 min), and an equal volume of 20% (vol/vol) trichloroacetic acid was added to remove proteins. After centrifugation (16,060 x g for 15 min) the supernatant was filtered (0.2-µm Minisart RC4 filters; Sartorius AG) before injection.

The amounts of mannitol, glycerol, and erythritol were determined by high-performance anion-exchange chromatography with pulsed amperometric detection (Dionex, Sunnyvale, CA) using a CarboPac PA-10 column. The mobile phase (flow rate, 1.0 ml min–1) consisted of ultrapure water (0.015 µS cm–1) (eluent A) and 250 mM NaOH (eluent B), with the following gradient: zero time, 85% (vol/vol) eluent A and 15% (vol/vol) eluent B; 10.0 min, 85% (vol/vol) eluent A and 15% (vol/vol) eluent B; 20.0 min, 75% (vol/vol) eluent A and 25% (vol/vol) eluent B; 30.0 min, 65% (vol/vol) eluent A and 35% (vol/vol) eluent B; 50.0 min, 65% (vol/vol) eluent A and 35% (vol/vol) eluent B; 51.0 min, 100% (vol/vol) eluent B; 56.0 min, 100% (vol/vol) eluent B; 57.0 min, 85% (vol/vol) eluent A and 15% (vol/vol) eluent B; and 75.0 min, 85% (vol/vol) eluent A and 15% (vol/vol) eluent B. The supernatant was treated with trichloroacetic acid as described above. The samples were filtered (0.2-µm Minisart RC4) prior to injection.

The amount of succinic acid was determined using a Waters 2695 high-performance liquid chromatograph (Waters) coupled to a Quattro Micro mass spectrometer (Waters). The column (Atlantis; Waters) was kept at 35°C. The mobile phase (flow rate, 0.2 ml min–1) was composed of ultrapure water (eluent A), acetonitrile (eluent B), and 10 mM ammonium acetate (pH 6.5) (eluent C). The gradient used was as follows: zero time, 85% (vol/vol) eluent A, 5% (vol/vol) eluent B, and 10% (vol/vol) eluent C; 15.0 min, 40% (vol/vol) eluent A, 50% (vol/vol) eluent B, and 10%(vol/vol) eluent C; 15.1 min, 10%(vol/vol) eluent A, 80% (vol/vol) eluent B, and 10% (vol/vol) eluent C; 23.0 min, 10% (vol/vol) eluent A, 80% (vol/vol) eluent B, and 10% (vol/vol) eluent C; 23.1 min, 85% (vol/vol) eluent A, 5% (vol/vol) eluent B, and 10% (vol/vol) eluent C; and 30.0 min, 85% (vol/vol) eluent A, 5% (vol/vol) eluent B, and 10%(vol/vol) eluent C. Samples were centrifuged (16,060 x g for 15 min), and 100 µl of an internal standard (3,4-dihydroxybenzoic acid) was added to 500 µl of supernatant. After this 600 µl of acetonitrile was added, and the samples were centrifuged again (16,060 x g for 15 min). The supernatant was filtered (0.2-µm Minisart RC4) and injected.

The amounts of short-chain fatty acids (SCFA) (acetic acid, propionic acid, butyric acid, valeric acid, and capronic acid) were determined by gas chromatography with an Agilent 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA) coupled to an Agilent 5973N mass spectrometer (Agilent Technologies). A capillary column (DB-WAXetr; Agilent Technologies) was used together with the following oven temperature program: zero time, 90°C; 5.0 min, 90°C; 7.3 min, 125°C; 12.3 min, 125°C; 16.0 min, 180°C; 31.3 min, 180°C; 34.3 min, 230°C; and 47.3 min, 230°C. Helium (Air Liquide) was used as the carrier gas at a flow rate of 1.1 ml min–1. The samples were centrifuged (16,060 x g for 15 min), and 100 µl of an internal standard (2,6-dimethylphenol) and 50 µl of H2SO4 were added to 500 µl of supernatant. After mixing for 15 s, 750 µl of diethyl ether was added to the sample and mixed thoroughly (30 min). After this, the organic phase was transferred into a vial. The extraction procedure with diethyl ether was performed twice, after which the samples were injected.

The amount of ethanol was also determined using the gas chromatography-mass spectrometry apparatus described above. The same column was used with the following temperature program: zero time, 40°C; 5.0 min, 40°C; 9.29 min, 100°C; 10.37 min, 230°C; and 15 min, 230°C. The procedure used for sample preparation was similar to the procedure used for SCFA, except that no H2SO4 was added, chloroform was used as the organic phase instead of diethyl ether, and methanol (0.5% [wt/vol] in ultrapure water) was used as the internal standard.

All samples were analyzed in triplicate, and the means ± standard deviations are given below. The level of carbon recovery (expressed as a percentage) was calculated by dividing the total amount of carbon recovered in the sugar metabolites by the total amount of carbon present in the added energy source. The amounts of acetic acid produced by conversion of acetyl-coenzyme A (acetyl-CoA) were calculated by subtracting the amount of acetic acid produced in the upper part of the metabolic pathway (3 mol of acetic acid are produced for every 2 mol of hexose consumed) from the total amount of acetic acid produced. NAD+ recovery was calculated by taking into account the fact that 1 mol of NADH + H+ was produced per mol of hexose consumed and the fact that 1 mol of NAD+ was regenerated per mol of lactic acid, while 2 mol of NAD+ was regenerated per mol of ethanol or succinic acid. The amount of acetyl-CoA converted was assumed to be equal to the sum of the amount of ethanol and the amount of acetic acid produced in the lower part of the pathway (1 mol of acetyl-CoA can be converted to 1 mol of ethanol or 1 mol of acetic acid). The specific sugar consumption rate was calculated by dividing the amount of hexose equivalents consumed by the time needed to consume this amount of sugar and by the biomass.


arrow
RESULTS
 
Production of succinic acid during pH free fermentation.
The metabolism of 10 Bifidobacterium strains was studied in detail using in vitro fermentations in MCB (Table 1). All strains were able to grow well in the medium used, and the final pH values were 3.63 to 4.35 after 24 h of anaerobic incubation at 37°C. The total cell counts varied from 8.00 x 106 to 1.75 x 109 CFU ml–1. The consumption of glucose resulted in production of mainly acetic acid (22.5 ± 0.2 to 72.7 ± 0.5 mM) and lactic acid (9.5 ± 0.1 to 49.3 ± 0.2 mM). The molar ratio of acetic acid to lactic acid varied from 1.5 to 2.4. B. animalis subsp. lactis Bb 12, B. bifidum LMG 11583, B. breve LMG 11084, B. pseudolongum subsp. globosum LMG 11614, and B. pseudocatenulatum LMG 10505 produced ethanol in addition to acetic acid and lactic acid. The highest ethanol concentration (3.5 ± 0.7 mM) was produced by B. pseudolongum subsp. globosum strain LMG 11614. All ethanol-producing strains produced formic acid as well, but the concentrations were too low (<10 mM) to quantify accurately by our high-performance liquid chromatography method. Other SCFA were not produced. Remarkably, all strains tested also produced succinic acid, although the concentrations were low (0.12 ± 0.03 to 0.76 ± 0.10 mM).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Growth of and metabolite production by 10 Bifidobacterium strains grown in MCB at 37°C for 24 h

Kinetic analysis of the growth of and production of metabolites by B. longum BB536 on different energy sources.
B. longum strain BB536 was able to metabolize glucose, fructose, lactose, and oligofructose (Table 2 and Fig. 1) within 48 h. The cell counts were always more than 1.0 x 109 CFU ml–1. Glucose was consumed fastest, with a specific sugar consumption rate of 3.5 mmol hexose equivalents g CDM–1 h–1. Oligofructose was consumed more slowly than all of the other substrates, with a specific sugar consumption rate of 1.3 mmol hexose equivalents g CDM–1 h–1. Acetic acid was the major metabolite produced, and the concentrations ranged from 130 ± 7 mM for the fermentation with glucose to 161 ± 2 mM for the fermentation with oligofructose. Lactic acid, formic acid, and ethanol were also produced during all fermentations. In the fermentation with glucose, high concentrations of lactic acid were produced (74 ± 4 mM), and only traces of ethanol and formic acid were found. Larger amounts of acetic acid, formic acid, and ethanol and smaller amounts of lactic acid were produced during growth on fructose, lactose, or oligofructose than during the fermentation with glucose. In general, larger amounts of acetic acid corresponded with larger amounts of formic acid and ethanol and smaller amounts of lactic acid and vice versa. The molar ratio of acetic acid to lactic acid varied from 1.75 ± 0.14 (fermentation with glucose) to 7.06 ± 0.10 (fermentation with oligofructose). During all four fermentations, low concentrations of succinic acid were produced, and the concentrations ranged from 0.88 ± 0.01 mM for the fermentation with oligofructose to 2.05 ± 0.03 mM for the fermentation with lactose. Succinic acid production was always growth associated and stopped when the substrate was completely consumed. Although the production of lactic acid and ethanol accounted for most of the NAD+ recuperation, the production of succinic acid still accounted for 2.0% (fermentation with oligofructose) to 4.6% (fermentation with lactose) of the total NAD+ recuperation. It appeared that higher yields of succinic acid corresponded to a higher specific sugar consumption rate for this strain. We calculated that less than 50% of the acetyl-CoA was converted into ethanol and that more than 50% of the acetyl-CoA was converted into acetic acid. Other possible electron sinks, such as mannitol, glycerol, and erythritol, were not produced. Performing the fermentation using glucose as the sole added energy source and only nitrogen instead of the N2/CO2 mixture to sparge the medium had no influence on the production of succinic acid as similar concentrations were obtained, indicating that external CO2 is not necessary for succinic acid production (data not shown).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Specific sugar consumption rates and metabolite production for B. longum BB536 after growth in MCB containing different energy sources


Figure 1
View larger version (22K):
[in this window]
[in a new window]
  		FIG. 1. B. longum BB536 fermentation in MCB with 15 g liter–1 glucose (A) or 15 g liter–1 lactose (B). The left axis indicates the glucose concentration (mM) (A) or the lactose concentration (mM) (B) (•), the acetic acid concentration (mM) ({circ}), the lactic acid concentration (mM) ({diamond}), the ethanol concentration (mM) ({blacklozenge}), and the formic acid concentration (mM) ({triangleup}). The right axis indicates the concentration of the organism (log CFU ml–1) ({blacktriangleup}) and the succinic acid concentration (mM) ({square}). The data for fructose or oligofructose (RaftiloseP95) as the sole added energy source are from reference 40.

Kinetic analysis of the growth of and production of metabolites by B. animalis subsp. lactis Bb 12 on different energy sources.
In contrast to B. longum strain BB536, B. animalis subsp. lactis Bb 12 was not able to grow on fructose. Therefore, only results for growth on glucose, lactose, and oligofructose are shown in Table 3 and Fig. 2. The cell counts were always more than 1.0 x 109 CFU ml–1. Lactose was consumed faster than glucose or oligofructose, and the accompanying specific sugar consumption rates were 3.9, 2.0, and 0.5 mmol hexose equivalents g CDM–1 h–1, respectively. The total amount of acetic acid produced was greatest for the fermentation with glucose (160 ± 4 mM), although the molar yield of acetic acid (i.e., moles of acetic acid per mole of hexose equivalents consumed) was greatest for the fermentation with oligofructose. As not all oligofructose was consumed within 48 h, the total amount of acetic acid produced was less than the amounts observed during the other fermentations. The concentration of lactic acid produced was highest for the fermentation with lactose (75 ± 2 mM) and lowest for the fermentation with oligofructose (10 ± 1 mM). Lower rates of specific sugar consumption corresponded well with larger amounts of acetic acid, formic acid, and ethanol and with smaller amounts of lactic acid (and vice versa), as was the case for B. longum BB536 (Fig. 3A). The molar ratio of acetic acid to lactic acid varied between 1.92 ± 0.06 (fermentation with lactose) and 8.97 ± 1.30 (fermentation with oligofructose). Growth-associated production of succinic acid was observed during all fermentations. Although the production of lactic acid and the production of ethanol were the main metabolic routes used to regenerate NAD+, the production of succinic acid still accounted for 2.1% (fermentation with lactose) to 3.3% (fermentation with oligofructose) of the total NAD+ recuperation. Higher yields of succinic acid seemed to correspond with a lower specific sugar consumption rate for this strain, which was the opposite of what was found for the B. longum BB536 strain (Fig. 3B). However, less than 50% of the acetyl-CoA was metabolized to ethanol, and more than 50% of the acetyl-CoA was metabolized to acetic acid. Mannitol, glycerol, and erythritol were not produced. This strain also produced similar amounts of succinic acid during fermentation with glucose as the sole added energy source and when pure nitrogen instead of the N2/CO2 mixture was used to sparge the medium (data not shown).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Specific sugar consumption rate and metabolite production for B. animalis subsp. lactis Bb 12 after growth in MCB containing different energy sources


Figure 2
View larger version (19K):
[in this window]
[in a new window]
  		FIG. 2. B. animalis subsp. lactis Bb 12 fermentation in MCB with 15 g liter–1 glucose (A), 15 g liter–1 lactose (B), or 15 g liter–1 oligofructose (RaftiloseP95) (C). The left axis indicates the glucose concentration (mM) (A), lactose concentration (mM) (B), or oligofructose concentration (expressed as mM fructose) (C) (•), acetic acid concentration (mM) ({circ}), lactic acid concentration (mM) ({diamond}), ethanol concentration (mM) ({blacklozenge}), and formic acid concentration (mM) ({triangleup}). The right axis indicates the concentration of the organism (log CFU ml–1) ({blacktriangleup}) and the succinic acid concentration (mM) ({square}).


Figure 3
View larger version (21K):
[in this window]
[in a new window]
  		FIG. 3. (A) Relationship between the specific sugar consumption rate and the yields of acetic acid ({circ}), lactic acid ({diamond}), formic acid ({triangleup}), and ethanol ({blacklozenge}) for B. longum BB536 and B. animalis subsp. lactis Bb 12. (B) Relationship between the specific sugar consumption rate and the yield of succinic acid for B. longum BB536 ({diamond}) and B. animalis subsp. lactis Bb 12 ({circ}). The yields are expressed in mol produced per mol of hexose equivalents consumed. The correlation coefficients (R2) are indicated.


arrow
DISCUSSION
 
Although bifidobacterial metabolism was discovered in the 1960s, it is often misinterpreted. For instance, it is often reported that only acetic acid and lactic acid are produced by these bacteria at a molar ratio of 3:2. However, researchers have described other features of bifidobacterial metabolism, such as the variation of the ratio of acetic acid to lactic acid and the production of other metabolites, such as formic acid and ethanol (1, 5, 19, 26, 29, 31, 39). It is clear from our study that the specific rate of sugar consumption plays an important role in the ratio of the final metabolites produced, as has been shown previously for a strain of the lactic acid bacterium Lactococcus lactis (11). If bifidobacteria consume the energy source fast, larger amounts of lactic acid and relatively smaller amounts of acetic acid, formic acid, and ethanol are produced. If the energy source is consumed slowly (e.g., the oligofructose used in this study), less lactic acid is produced and more acetic acid, formic acid, and ethanol are produced. This is clear proof of the production of formic acid, acetic acid, and ethanol by bifidobacteria through dissipation of pyruvate (Fig. 4). Several factors related to the specific sugar consumption rate, such as the sugar uptake systems and the conversions necessary to incorporate a specific sugar into the fructose-6-phosphate pathway, can have great effects on which metabolic route is used (3, 17). It has also been reported that in L. lactis the NADH + H+/NAD+ ratio (11) or the pool of ADP and ATP (30) can have an effect on changes in end product formation. However, the fact that B. longum BB536 had almost the same specific rate of sugar consumption on fructose and on lactose, although the amounts and ratios of the end products differed, proved that the specific sugar consumption rate was not the only determining factor. Other factors can also be important, such as the amount or the regulation of the production of certain enzymes, as has been reported for L. lactis (23). Although it is still not clear how sugar metabolism is controlled in bifidobacteria and lactic acid bacteria, this metabolism is certainly regulated by a complex interaction of different factors (27).


Figure 4
View larger version (23K):
[in this window]
[in a new window]
  		FIG. 4. Schematic diagram of bifidobacterial sugar metabolism. 1, Enzymes of the bifidobacterial fructose-6-phosphate shunt; 2, pyruvate kinase (pyk); 3, lactate dehydrogenase (ldh); 4, pyruvate formate lyase (pfl); 5, phosphotransacetylase (pta) and acetate kinase (ack); 6, acetaldehyde dehydrogenase (adh) and alcohol dehydrogenase (adh); 7, phosphoenolpyruvate carboxylase (ppc); 8, malate dehydrogenase (coding sequence not found in either B. longum genome); 9, fumarase (coding sequence not found in either B. longum genome); 10, succinate dehydrogenase (sdh). The values in boxes are the percentages of conversion of acetyl-CoA.

If less lactic acid is produced, ethanol production is the main pathway for regenerating NAD+. As 2 mol of NAD+ is regenerated per mol of ethanol produced (Fig. 4), only part of the acetyl-CoA (theoretically 50%) has to be metabolized in this pathway. The remaining part can be used for additional ATP production through conversion into acetic acid. This results in a more efficient use of the energy source, as was observed for a bile-adapted strain of B. animalis by Ruas-Madiedo et al. (34). In this study, we found that both B. longum BB536 and B. animalis subsp. lactis Bb 12 converted more than 50% of acetyl-CoA into acetic acid during all fermentations. As this percentage is higher than the percentage that is theoretically possible, there must be other ways to regenerate NAD+; otherwise, the redox balance would remain out of equilibrium. As observed for other lactic acid bacteria, the reduction of fructose to mannitol is a possible pathway (43), but mannitol was not produced (detection limit, 50 µM). The production of erythritol or glycerol can also be an alternative pathway for regenerating NAD+ (42), but neither of these compounds was found (detection limits, 80 µM and 100 µM, respectively). However, production of succinic acid can be an alternative way to equilibrate the redox balance (Fig. 4).

The production of succinic acid by Bifidobacterium strains was observed by Lauer and Kandler (19) and Wolin et al. (44). All 10 Bifidobacterium strains tested in our study, which belonged to different species, produced small amounts of succinic acid (<1 mM during pH free fermentations). The two Bifidobacterium strains that were studied in more detail produced succinic acid during growth on all carbohydrate sources tested, and we observed that the production of succinic acid was growth associated and stopped when the energy source was depleted. The production of succinic acid probably occurred through the conversion of PEP into oxaloacetate with the incorporation of CO2, due to the action of a PEP carboxylase. The gene coding for PEP carboxylase has recently been found in the genomes of two B. longum strains (37) (GenBank accession no. NC_004307 and NZ_AABM00000000). However, external CO2 is not incorporated during the conversion of PEP into oxaloacetate, as has been observed by Chiappini (4). Indeed, we found no differences in succinic acid production between fermentations that were sparged with external CO2 and fermentations that were not sparged with external CO2. When we examined the genome information, only fumarate reductase, which is responsible for the conversion of fumarate into succinate, was found, but both malate dehydrogenase and fumarase, which are responsible for the conversion of oxaloacetate into fumarate, were not found. However, minor activity of the malate dehydrogenase enzyme in bifidobacteria was found (data not shown), as reported previously (4). Furthermore, similarity-based tools may not successfully annotate many genes, thereby creating gaps in certain metabolic pathways (28). Thus, it is possible that these genes are indeed present but are not detected due to low degrees of homology with known genes. Sequencing more Bifidobacterium genomes or a search on the molecular level for these enzymes may provide an answer to this question.

The reason why small amounts of succinic acid are produced is still unclear. As the conversion of PEP into oxaloacetate is mediated through the PEP carboxylase, as it is in Escherichia coli (25), the energy conserved in PEP is dissipated when the molecule is converted into succinic acid. In this way, energy is lost, which may explain why only small amounts of succinic acid are produced. Bacteria that produce large amounts of succinic acid (e.g., the colon bacterium Bacteroides) use a PEP carboxykinase which conserves the energy of PEP through production of ATP during conversion of PEP into oxaloacetate (21). Nevertheless, succinic acid is always produced by bifidobacteria, which was clear from our study. The metabolic route shown in Fig. 4 indicates that 2 mol of NAD+ is regenerated when PEP is converted into succinic acid. When theoretically calculating the NADH + H+/NAD+ balance, we noticed that a small percentage of NAD+ recuperation occurs through this pathway, which may explain why succinic acid is produced. However, the question is why not all NAD+ is regenerated through the production of ethanol, since this pathway involves the conversion of PEP into pyruvate, enabling the production of ATP, which is not the case during the conversion of PEP into oxaloacetate in bifidobacteria. A clear relationship between the yield of succinic acid and the specific sugar consumption rate could not be detected, since it was observed that in B. longum strain BB536 higher yields of succinic acid corresponded with a higher specific sugar consumption rate, whereas the opposite was observed for B. animalis subsp. lactis Bb 12 strain. Detailed kinetic analysis of more Bifidobacterium strains may reveal a possible relationship.

The low specific rate of sugar consumption during growth of the Bifidobacterium strains on the prebiotic oligofructose may indicate that mainly acetic acid and to a lesser extent formic acid, ethanol, and lactic acid are produced upon degradation of this prebiotic in vivo. Moreover, we have previously shown that bifidobacteria preferentially use the short fractions of oligofructose rapidly, resulting in production of acetic acid and lactic acid during growth on these shorter fractions (39, 40). Therefore, acetic acid and lactic acid are the main metabolites produced by bifidobacteria upon prebiotic stimulation with oligofructose, because bifidobacteria probably profit from the degradation of the longer oligofructose fractions by other colon bacteria (40). In this way, ingestion of oligofructose may indirectly stimulate the production of butyric acid in the colon through cross-feeding of other colon bacteria on the bifidobacterial metabolites (2, 7, 8).

In conclusion, we found that the changes in end product formation in strains of Bifidobacterium can be related to the specific rate of sugar consumption. When the latter increased, relatively more lactic acid and less acetic acid, formic acid, and ethanol were produced, and vice versa. Furthermore, we found that succinic acid production occurs in all Bifidobacterium strains tested and that this production is growth associated. In general, the concentrations of succinic acid produced were low, usually 1 mM to a few millimolar. Although only small amounts of succinic acid were produced, succinic acid production contributed to the regeneration of NAD+, which in Bifidobacterium strains occurs mainly through the production of lactic acid or ethanol.


arrow
ACKNOWLEDGMENTS
 
This work was supported by the Research Council of the Vrije Universiteit Brussel, the Fund for Scientific Research-Flanders, the European Community (Quality of Life grant QLK1-CT-2001-01179), and the Flemish Institute for the Encouragement of Scientific and Technological Research in Industry (GBOU project 10054).


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium. Phone: 32 2 6293245. Fax: 32 2 6292720. E-mail: ldvuyst{at}vub.ac.be. Back


arrow
REFERENCES
 
    1
  1. Ballongue, J. 1998. Bifidobacteria and probiotic action, p. 519-587. In S. Salminen and A. von Wright (ed.), Lactic acid bacteria: microbiology and functional aspects. Marcel Dekker Inc., New York, N.Y.
    2. 2
  2. Bourriaud, C., R. J. Robins, L. Martin, F. Kozlowski, E. Tenailleau, C. Cherbut, and C. Michel. 2005. Lactate is mainly fermented to butyrate by human intestinal microfloras but inter-individual variation is evident. J. Appl. Microbiol. 99:201-212.[CrossRef][Medline]
    3. 3
  3. Caescu, C. I., O. Vidal, F. Krzewinski, V. Artenie, and S. Bouquelet. 2004. Bifidobacterium longum requires a fructokinase (Frk; ATP:D-fructose 6-phosphotransferase; EC 2.7.1.4) for fructose catabolism. J. Bacteriol. 186:6515-6525.[Abstract/Free Full Text]
    4. 4
  4. Chiappini, M. G. 1966. Carbon dioxide fixation in some strains of the species Bifidobacterium bifidum, Bifidobacterium constellatum, Actinomyces bovis and Actinomyces israelii. Ann. Microbiol. 16:25-32.
    5. 5
  5. De Vries, W., and H. Stouthamer. 1968. Fermentation of glucose, lactose, galactose, mannitol, and xylose by bifidobacteria. J. Bacteriol. 96:472-478.[Abstract/Free Full Text]
    6. 6
  6. De Vries, W., and H. Stouthamer. 1967. Pathway of glucose fermentation in relation to the taxonomy of bifidobacteria. J. Bacteriol. 93:574-576.[Abstract/Free Full Text]
    7. 7
  7. Duncan, S. H., A. Barcenilla, C. S. Stewart, S. E. Pryde, and H. J. Flint. 2002. Acetate utilization and butyryl coenzyme A (CoA): acetate-CoA transferase in butyrate-producing bacteria from the human large intestine. Appl. Environ. Microbiol. 68:5186-5190.[Abstract/Free Full Text]
    8. 8
  8. Duncan, S. H., P. Louis, and H. J. Flint. 2004. Lactate-utilizing bacteria, isolated from human feces, that produce butyrate as a major fermentation product. Appl. Environ. Microbiol. 70:5810-5817.[Abstract/Free Full Text]
    9. 9
  9. Fooks, L. J., R. Fuller, and G. R. Gibson. 1999. Prebiotics, probiotics and human gut microbiology. Int. Dairy J. 9:53-61.
    10. 10
  10. Franks, A. H., H. J. M. Harmsen, G. C. Raangs, G. J. Jansen, F. Schut, and G. W. Welling. 1998. Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Appl. Environ. Microbiol. 64:3336-3345.[Abstract/Free Full Text]
    11. 11
  11. 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]
    12. 12
  12. Gibson, G. R., H. M. Probert, J. Van Loo, R. A. Rastall, and M. B. Roberfroid. 2004. Dietary modulation of the human colonic microbiota: updating the concept of prebiotics. Nutr. Res. Rev. 17:259-275.[CrossRef]
    13. 13
  13. Hopkins, M. J., J. H. Cummings, and G. T. Macfarlane. 1998. Inter-species differences in maximum specific growth rates and cell yields of bifidobacteria cultured on oligosaccharides and other simple carbohydrate sources. J. Appl. Microbiol. 85:381-386.[CrossRef]
    14. 14
  14. Hopkins, M. J., R. Sharp, and G. T. Macfarlane. 2001. Age and disease related changes in intestinal bacterial populations assessed by cell culture, 16S rRNA abundance, and community cellular fatty acid profiles. Gut 48:198-205.[Abstract/Free Full Text]
    15. 15
  15. Kaplan, H., and R. W. Hutkins. 2000. Fermentation of fructooligosaccharides by lactic acid bacteria and bifidobacteria. Appl. Environ. Microbiol. 66:2682-2684.[Abstract/Free Full Text]
    16. 16
  16. Klijn, A., A. Mercenier, and F. Arigoni. 2005. Lessons from the genomes of bifidobacteria. FEMS Microbiol. Rev. 29:491-509.[CrossRef][Medline]
    17. 17
  17. Krzewinski, F., C. Brassart, F. Gavini, and S. Bouquelet. 1996. Characterization of the lactose transport system in the strain Bifidobacterium bifidum DSM 20082. Curr. Microbiol. 32:301-307.[CrossRef][Medline]
    18. 18
  18. Langendijk, P. S., F. Schut, G. J. Jansen, G. C. Raangs, G. R. Kamphuis, M. H. F. Wilkinson, and G. W. Welling. 1995. Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S ribosomal-RNA-targeted probes and its application in fecal samples. Appl. Environ. Microbiol. 61:3069-3075.[Abstract]
    19. 19
  19. Lauer, E., and O. Kandler. 1976. Mechanism of variation of acetate/lactate ratio during glucose fermentation by bifidobacteria. Arch. Microbiol. 110:271-277.[CrossRef][Medline]
    20. 20
  20. Leahy, S. C., D. G. Higgins, G. F. Fitzgerald, and D. van Sinderen. 2005. Getting better with bifidobacteria. J. Appl. Microbiol. 98:1303-1315.[CrossRef][Medline]
    21. 21
  21. Macy, J. M., L. G. Ljungdahl, and G. Gottschalk. 1978. Pathway of succinate and propionate formation in Bacteroides fragilis. J. Bacteriol. 134:84-91.[Abstract/Free Full Text]
    22. 22
  22. Matsuki, T., K. Watanabe, J. Fujimoto, Y. Miyamoto, T. Takada, K. Matsumoto, H. Oyaizu, and R. Tanaka. 2002. Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces. Appl. Environ. Microbiol. 68:5445-5451.[Abstract/Free Full Text]
    23. 23
  23. Melchiorsen, C. R., K. V. Jokumsen, J. Villadsen, H. Israelsen, and J. Arnau. 2002. The level of pyruvate-formate lyase controls the shift from homolactic to mixed-acid product formation in Lactococcus lactis. Appl. Microbiol. Biotechnol. 58:338-344.[CrossRef][Medline]
    24. 24
  24. Menne, E., N. Guggenbuhl, and M. Roberfroid. 2000. Fn-type chicory inulin hydrolysate has a prebiotic effect in humans. J. Nutr. 130:1197-1199.[Abstract/Free Full Text]
    25. 25
  25. Millard, C. S., Y. P. Chao, J. C. Liao, and M. I. Donnelly. 1996. Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase in Escherichia coli. Appl. Environ. Microbiol. 62:1808-1810.[Abstract]
    26. 26
  26. Mlobeli, N. T., N. A. Gutierrez, and I. S. Maddox. 1998. Physiology and kinetics of Bifidobacterium bifidum during growth on different sugars. Appl. Microbiol. Biotechnol. 50:125-128.[CrossRef]
    27. 27
  27. Neves, A. R., W. A. Pool, J. Kok, O. P. Kuipers, and H. Santos. 2005. Overview on sugar metabolism and its control in Lactococcus lactis—the input from in vivo NMR. FEMS Microbiol. Rev. 29:531-554.[CrossRef][Medline]
    28. 28
  28. Osterman, A., and R. Overbeek. 2003. Missing genes in metabolic pathways: a comparative genomics approach. Curr. Opin. Chem. Biol. 7:238-251.[CrossRef][Medline]
    29. 29
  29. Palframan, R., G. R. Gibson, and R. A. Rastall. 2003. Carbohydrate preferences of Bifidobacterium species isolated from the human gut. Curr. Issues Intest. Microbiol. 4:71-75.[Medline]
    30. 30
  30. Palmfeldt, J., M. Paese, B. Hahn-Hägerdal, and E. W. J. Van Niel. 2004. The pool of ADP and ATP regulates anaerobic product formation in resting cells of Lactococcus lactis. Appl. Environ. Microbiol. 70:5477-5484.[Abstract/Free Full Text]
    31. 31
  31. Perrin, S., M. Warchol, J. P. Grill, and F. Schneider. 2001. Fermentations of fructo-oligosaccharides and their components by Bifidobacterium infantis ATCC 15697 on batch culture in semi-synthetic medium. J. Appl. Microbiol. 90:859-865.[CrossRef][Medline]
    32. 32
  32. Poupard, J. A., I. Husain, and R. F. Norris. 1973. Biology of bifidobacteria. Bacteriol. Rev. 37:136-165.[Free Full Text]
    33. 33
  33. Rao, V. A. 2001. The prebiotic properties of oligofructose at low intake levels. Nutr. Res. 21:843-848.[CrossRef]
    34. 34
  34. Ruas-Madiedo, P., A. Hernandez-Barranco, A. Margolles, and C. G. de los Reyes-Gavilan. 2005. A bile salt-resistant derivative of Bifidobacterium animalis has an altered fermentation pattern when grown on glucose and maltose. Appl. Environ. Microbiol. 71:6564-6570.[Abstract/Free Full Text]
    35. 35
  35. Scardovi, V. 1986. Genus Bifidobacterium Orla-Jensen, p. 1418-1434. In J. G. Holt, P. H. A. Sneath, N. S. Mair, and M. E. Sharpe (ed.), Bergey's manual of systematic bacteriology, vol. 2. Williams & Wilkins, Baltimore, MD.
    36. 36
  36. Scardovi, V., and L. D. Trovatelli. 1965. The fructose-6-phosphate shunt as a peculiar pattern of hexose degradation in the genus Bifidobacterium. Ann. Microbiol. 15:19-29.
    37. 37
  37. Schell, M. A., M. Karmirantzou, B. Snel, D. Vilanova, B. Berger, G. Pessi, M. C. Zwahlen, F. Desiere, P. Bork, M. Delley, R. D. Pridmore, and F. Arigoni. 2002. The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract. Proc. Natl. Acad. Sci. USA 99:14422-14427.[Abstract/Free Full Text]
    38. 38
  38. Tamura, Z. 1983. Nutriology of bifidobacteria. Bifidobacteria Microflora 2:3-16.
    39. 39
  39. Van der Meulen, R., L. Avonts, and L. De Vuyst. 2004. Short fractions of oligofructose are preferentially metabolized by Bifidobacterium animalis DN-173 010. Appl. Environ. Microbiol. 70:1923-1930.[Abstract/Free Full Text]
    40. 40
  40. Van der Meulen, R., L. Makras, K. Verbrugghe, T. Adriany, and L. De Vuyst. 2006. In vitro kinetic analysis of oligofructose consumption by Bacteroides and Bifidobacterium spp. indicates different degradation mechanisms. Appl. Environ. Microbiol. 72:1006-1012.[Abstract/Free Full Text]
    41. 41
  41. Van Loo, J., J. Cummings, N. Delzenne, H. Englyst, A. Franck, M. Hopkins, N. Kok, G. Macfarlane, D. Newton, M. Quigley, M. Roberfroid, T. van Vliet, and E. van den Heuvel. 1999. Functional food properties of non-digestible oligosaccharides: a consensus report from the ENDO project (DGXII AIRII-CT94-1095). Br. J. Nutr. 81:121-132.[Medline]
    42. 42
  42. Veiga-da-Cunha, M., H. Santos, and E. Van Schaftingen. 1993. Pathway and regulation of erythritol formation in Leuconostoc oenos. J. Bacteriol. 175:3941-3948.[Abstract/Free Full Text]
    43. 43
  43. Wisselink, H. W., R. A. Weusthuis, G. Eggink, J. Hugenholtz, and G. J. Grobben. 2002. Mannitol production by lactic acid bacteria: a review. Int. Dairy J. 12:151-161.[CrossRef]
    44. 44
  44. Wolin, M. J., Y. C. Zhang, S. Bank, S. Yerry, and T. L. Miller. 1998. NMR detection of (CH3COOH)-C-13-C-13 from 3-C-13-glucose: a signature for Bifidobacterium fermentation in the intestinal tract. J. Nutr. 128:91-96.[Abstract/Free Full Text]

Applied and Environmental Microbiology, August 2006, p. 5204-5210, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00146-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Cleenwerck, I., De Wachter, M., Gonzalez, A., De Vuyst, L., De Vos, P. (2009). Differentiation of species of the family Acetobacteraceae by AFLP DNA fingerprinting: Gluconacetobacter kombuchae is a later heterotypic synonym of Gluconacetobacter hansenii. Int. J. Syst. Evol. Microbiol. 59: 1771-1786 [Abstract] [Full Text]  
  • Falony, G., Calmeyn, T., Leroy, F., De Vuyst, L. (2009). Coculture Fermentations of Bifidobacterium Species and Bacteroides thetaiotaomicron Reveal a Mechanistic Insight into the Prebiotic Effect of Inulin-Type Fructans. Appl. Environ. Microbiol. 75: 2312-2319 [Abstract] [Full Text]  
  • Falony, G., Lazidou, K., Verschaeren, A., Weckx, S., Maes, D., De Vuyst, L. (2009). In Vitro Kinetic Analysis of Fermentation of Prebiotic Inulin-Type Fructans by Bifidobacterium Species Reveals Four Different Phenotypes. Appl. Environ. Microbiol. 75: 454-461 [Abstract] [Full Text]  
  • Cleenwerck, I., Gonzalez, A., Camu, N., Engelbeen, K., De Vos, P., De Vuyst, L. (2008). Acetobacter fabarum sp. nov., an acetic acid bacterium from a Ghanaian cocoa bean heap fermentation. Int. J. Syst. Evol. Microbiol. 58: 2180-2185 [Abstract] [Full Text]  
  • Van der Meulen, R., Scheirlinck, I., Van Schoor, A., Huys, G., Vancanneyt, M., Vandamme, P., De Vuyst, L. (2007). Population Dynamics and Metabolite Target Analysis of Lactic Acid Bacteria during Laboratory Fermentations of Wheat and Spelt Sourdoughs. Appl. Environ. Microbiol. 73: 4741-4750 [Abstract] [Full Text]  
  • Cleenwerck, I., Camu, N., Engelbeen, K., De Winter, T., Vandemeulebroecke, K., De Vos, P., De Vuyst, L. (2007). Acetobacter ghanensis sp. nov., a novel acetic acid bacterium isolated from traditional heap fermentations of Ghanaian cocoa beans. Int. J. Syst. Evol. Microbiol. 57: 1647-1652 [Abstract] [Full Text]  
  • Amaretti, A., Bernardi, T., Tamburini, E., Zanoni, S., Lomma, M., Matteuzzi, D., Rossi, M. (2007). Kinetics and Metabolism of Bifidobacterium adolescentis MB 239 Growing on Glucose, Galactose, Lactose, and Galactooligosaccharides. Appl. Environ. Microbiol. 73: 3637-3644 [Abstract] [Full Text]  
  • Camu, N., De Winter, T., Verbrugghe, K., Cleenwerck, I., Vandamme, P., Takrama, J. S., Vancanneyt, M., De Vuyst, L. (2007). Dynamics and Biodiversity of Populations of Lactic Acid Bacteria and Acetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans in Ghana. Appl. Environ. Microbiol. 73: 1809-1824 [Abstract] [Full Text]  
  • Falony, G., Vlachou, A., Verbrugghe, K., Vuyst, L. D. (2006). Cross-Feeding between Bifidobacterium longum BB536 and Acetate-Converting, Butyrate-Producing Colon Bacteria during Growth on Oligofructose. Appl. Environ. Microbiol. 72: 7835-7841 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Van der Meulen, R.
Right arrow Articles by De Vuyst, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Van der Meulen, R.
Right arrow Articles by De Vuyst, L.
Agricola
Right arrow Articles by Van der Meulen, R.
Right arrow Articles by De Vuyst, L.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»