Anaerobic conversion of lactic acid to acetic acid and 1, 2-propanediol by Lactobacillus buchneri.
ABSTRACT The degradation of lactic acid under anoxic conditions was studied in several strains of Lactobacillus buchneri and in close relatives such as Lactobacillus parabuchneri, Lactobacillus kefir, and Lactobacillus hilgardii. Of these lactobacilli, L. buchneri and L. parabuchneri were able to degrade lactic acid under anoxic conditions, without requiring an external electron acceptor. Each mole of lactic acid was converted into approximately 0.5 mol of acetic acid, 0.5 mol of 1,2-propanediol, and traces of ethanol. Based on stoichiometry studies and the high levels of NAD-linked 1, 2-propanediol-dependent oxidoreductase (530 to 790 nmol min(-1) mg of protein(-1)), a novel pathway for anaerobic lactic acid degradation is proposed. The anaerobic degradation of lactic acid by L. buchneri does not support cell growth and is pH dependent. Acidic conditions are needed to induce the lactic-acid-degrading capacity of the cells and to maintain the lactic-acid-degrading activity. At a pH above 5.8 hardly any lactic acid degradation was observed. The exact function of anaerobic lactic acid degradation by L. buchneri is not certain, but some results indicate that it plays a role in maintaining cell viability.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: The changes in yields and nutritive composition of whole crop wheat (Triticum aestivum L.) during maturation and effects of maturity stage and lactic acid bacteria (LAB) inoculants on the fermentation quality and aerobic stability were investigated under laboratory conditions. Whole crop wheat harvested at three maturation stages: flowering stage, milk stage and dough stage. Two strains of LAB (Lactobacillus plantarum: LAB1, Lactobacillus parafarraqinis: LAB2) were inoculated for wheat ensiling at 1.0×10(5) colony forming units per gram of fresh forage. The results indicated that wheat had higher dry matter yields at the milk and dough stages. The highest water-soluble carbohydrates content, crude protein yields and relative feed value of wheat were obtained at the milk stage, while contents of crude fiber, neutral detergent fiber and acid detergent fiber were the lowest, compared to the flowering and dough stages. Lactic acid contents of wheat silage significantly decreased with maturity. Inoculating homofermentative LAB1 markedly reduced pH values and ammonia-nitrogen (NH3-N) content (p<0.05) of silages at three maturity stages compared with their corresponding controls. Inoculating heterofermentative LAB2 did not significantly influence pH values, whereas it notably lowered lactic acid and NH3-N content (p<0.05) and effectively improved the aerobic stability of silages. In conclusion, considering both yields and nutritive value, whole crop wheat as forage should be harvested at the milk stage. Inoculating LAB1 improved the fermentation quality, while inoculating LAB2 enhanced the aerobic stability of wheat silages at different maturity stages.Asian Australasian Journal of Animal Sciences 10/2012; 25(10):1374-80. · 0.64 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Three microorganisms and one chemical preservative were tested for their effects on the fermentation and aerobic stability of whole-crop wheat, sorghum and maize silages. Wheat at the early dough stage, sorghum at the late milk stage and maize at the one-third milk line stage were harvested and ensiled in 1.5-l anaerobic jars untreated or after the following treatments: control (no additives); Lactobacillus plantarum (LP) at 1.0??06 colony-forming units (CFU)/g of fresh forage; L. buchneri (LB) at 1.0??06 CFU/g; Propionibacterium acidipropionici (PA) at 1.0??06 CFU/g; and a formic acid-based preservative (FAP) at 3 ml/kg of fresh forage weight. Three jars per treatment were sampled on d 90 after ensiling, for chemical and microbiological analysis. At the end of the ensiling period, 90 d, the silages were subjected to an aerobic stability test lasting 5 d. In this test, CO2 produced during aerobic exposure was measured along with chemical and microbiological parameters which serve as spoilage indicators. The silages inoculated with LP had higher concentration of lactic acid compared with the controls and the other treated silages (pAsian Australasian Journal of Animal Sciences 03/2007; 20(3). · 0.64 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: This study examined the effects of bacterial inoculants on chemical composition and fermentation indices of barley silage. Barley forage (Youngyang) was harvested at 24% dry matter (DM) and wilted to 47.9% DM. The wilted barley forage was chopped to 3-5 cm length and applied with no inoculant (CON), L. plantarum (1×10(10) cfu/g, LP) or Effective Microorganisms (0.5×10(9) cfu/g, EM). Then the forages were ensiled in four replications for each treatment in 20 L mini silos and stored for 100 days. The contents of crude protein and ether extract were higher in CON silage ensiled for 100-d, while the contents of DM and crude ash were higher in EM silage (p<0.05). The contents of ADF, NDF and hemicellulose as well as the in vitro DM digestibility were not affected by microbial inoculation (p>0.05). The pH, ammonia-N concentration and lactate to acetate ratio were higher (p<0.05) in CON silage, while lactate concentrations were higher (p<0.05) in CON and LP silage. Acetate concentration and lactic acid bacteria was increased (p<0.05) by both inoculants (LP and EM), but propionate concentration and yeast was increased (p<0.05) by EM and LP, respectively. These results indicated that the fermentation quality of barley silage was improved by the application of bacterial inoculants.Asian Australasian Journal of Animal Sciences 04/2014; 27(4):511-7. · 0.64 Impact Factor
APPLIED AND ENVIRONMENTAL MICROBIOLOGY,
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Jan. 2001, p. 125–132 Vol. 67, No. 1
Anaerobic Conversion of Lactic Acid to Acetic Acid and
1,2-Propanediol by Lactobacillus buchneri
STEFANIE J. W. H. OUDE ELFERINK,1* JANNEKE KROONEMAN,2JAN C. GOTTSCHAL,2
SIERK F. SPOELSTRA,1FOLKERT FABER,2AND FRANK DRIEHUIS1
ID TNO Animal Nutrition, Lelystad,1and Department of Microbiology, University of Groningen, Haren,2
Received 15 May 2000/Accepted 11 October 2000
The degradation of lactic acid under anoxic conditions was studied in several strains of Lactobacillus buchneri
and in close relatives such as Lactobacillus parabuchneri, Lactobacillus kefir, and Lactobacillus hilgardii. Of these
lactobacilli, L. buchneri and L. parabuchneri were able to degrade lactic acid under anoxic conditions, without
requiring an external electron acceptor. Each mole of lactic acid was converted into approximately 0.5 mol of
acetic acid, 0.5 mol of 1,2-propanediol, and traces of ethanol. Based on stoichiometry studies and the high levels
of NAD-linked 1,2-propanediol-dependent oxidoreductase (530 to 790 nmol min?1mg of protein?1), a novel
pathway for anaerobic lactic acid degradation is proposed. The anaerobic degradation of lactic acid by L.
buchneri does not support cell growth and is pH dependent. Acidic conditions are needed to induce the
lactic-acid-degrading capacity of the cells and to maintain the lactic-acid-degrading activity. At a pH above 5.8
hardly any lactic acid degradation was observed. The exact function of anaerobic lactic acid degradation by L.
buchneri is not certain, but some results indicate that it plays a role in maintaining cell viability.
Although lactic acid bacteria are named after their ability to
form lactic acid, many are able to degrade lactic acid as well,
especially if O2is available as electron acceptor (21, 23). Some
lactic acid bacteria are also able to degrade lactic acid under
anoxic conditions in the presence of alternative electron ac-
ceptors. For example, Lactobacillus plantarum and L. pentosus
can use citrate as electron acceptor (11, 19). The products of
this cofermentation of lactic acid and citrate are succinic acid,
acetate, formate, and CO2. Other lactic acid bacteria, such as
L. brevis and L. buchneri, can degrade lactic acid by using
glycerol as an electron acceptor, while producing acetate, 1,3-
propanediol, and CO2(26). L. bifermentans is thus far the only
species known to ferment lactic acid, i.e., without requiring an
external electron acceptor. This bacterium can form acetic
acid, ethanol, CO2and H2from lactic acid at a pH of ?4.0
From silage studies we obtained indications that yet another
pathway for the anaerobic lactic acid degradation by lactic acid
bacteria existed. In studies in which L. buchneri was used as an
inoculant for silage fermentation, we observed an anaerobic
degradation of lactic acid, and no production of succinic acid,
formic acid, or H2was observed (13). The aim of the present
study was to determine whether L. buchneri could indeed de-
grade lactic acid to acetic acid under anoxic conditions and to
obtain more information on the possible degradation pathway.
Furthermore, this study aims at investigating whether besides
L. buchneri other Lactobacillus species are able to carry out
anaerobic lactic acid degradation and how this degradation is
influenced by such environmental conditions as pH and tem-
perature. It was found that L. buchneri and relatives such as L.
parabuchneri are indeed able to degrade lactic acid to acetic
acid with the concomitant production of 1,2-propanediol, as
well as traces of ethanol, under anoxic conditions without re-
quiring an external electron acceptor. Based on stoichiometry
and the high levels of NAD-linked 1,2-propanediol-dependent
oxidoreductase (530 to 790 nmol min?1mg of protein?1), a
novel lactic acid fermentation pathway for L. buchneri is pro-
posed. Furthermore, it is shown that the occurrence of lactic
acid degradation and its rate depend on pH and temperature.
MATERIALS AND METHODS
Organisms and culture conditions. L. buchneri (LMG 6892T), L. bifermentans
(LMG 9845T), L. brevis (LMG 7944T), L. hilgardii (LMG 9895T), L. kefir (LMG
9480T), L. parabuchneri (LMG 11457T), and L. plantarum (LMG 6907T) were
obtained from the culture collection of the Laboratorium voor Microbiologie
(LMG), Gent, Belgium. L. buchneri strain PW01 (NCIMB 40788) and L. buch-
neri strain PW07 were both isolated from separate batches of maize silage and
were from our own laboratory collection. Stock cultures of the bacteria were
maintained in 20-ml culture tubes with loose plastic caps, containing 10 ml of
MRS-Broth (Oxoid). To test anaerobic lactic acid utilization, the bacteria were
cultured in 120-ml serum vials closed with butyl rubber stoppers and aluminum
crimp seals and in 1.2-liter glass bottles closed with butyl rubber stoppers and
aluminum caps. The vials contained 50 ml, and the bottles contained 500 ml of
modified MRS-Broth (MRS-MOD medium) with the following composition (per
liter): peptone (5.0 g), Lab-Lemco powder (Oxoid, 4.0 g), yeast extract (2.0 g),
Tween 80 (0.5 ml), K2HPO4(1.0 g), NaH2PO4? H2O (3.0 g), sodium acetate (0.6
g), MgSO4? 7H2O (0.2 g), and MnSO4? H2O (0.04 g). The vials were made
anoxic by flushing with nitrogen gas and were autoclaved for 15 min at 120°C.
Lactic acid and glucose were added separately to the medium from filter-steril-
ized stock solutions (1 M). Fermentation of lactic acid (45 mM) was tested with
or without the addition of a small amount of glucose (5 mM). If required, the pH
was adjusted by adding HCl from a sterile stock solution (1 M). The inoculum
size was 1%. Unless stated otherwise, all cultures were incubated at pH 3.8, at
30°C in the dark, in a rotary shaker (100 rpm). All batch experiments were done
The basic medium used for continuous cultivation in a chemostat (500 ml,
working volume) was the same as described above. Lactic acid (25 mM) and
glucose (25 mM) were added from filter-sterilized stock solutions (1 M). The
chemostat culture was magnetically stirred and was kept anoxic by passing a
100% N2gas flow (20 ml h?1) over the culture continuously. The dilution rate of
the chemostat was 0.025 h?1. Traces of oxygen had been removed from the N2
gas by passing it over reduced copper curls, which were kept at a temperature of
* Corresponding author. Mailing address: ID TNO Animal Nutri-
tion, P.O. Box 65, 8200 AB Lelystad, The Netherlands. Phone: 31-320-
237-359. Fax: 31-320-237-320. E-mail: firstname.lastname@example.org.
150 to 180°C. The pH was maintained at the required set point by automatic
titration with sterile solutions of 0.5 M HCl or 0.5 M NaOH.
Analytical and microbiological procedures. Organic acids, alcohols, and sugars
were determined by high-performance liquid chromatography (HPLC) (Beck-
man Instruments B.V., Mijdrecht, The Netherlands), with a prepacked cation-
exchange resin column (Polyspher OA HY, 300 by 6.5 mm; Merck, Darmstadt,
Germany) at a temperature of 45°C and a flow rate of 0.5 ml min?1with 2.5 mM
H2SO4as the eluent. The injection volume was 20 ?l. Detection occurred on a
refractometer. Volatile fatty acids and alcohols were additionally determined by
gas chromatography (GC) with a Hewlett-Packard 5890 gas chromatograph
equipped with a Chrompack CP-Sil-5CB column (25 m by 0.32 mm [inner
diameter] by 5-?m film thickness; Chrompack, Middelburg, The Netherlands)
and a flame ionization detector. The injection volume was 1 ?l. The injector was
held at 250°C, the detector was kept at 300°C, and the He inlet pressure was 50
kPa. The temperature program for the column was 35°C for 0.25 min, followed
by a 5°C min?1rise to 150°C, followed by a 10°C min?1rise to 225°C, which was
held for 14.3 min. 1,2-Propanediol eluted from the GC and HPLC column as one
peak and was separate from controls such as 1,3-propanediol, 1-propanol, 2-pro-
panol, and 1,2-butanediol. Hydrogen was determined by GC (13).
Cell densities were measured by detecting the culture turbidity using a spec-
trophotometer at a wavelength of 660 nm. Cell numbers were determined by
plate counts using double-layered MRS-Agar pour plates (Oxoid, Basingstoke,
United Kingdom), i.e., after the inoculated medium had solidified, a second layer
of medium was added. Cell numbers were expressed as CFU per milliliter.
Protein concentrations in cell extracts were determined according to Bradford
Resting-cell experiments. The rates of lactic acid metabolism by resting-cell
suspensions of L. buchneri (LMG 6892T) were determined by taking 100-ml
aliquots of a glucose-limited chemostat culture and incubating them anoxically
under an N2atmosphere in the dark at 30°C. Chloramphenicol (30 mg liter?1)
was added to prevent protein synthesis during the incubation period. Lactic acid
was added to the cell suspensions to a final concentration of approximately 50
mM. From duplicate incubations the disappearance of the substrate was moni-
tored over time by HPLC analyses.
Preparation cell extracts and enzyme measurements. Aliquots (100 ml) taken
from a glucose-limited chemostat culture were centrifuged (10 min at 4°C and
11,000 ? g), washed twice in 25 mM phosphate buffer (pH 5.8 for cells grown at
pH 5.8 and pH 3.8 for cells grown at pH 3.8), and concentrated 25-fold. Crude
cell extracts were obtained by using a French pressure cell (10 times at 6.9 MPa).
Cell debris was removed by centrifugation (30 min, 11,000 ? g at 4°C). The
resulting cell extract was stored on ice. Reverse 1,2-propanediol-dependent
NADH-linked oxidoreductase activities were measured in cell extracts at 30°C
(26). The 1,2-propanediol-dependent formation of NADH was monitored in
anaerobic cuvettes in an assay mixture containing 1 ml of 100 mM Tris-HCl
buffer (pH 9.0) in which 1 mM MnCl2, 10 mM 1,2-propanediol, 5 mM NAD, and
5 to 50 ?l of cell extract were present. The forward reaction was measured by
monitoring the disappearance of NADH in an assay mixture containing 50 mM
Tris-HCl (pH 7.5), 0.2 mM NADH, and 5 to 50 ?l of cell extract. The reaction
was started by adding 2 mM lactaldehyde.
Anaerobic lactic acid degradation by L. buchneri and rela-
tives. To investigate whether the disappearance of lactic acid in
silage inoculated with L. buchneri was due to the metabolic
activity of this Lactobacillus strain, the conversion of lactic acid
was monitored in pure cultures of L. buchneri (strain LMG
6892T, PW01, and PW07), under acidic and anoxic conditions
closely resembling those that prevail in silage. Furthermore, to
investigate whether, besides L. buchneri, other related lacto-
bacilli (15) are also able to degrade lactic acid under “silage
conditions,” lactic acid conversion was monitored in pure cul-
tures of L. parabuchneri, L. brevis, L. hilgardii, L. kefir, and L.
plantarum. L. buchneri and the other lactobacilli were grown in
anoxic batch cultures, at pH 4, with 45 mM lactic acid as a
substrate, with or without 5 mM glucose as a second substrate
to enhance initial growth. The disappearance of lactic acid,
product formation, the pH, and the optical density of each of
the cultures were monitored over time. In the series with lactic
acid with the additional 5 mM glucose, all strains except L. kefir
degraded the glucose (i.e., within 50 to 100 h). However, only
L. buchneri and L. parabuchneri were also able to slowly de-
grade lactic acid. During glucose degradation significant
growth, measured as an increase in cell density, was observed.
During the degradation of lactic acid only a slight increase in
cell density was observed. This suggests that growth mainly
occurred at the expense of glucose and possibly small amounts
of other carbon compounds initially present in the medium and
was not due or was only slightly due to the degradation of lactic
In the series with lactic acid without additional glucose, only
L. buchneri and L. parabuchneri showed a small but detectable
increase in cell density and were able to degrade the lactic acid.
For L. buchneri (LMG 6892T), the dynamics of growth and the
formation of fermentation products in basic MRS-MOD me-
dium with or without added lactic acid are depicted in Fig. 1.
During the degradation of lactic acid (from 43.5 to 27.0 mM),
acetic acid increased from 8.0 to 15.5 mM, 1,2-propanediol
increased from 0 to 7.5 mM, and ethanol increased from 0 to
0.7 mM. The 1:1 molar ratio of acetic acid and 1,2-propanediol
production during lactic acid degradation suggests that their
production is linked together. The stoichiometry of lactic acid
degradation by L. buchneri (Fig. 1A) and L. parabuchneri (data
not shown) was in good agreement with the stoichiometry of
the complete degradation of lactic acid according to the fol-
lowing equation (CO2was calculated based on the C and O
atom balance): 1 lactic acid 3 0.48 acetic acid ? 0.48 1,2-
propanediol ? 0.04 ethanol ? 0.52 CO2.
The L. buchneri culture showed a small increase in cell
density during lactic acid degradation (Fig. 1A). However,
during the first 3 days almost the same increase in cell density
was observed in basic MRS-MOD medium without lactic acid
(Fig. 1B). The protein concentration in the culture with lactic
acid was 4.0 ?g ml?1after 3 days of incubation and increased
to a maximum of 5.5 ?g ml?1after 17 days of incubation. In
the culture without lactic acid the protein concentration was
3.7 ?g ml?1after 3 days of incubation and decreased to 2.1 ?g
ml?1after 17 days of incubation. This suggests that the initial
growth was probably mainly due to the degradation of carbon
compounds initially present in the medium and not to the
degradation of lactic acid. The MRS-MOD medium was buff-
ered at pH 4.0, but the pH did rise to pH 4.4 at 55 days,
probably due to the production of acetate, which has a pKa
value of 4.77, at the expense of lactic acid, which has a pKa
value of 3.86.
A second batch experiment was carried out to further inves-
tigate the role of lactic acid degradation on growth. L. buchneri
LMG 6892Twas cultured in anoxic batch cultures at pH 4, with
or without 45 mM lactic acid as a substrate, and the optical
density, the protein concentration, and cell numbers were
monitored over time. During the first 3 days of incubation the
cell numbers in the cultures with or without lactic acid in-
creased from 3.6 ? 106CFU ml?1to 2.0 ? 108or 1.3 ? 108
CFU ml?1, respectively. From day 3 to day 14, the cell num-
bers in the cultures with lactic acid slightly decreased from
2.0 ? 108to 1.9 ? 108CFU ml?1, while the protein concen-
tration increased almost twofold from 7.1 to 11.9 ?g ml?1. In
the cultures without lactic acid, the cell numbers decreased
from day 3 to day 14, from 1.3 ? 108to 8.1 ? 107CFU ml?1,
126OUDE ELFERINK ET AL.APPL. ENVIRON. MICROBIOL.
while the protein concentration decreased slightly from 5.1 to
4.9 ?g ml?1. These results indicate that cell growth, as repre-
sented by increasing cell numbers, indeed only occurs during
the first few days of incubation. Lactic acid does not seem to
support detectable growth.
Anaerobic lactic acid degradation at different temperatures.
To investigate the effect of temperature on the degradation
rate of lactic acid, L. buchneri (strains LMG 6892T, PW01, and
PW07) and L. parabuchneri were incubated for 50 days in basic
MRS-MOD medium with lactic acid (45 mM), at 15, 20, 25, 30,
and 37°C. The disappearance of lactic acid, product formation,
the pH, and the optical density of the cultures were monitored
over time. The protein concentrations of the cultures were
estimated on the basis of a calibration curve of protein content
versus optical density of a culture grown at 30°C (data not
shown). The dynamics of growth and the stoichiometry of the
lactic acid degradation for the different cultures were similar to
those depicted for L. buchneri (LMG 6892T) in Fig. 1. Between
days 10 and 50 a linear decrease in lactic acid concentration in
the culture medium could be observed, and the optical density
during this period remained almost constant. The rate of lactic
acid degradation was calculated for this period for all cultures.
The rate of lactic acid degradation (in millimoles per day per
gram of protein) varied between the different strains. Further-
more, the rate of lactic acid degradation also varied with tem-
perature (Fig. 2). Lactic acid could be degraded at tempera-
tures between 15 and 37°C by all strains. The optimum
temperature for lactic acid conversion for the different strains
appeared to be between 20 and 30°C (Fig. 2), except for L.
buchneri strain (LMG 6892T), which had the highest degrada-
tion rate at 15°C.
Anaerobic lactic acid conversion at different pH values. To
investigate the influence of the pH on the lactic acid conver-
sion and to obtain information on the moment at which the
FIG. 1. Dynamics of growth and formation of fermentation products by L. buchneri LMG 6892Tcultured in basic MRS-MOD medium with (A)
or without (B) added lactic acid. Lactic acid (}) acetic acid (?), 1,2-propanediol (Œ), ethanol (F), and biomass (—) are shown. At t ? 0 days the
pH was set at 4.0, at t ? 55 days the pH had increased to pH 4.4.
VOL. 67, 2001 DEGRADATION OF LACTIC ACID BY L. BUCHNERI127
process starts within treated silages, the effect of the pH on the
process was studied in resting-cell suspensions of L. buchneri
(LMG 6892T) that were grown under glucose limitation con-
ditions in continuous culture at three different pH values, i.e.,
5.8, 4.3, and 3.8. From each steady state (pH 5.8, 4.3, and 3.8),
triplicate samples were taken, and the lactic acid conversion
rate was subsequently monitored over time under anoxic con-
ditions at a pH equal to those in the continuous culture (Fig.
3). It appeared that cells grown at pH 5.8 hardly utilized any
lactic acid during a time course of approximately 200 h. Cells
grown at pH 4.3 showed an increased conversion rate of lactic
acid, and even higher conversion rates were obtained with cells
that were grown at pH 3.8 (Fig. 3). Both acetic acid and
1,2-propanediol accumulated in equimolar amounts (data not
shown). In conclusion, an increasing lactic acid conversion rate
was observed with decreasing pH.
Induction of the anaerobic lactic acid converting capacity.
The fact that anoxically grown L. buchneri (LMG 6892T) is
able to utilize lactic acid only at pH values below approxi-
mately 5.8 raises the question as to whether induction of this
lactic acid converting ability is also pH dependent. To answer
this question cells were grown glucose limited in continuous
culture at pH 3.8 and, once a steady state was obtained, trip-
licate samples were taken and anoxically incubated with lactic
acid at pH 3.8 or 5.8, in the presence of the protein synthesis
inhibitor chloramphenicol (Fig. 4A). In a second experiment,
cells were taken from a glucose-limited chemostat grown at pH
5.8 and anoxically incubated with lactic acid at pH 5.8 and 3.8,
FIG. 2. Rate of lactic acid degradation by L. buchneri LMG 6892T(F), PW01 (}), and PW07 (?) and L. parabuchneri (Œ) in MRS-MOD
medium with 45 mM lactic acid at different temperatures.
FIG. 3. Lactate utilization in time by anoxic resting-cell suspensions of L. buchneri pregrown in glucose-limited conditions at pH 5.8 (E), pH
4.3 (?), and pH 3.8 (‚). The data shown data are mean values of triplicate incubations, and the standard errors of the means are shown as error
128OUDE ELFERINK ET AL.APPL. ENVIRON. MICROBIOL.
both in the absence and in the presence of chloramphenicol
(Fig. 4B). L. buchneri cells pregrown at pH 3.8 (Fig. 4A)
showed a rapid lactic acid utilization at pH 3.8 and a much
lower rate of conversion at pH 5.8. In accordance with the
experiments described above, the conversion of lactic acid
yielded equimolar amounts of acetic acid and 1,2-propanediol
(data not shown). In the presence of the protein synthesis
inhibitor chloramphenicol, L. buchneri cells pregrown at pH
5.8 (Fig. 4B) showed no lactic acid utilization in cell suspen-
sions maintained at pH 5.8 or in cell suspensions incubated at
pH 3.8. However, in the absence of chloramphenicol lactic acid
utilization commenced after approximately 50 h in those sus-
pensions which were kept at pH 3.8 (Fig. 4B). These results
show that the ability to convert lactic acid anoxically into acetic
acid and 1,2-propanediol is only expressed at pH values well
Propanediol-dependent NADH-linked oxidoreductase activ-
ities in cells of L. buchneri. If the anaerobic degradation of
lactic acid to acetic acid is indeed coupled to the reduction of
lactic acid into 1,2-propanediol as presented above, this reduc-
tion could proceed via lactaldehyde as an intermediate. To
support this hypothesis, the activities of an NADH-linked ox-
idoreductase that catalyzes the conversion of lactaldehyde into
1,2-propanediol were measured. L. buchneri (LMG 6892T)
cells pregrown under glucose-limited conditions in the pres-
ence of lactate at pH 5.8 indeed possessed high activities of this
FIG. 4. (A) Anaerobic lactic acid conversion at pH 3.8 (‚) and pH 5.8 (E), in the presence of the protein synthesis inhibitor chloramphenicol,
by resting cells of L. buchneri that were pregrown in glucose-limited conditions at pH 3.8. (B) Anaerobic lactic acid conversion at pH 5.8 (E) and
pH 3.8 (?) in the presence of chloramphenicol and at pH 3.8 in the absence of chloramphenicol (‚) by resting cells of L. buchneri that were
pregrown in glucose-limited conditions at pH 5.8. The data shown are mean values of triplicate incubations, with the standard errors of the means
shown as error bars.
VOL. 67, 2001DEGRADATION OF LACTIC ACID BY L. BUCHNERI 129
enzyme of 534 nmol min?1mg of protein?1. Cells pregrown at
pH 3.8 possessed even higher activities of 788 nmol min?1mg
Bacterial inoculants are a popular means to increase the
quality of preserved plant materials and to enhance the aerobic
stability of silages. Thus far, most commercial silage inoculants
contain homofermentative or facultatively heterofermentative
lactic acid bacteria (e.g., Enterococcus spp., Pediococcus spp.,
and Lactobacillus plantarum). These lactic acid bacteria have a
positive effect on the extent and rate of lactic acid production
in the silage, thus stimulating a rapid drop in silage pH and
suppressing the growth of clostridia and other undesired an-
aerobic organisms in silage. However, these lactic acid bacteria
sometimes impair the silage aerobic stability (27). This is prob-
ably due to the fact that yeasts, which generally cause the onset
of aerobic silage spoilage, are inhibited more by acetic and
propionic acid than by lactic acid (20, 27).
Recently, silage studies with whole crop maize, using the
obligately heterofermentative lactic acid bacterium L. buchneri
as an inoculant, showed a 20-fold increase in the aerobic sta-
bility of the silage, which increased from approximately 40 h
for nontreated silages to more than 790 h for the inoculated
silages (13). Unexpectedly, it was demonstrated that in these
treated silages the lactic acid concentration was significantly
lower and the acetic acid concentration was significantly higher
than with nontreated silage. It was suggested that this could be
due to the capacity of L. buchneri to degrade lactic acid to
acetic acid under anoxic conditions.
So far the anoxic degradation of lactic acid to acetic acid
without an external electron acceptor has only been described
for L. bifermentans. L. bifermentans produces hydrogen gas to
get rid of its excess of reducing equivalents (16). However, the
present study shows that L. buchneri and L. parabuchneri do
not produce hydrogen gas during lactic acid degradation. In-
stead, they produce large quantities of 1,2-propanediol.
The formation of 1,3-propanediol under anoxic conditions
by lactic acid bacteria has been shown before. Veiga da Cunha
and Foster (26) reported the reduction of glycerol to 1,3-
propanediol, coupled to the oxidation of lactate to acetate via
pyruvate, by L. brevis B22 and L. buchneri B190. Anaerobic
cultures of Lactobacillus reuteri grown on a mixture of maltose
and glycerol were also shown to produce lactate, acetate, eth-
anol, and 1,3-propanediol (12).
However, production of 1,2-propanediol from sugars has
only been found for some non-lactic acid bacteria such as
Escherichia coli (6), Clostridium sphenoides DSM 614 (24), and
esterveld and colleagues (2, 3) demonstrated the formation of
traces of 1,2-propanediol by Bacteroides xylanolyticus X5-1 dur-
ing growth on xylose, whereas significant amounts of 1,2-pro-
panediol were only produced in the presence of acetol as an
external electron acceptor during growth on xylose.
The pathway of lactic acid formation from 1,2-propanediol
has been elucidated for E. coli (9). In this is process, 2 mol of
NAD?is converted to NADH ? H?per mol of lactic acid
formed (9, 28). From the detailed pathways for mixed acid
fermentation of sugars by lactic acid bacteria, it is expected
that for each mole of lactic acid that is degraded to acetic acid,
2 mol of NAD?is converted to NADH ? H?(8). Based on
this knowledge and on the results we obtained in the present
study, we propose a novel pathway of anoxic lactic acid deg-
radation by L. buchneri and relatives (Fig. 5) in which 2 mol of
lactic acid is degraded to 1 mol of acetic acid and 1 mol of
The presence of NAD-linked 1,2-propanediol-dependent
oxidoreductase activity within cells of L. buchneri suggests that
during the anaerobic conversion of two molecules of lactic
acid, one lactic acid molecule is indeed reduced via lactalde-
hyde to 1,2-propanediol. Simultaneous anaerobic oxidation of
another molecule of lactate into acetate could provide the
required amount of hydrogen equivalents in the form of
NADH for the reduction of lactate to one molecule of 1,2-
propanediol. However, to conclude unambiguously that this
pathway is used by lactic acid utilizing cells of L. buchneri,
further evidence is needed based on the detection of interme-
diates and the activities of all enzymes involved in this pathway.
Furthermore, it is not clear yet which enzymes, necessary for
the anaerobic degradation of lactic acid, are lacking at pH 5.8
and are only induced at low pH. It appears not to be NADH-
linked 1,2-propanediol-dependent oxidoreductase, because
this enzyme is present in cells grown at pH 5.8 or 3.8, although
cells grown at pH 5.8 possess a 30% lower enzyme activity than
cells grown at pH 3.8.
In the proposed pathway, the degradation of 2 mol of lactic
acid yields 1 mol of ATP. Yet, in batch culture studies the
conversion of lactic acid did not result in significant growth of
L. buchneri. This could point toward an alternative function of
the lactic acid degradation process, especially in view of the
facts that the ability of L. buchneri to degrade lactic acid is
strongly influenced by the pH and that acidic conditions are
needed to induce the lactic acid degradation. This response
may in fact represent a protective mechanism against sur-
rounding low pH. In acidic environments, many organic acids
occur in undissociated form. It is known that undissociated
organic acids readily penetrate cell membranes and then ac-
cumulate within the cytoplasm of the cells, thereby causing the
loss of viability and cell destruction (4, 10, 17, 18). Therefore,
degradation of lactic acid into an alcohol and a fatty acid with
a higher pKamay have survival value by decreasing the con-
centration of undissociated acids. Our observation in previous
silage studies that the number of lactobacilli in silages inocu-
lated with L. buchneri only decreased very slowly in contrast to
uninoculated silages also indicates a positive effect of lactic
acid degradation on cell viability (13). Even in maize silages
that were stored for 2 years, relatively high numbers (6 log
CFU g?1) of lactobacilli were still present, while in the unin-
oculated silages no lactobacilli could be detected anymore
(S. J. W. H. Oude Elferink, unpublished data).
Protective mechanisms against acidic environments with a
fermentation product shift to less-acidic compounds have been
implicated previously. Tsau et al. (25) reported that L. planta-
rum accumulated pyruvic acid at neutral pH values and that
this strain shifted its fermentation at lower environmental pH
values to the production of acetoin, a pH-neutral compound. A
similar response was described for a Clostridium acetobutyli-
cum (14). It was demonstrated that if this Clostridium sp. was
grown in glucose-limited conditions at a neutral pH it pro-
130OUDE ELFERINK ET AL.APPL. ENVIRON. MICROBIOL.
duced acetate, butyrate, carbon dioxide, and hydrogen (1, 14).
However, at pH values of ?5.0, the organism produced the
pH-neutral components acetone and butanol. Additions of
butyric acid to the growth medium at pH 4.3 resulted in a shift
from acid to solvent production, probably since butyric acid
entered the cells by diffusion at the low pH values. In the case
of L. buchneri, lactic acid may also freely enter the cells at low
pH values. This results in a decrease of the internal pH. As a
consequence, the bacteria probably need to expel protons by
ATP hydrolysis in order to maintain a proton motive force as
stated by Ten Brink and Konings (22). Maintaining a proton
motive force at low pH is an energy-requiring process which
may explain the “marginal growth” of L. buchneri with lactate
as the substrate, as shown in our experiments.
In summary, we have shown that L. buchneri and some close
relatives are capable of converting lactic acid into equimolar
amounts of 1,2-propanediol and acetic acid and small amounts
of ethanol under anoxic conditions. In addition, the lactate-
converting ability is strongly influenced by the pH, and acidic
conditions are even needed to induce this lactic-acid-convert-
ing capacity. Thus, the anaerobic conversion of lactic acid
within silages that are inoculated with L. buchneri probably
starts after initial fermentation of the water-soluble carbohy-
drates, which leads to the acidification of the environment.
Once the environmental conditions are acidic, anaerobic lactic
acid conversion by L. buchneri will start. Based on the batch
culture studies presented here, it can be concluded that the
lactic acid conversion rate will be influenced by the tempera-
ture of the silage, the number of L. buchneri or relatives
present, and the strain used.
Surprisingly, 1-propanol and not 1,2-propanediol was mea-
sured as a dominant fermentation product in the L. buchneri-
treated maize silages described by Driehuis et al. (13). This
suggests that 1,2-propanediol is further degraded within si-
lages. The fact that L. buchneri is unable to degrade 1,2-pro-
panediol in pure cultures or in the heterogeneous silage hab-
itat strongly suggests that other bacteria are involved in the
further degradation of 1,2-propanediol. Recently, anaerobic
1,2-propanediol-degrading bacteria have been isolated from
silages inoculated with L. buchneri. There are indications that
1,2-propanediol is indeed anaerobically degraded by anaerobic
bacteria that are distinctly different from L. buchneri. These
new isolates produce 1-propanol as a major fermentation prod-
uct from 1,2-propanediol (F. Faber, unpublished data).
This research was financially supported by The Netherlands Tech-
nology Foundation (STW) and was coordinated by the Earth and Life
Sciences Foundation (ALW).
J. Krooneman and S. J. W. H. Oude Elferink contributed equally to
1. Bahl, H., W. Andersch, K. Braun, and G. Gottschalk. 1982. Effect of pH and
butyrate concentration on the production of acetone and butanol by Clos-
FIG. 5. Proposed pathway for anaerobic degradation of lactic acid by L. buchneri into equimolar amounts of 1,2-propanediol and acetic acid
and trace amounts of ethanol.
VOL. 67, 2001DEGRADATION OF LACTIC ACID BY L. BUCHNERI131
tridium acetobutylicum grown in continuous culture. Eur. J. Appl. Microbiol.
2. Biesterveld, S., M. D. Kok, C. Dijkema, A. J. B. Zehnder, and A. J. M. Stams.
1994. D-Xylose catabolism in Bacteroides xylanolyticus X5–1. Arch. Micro-
3. Biesterveld, S., H. C. M. Scholten, A. J. B. Zehnder, and A. J. M. Stams.
1994. Influence of external electron acceptors and of CO and H2on the
xylose metabolism of Bacteroides xylanolyticus X5–1. Appl. Microbiol. Bio-
4. Booth, I. R. 1985. Regulation of cytoplasmic pH in bacteria. Microbiol. Rev.
5. Bradford, M. M. 1976. A rapid and sensitive method for the quantification of
microgram quantities of protein utilizing the principle of protein-dye bind-
ing. Anal. Biochem. 72:248–254.
6. Cabiscol, E., J. Badia, L. Baldoma, E. Hidalgo, J. Aguilar, and J. Ros. 1992.
Inactivation of propanediol oxidoreductase of Escherichia coli by metal-
catalyzed oxidation. Biochim. Biophys. Acta 1118:155–160.
7. Cameron, D. C., N. E. Altaras, M. L. Hoffman, and A. J. Shaw. 1998.
Metabolic engineering of propanediol pathways. Biotechnol. Prog. 14:116–
8. Cocaign-Bousquet, M., C. Garrigues, P. Loubiere, and N. D. Lindley. 1996.
Physiology of pyruvate metabolism in Lactococcus lactis. Antonie Leeuwen-
9. Cocks, G. T., J. Aguilar, and E. C. C. Lin. 1974. Evolutions of L-1,2-pro-
panediol catabolism in Escherichia coli by recruitment of enzymes for L-
fucose and L-lactate metabolism. J. Bacteriol. 118:83–88.
10. Cramer, J. A., and J. H. Prestegard. 1977. NMR studies of pH-induced
transport of carboxylic acids across phospholipid vesicle membranes. Bio-
chem. Biophys. Res. Commun. 75:295–301.
11. Cselovszky, J., G. Wolf, and W. P. Hammes. 1992. Production of formate,
acetate, and succinate by anaerobic fermentation of Lactobacillus pentosus in
the presence of citrate. Appl. Microbiol. Biotechnol. 37:94–97.
12. De Valdez, G. F., A. Ragout, J. M. Bruno-Ba ´rcena, H. Diekmann, and F.
Sin ˜eriz. 1997. Shifts in pH affect the maltose/glycerol co-fermentation by
Lactobacillus reuteri. Biotechnol. Lett. 19:645–649.
13. Driehuis, F., S. J. W. H. Oude Elferink, and S. F. Spoelstra. 1999. Anaerobic
lactic acid degradation during ensilage of whole crop maize inoculated with
Lactobacillus buchneri inhibits yeast growth and improves aerobic stability.
J. Appl. Microbiol. 87:583–594.
14. Gottschal, J. C., and J. G. Morris. 1981. The induction of acetone and
butanol production in cultures of Clostridium acetobutylicum by elevated
concentrations of acetate and butyrate. FEMS Microbiol. Lett. 12:385–389.
15. Hammes, W. P., N. Weiss, and W. Holzapfel. 1992. The genera Lactobacillus
and Carnobacterium, p. 1535–1594. In A. Balows, H. G. Tru ¨per, M. Dworkin,
W. Harder, and K.-H. Schleifer (ed.), The prokaryotes, 2nd ed. Springer-
Verlag, New York, N.Y.
16. Kandler, O., U. Schillinger, and N. Weiss. 1983. Lactobacillus bifermentans
sp. nov., nom. rev., an organism forming CO2and H2from lactic acid.
System. Appl. Microbiol. 4:408–412.
17. Kashnet, E. R. 1987. The proton motive force in bacteria: a critical assess-
ment of methods. Annu. Rev. Microbiol. 39:219–242.
18. Kell, D. B., M. W. Peck, G. Rodger, and J. G. Morris. 1981. On the perme-
ability to weak acids and bases of the cytoplasmic membrane of Clostridium
pasteurianum. Biochem. Biophys. Res. Commun. 99:81–88.
19. Lindgren, S. E., L. T. Axelsson, and R. F. McFeeters. 1990. Anaerobic
L-lactate degradation by Lactobacillus plantarum. FEMS Microbiol. Lett.
20. Moon, N. J. 1983. Inhibition of the growth of acid-tolerant yeasts by acetate,
lactate and propionate and their synergistic mixtures. J. Appl. Bacteriol.
21. Murphy, M. G., L. O’Connor, D. Walsh, and S. Condon. 1985. Oxygen-
dependent lactate utilization by Lactobacillus plantarum. Arch. Microbiol.
22. Ten Brink, B., and W. N. Konings. 1980. Generation of an electrochemical
proton gradient by lactate efflux in membrane vesicles of Escherichia coli.
Eur. J. Biochem. 111:59–66.
23. Thomas, T. D., L. L. McKay, and H. A. Morris. 1985. Lactate metabolism by
pediococci isolated from cheese. Appl. Environ. Microbiol. 49:908–913.
24. Tran-Din, K., and G. Gottschalk. 1985. Formation of D-1,2-propanediol and
D-lactate from glucose by Clostridium sphenoides under phosphate limitation.
Arch. Microbiol. 142:87–92.
25. Tsau, J. L., A. A. Guffanti, and T. J. Montville. 1992. Conversion of pyruvate
to acetoin helps to maintain pH homeostasis in Lactobacillus plantarum.
Appl. Environ. Microbiol. 58:891–894.
26. Veiga da Cunha, M., and M. A. Foster. 1992. Sugar-glycerol cofermentations
in lactobacilli: the fate of lactate. J. Bacteriol. 174:1013–1019.
27. Weinberg, Z. G., and R. G. Muck. 1996. New trends and opportunities in the
development and use of inoculants for silage. FEMS Microbiol. Rev. 19:53–
28. Zhu, Y., and E. C. C. Lin. 1989. L-1,2-Propanediol exits more rapidly than
L-lactaldehyde from Escherichia coli. J. Bacteriol. 171:862–867.
132OUDE ELFERINK ET AL.APPL. ENVIRON. MICROBIOL.