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Applied and Environmental Microbiology, October 1999, p. 4582-4585, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Effect of Organic Complex Compounds on Bacillus
thermoamylovorans Growth and Glucose Fermentation
Y.
Combet-Blanc,1,2,*
M. C.
Dieng,2 and
P. Y.
Kergoat2
Laboratoire de Microbiologie IRD,
Université de Provence, 13288 Marseille Cedex 9, France,1 and Laboratoire de
Biotechnologie et d'Energétique, ENSUT, Dakar,
Sénégal2
Received 25 January 1999/Accepted 4 August 1999
 |
ABSTRACT |
The effect of the concentration of a mixture (1/1 [wt/wt]) of
yeast extract and bioTrypcase (YE+bT) on the growth and physiology of a
new species, Bacillus thermoamylovorans, a moderately
thermophilic, non-spore-forming, lactic acid-producing bacterium
isolated from palm wine, was studied. At an initial glucose
concentration of 100 mM, B. thermoamylovorans growth was
limited when the concentration of YE+bT was lower than 5.0 g
liter
1; under these conditions, cellular yield reached a
maximum value of 0.4 g of cells per g of YE+bT. Growth limitation
due to deficiency in growth factors led to a significant shift in
glucose metabolism towards lactate production. Lactate constituted 27.5 and 76% of the end products of glucose fermentation in media
containing YE+bT at 20.0 and 1.0 g liter1,
respectively. This result markedly differed from published data for
lactic bacteria, which indicated that fermentative metabolism remained
homolactic regardless of the concentration of YE. Our results showed
that the ratio between cellular synthesis and energy production
increased with the concentration of YE+bT in the culture medium. They
indicate that the industrial production of lactic acid through glucose
fermentation by B. thermoamylovorans can be optimized by
using a medium where glucose is present in excess and the organic
additives are limiting.
| |
INTRODUCTION |
We isolated from palm wine
Bacillus thermoamylovorans sp. nov., a nonsporulating
bacterium which produces lactate, acetate, ethanol, and formate by
glucose fermentation. Its phenotypic traits resemble those of
lactobacilli (6). In lactic bacteria
lactobacilli and
lactococcispecific modifications of culture conditions resulted in a
change in the fermentation balance (17, 18, 23). A switch
from homo- to heterolactic fermentation was observed upon a change from
acidic to alkaline pH (12, 15, 19, 21, 22) or from excessive
glucose to glucose-limiting culture conditions (3, 4, 8-10, 20,
26, 28). In contrast, the effects of nutrients such as peptides,
amino acids, and vitamins on growth and fermentation balance have been
poorly studied. However, several authors have shown that the kinetics
of milk acidification by a number of lactic strains was enhanced by the
addition of various nutrients, e.g., corn steep (16), yeast
extract (YE) (24), cell extracts of lactobacilli
(13), and amino acids (14).
In a previous paper, we reported the effect of pH on end products of
glucose fermentation and the growth kinetics of B. thermoamylovorans. Our results showed that, as with lactobacilli
and lactococci, a pH change from neutral to acidic resulted in a switch
in glucose metabolism towards lactate production (lactate constituted
62.6 and 23.5% of the products of fermentation at pH 5.6 and 7.0, respectively) (7).
Considering the high biotechnological potential of B. thermoamylovorans for lactic acid production (5), we
studied the effects of different concentrations of YE and peptides on
its growth and the end-product spectrum of glucose fermentation.
| |
MATERIALS AND METHODS |
Organism.
B. thermoamylovorans (type strain DKP [CNCM
I-1378]) was revived from cultures stored at 
80°C and grown as
described by Combet-Blanc et al. (6).
Culture methods and medium.
Batch cultures (run in
duplicate) were performed in a 2-liter fermentor (Labo 2000 Interscience, St.-Nom-La-Bretèche, France) at 50°C, with
stirring at 200 rpm. pH was maintained at 7.0 by using an automatic pH
regulator (Interscience) and 3 N sodium hydroxide. Anaerobiotic
conditions were maintained by passing a stream of O2-free
N2 over the headspace of the culture vessel. The fermentor,
containing 1,000 ml of culture medium, was autoclaved for 45 min at
110°C. The basic medium contained the following (per liter):
NH4Cl, 3.06 g; KH2PO4,
3.15 g; MgCl2 · 6H2O, 0.47 g;
NaCl, 0.3 g; FeSO4 · 7H2O, 5 mg;
CaCl2 · 2H2O, 0.4 mg; trace element
solution (1), 1 ml; and Tween 80, 1 g. Glucose, used as
an energy source, was filter sterilized separately and added to a final
concentration of 100 mM. The inoculum was grown overnight at 50°C in
90 ml of basic medium containing (per liter) 2.0 g of yeast
extract (Difco Laboratories, Detroit, Mich.), 2.0 g of bioTrypcase
(bT) (bioMérieux, Craponne, France), and 10.0 g of glucose.
Batch cultures were run in duplicate.
Cellular concentration.
Growth was monitored by turbidity
measurements (660 nm) at 30-min intervals during the fermentation in a
spectrophotometer (Shimadzu UV 160A; Shimadzu Co., Kyoto, Japan)
calibrated in grams of cells (dry weight) per liter. To determine the
cell dry weight, cells were harvested by centrifugation at 10,000 × g for 10 min, washed three times with a solution of NaCl
at 0.9%, and dried to constant weight at 105°C.
Analyses.
Lactic, formic, and acetic acids, ethanol, and
glucose were quantified by high-performance liquid chromatography,
using an Analprep 93 pump (Touzart et Matignon, Vitry sur Seine,
France), an ORH 801 type column (Interaction Chemicals, Inc., Mountain View, Calif.), and a differential refractometer detector (Shimadzu RID
6 A; Shimadzu Co.). Samples (20 µl) were injected into the column,
which was maintained at 35°C. A 25 mM H2SO4
solution was used as the eluant, at a flow rate of 0.7 ml
min1.
Fermentation parameters.
Fermentation parameters were
calculated at the end of the fermentation, when glucose had been fully
consumed. The yields of lactate (Ylac/s),
acetate (Yace/s), ethanol
(Yethan/s), and formate (Yform/s) and the energy yield derived from
glucose (YATP/s) were expressed in moles of
product per mole of glucose catabolized. Cellular yields derived from
glucose (Yx/s) and ATP
(Yx/ATP) were expressed in grams of cells (dry
weight) per mole. Cellular yield derived from YE+bT
(Rx/gf) was expressed in grams of cells (dry weight) per gram of mixture (1/1 [wt/wt]) of YE and bT. The average hourly growth rate (
) was calculated according to the
following equation: = ln (ODfinal × ODinitial
1) × (tferment1), where ODfinal
and ODinitial are the optical densities at 660 nm measured
at the end and the beginning of the fermentation, respectively, and
tferment is the time needed to completely
ferment the glucose (100 mM).
The specific consumption rates of glucose (qS),
glucose fermented into lactate (qS-L), and
glucose fermented into acetate, ethanol, and formate
(qS-AEF) were expressed in millimoles of glucose
per gram of cells (dry weight) per hour and were calculated according
to the following equations: qS = × (Yx/s)1,
qS-L = 0.5 × (Ylac/s) × × (Yx/s)1, and
qS-AEF = [0.333 × (Yacet/s + Yethan/s) + 0.167 × (Yform/s)] × × (Yx/s)1, where 0.5, 0.333, and
0.167 are the quantities (moles) of glucose needed for the production
of one mole of lactate, acetate or ethanol, and formate, respectively.
| |
RESULTS |
Effect of the concentration of YE+bT on B. thermoamylovorans growth.
In batch cultures, conducted with
an initial glucose concentration of 100 mM,
Rx/gf decreased from 0.32 to 0.17 g
g1 when the concentration of YE+bT was increased from 5.0 to 20.0 g liter1 (Table
1). At concentrations lower than 5.0 g liter1, Rx/gf was maximum and
constant, reaching 0.39 to 0.41 g g1. These results
indicated that, under the experimental conditions used, some unknown
growth factor(s) present in YE or bT limited the growth of B. thermoamylovorans at YE+bT concentrations lower than 5.0 g
liter1.
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TABLE 1.
Effect of the concentration of YE+bT on the yields of end
products of glucose fermentation
by B. thermoamylovoransa
|
|
When growth was not limited by growth factor(s), i.e., at YE+bT
concentrations higher than 5.0 g liter
1, a classical
growth response consisting of an exponential phase
followed by a
stationary phase was observed. Under conditions
where growth factors
were limiting, growth rates were drastically
reduced (Table
2). For the culture conducted with YE+bT
at 1
g liter
1, the exponential growth phase rapidly
became linear (Fig.
1).
Similar effects
on
Lactobacillus delbrueckii growth due to limitation
of
some unknown nutrients contained in YE were observed by Tsao
and Hanson
(
27), who suggested that this phenomenon was complex
and
probably implied the existence of several growth factors.
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FIG. 1.
Effect of the concentration of YE+bT on B. thermoamylovorans growth kinetics. Arrows indicate the boundaries
of the linear part of the growth phase. Data reported are the averages
of data obtained from batch cultures run in duplicate.
|
|
Effect of the concentration of YE+bT on end products of glucose
fermentation.
In experiments conducted in the fermentor, almost
all carbon from fermented glucose was recovered as lactate, acetate,
ethanol, and formate. In addition, the ratio between oxidized and
reduced carbon products (O/R balance) was close to 1.0 (Table 1). This suggested that no other metabolite was produced in significant concentration.
With an initial glucose concentration of 100 mM, the decrease of the
YE+bT concentration led to a significant shift in glucose
metabolism
towards lactate production (Table
1). At the end of
glucose
fermentation, lactate constituted 27.5 and 76% of the
yield in media
containing 20.0 and 1.0 g of YE+bT per liter, respectively.
On the
other hand, kinetic parameters of the fermentations showed
that
qS was optimal at YE+bT concentrations higher
than 2.0 g
liter
1 and was drastically reduced at
lower concentrations (Table
2 and Fig.
2). The dynamics of the fluxes of
qS-L and
qS-AEF showed
that
qS-L occurred optimally at YE+bT
concentrations ranging between
2.0 and 5.0 g liter
1
and that
qS-AEF increased with YE+bT
concentration, reaching
an optimum at concentrations higher than
10 g liter
1 (Table
2 and Fig.
2). These results
indicated that the activities
of these metabolic pathways depended
closely on YE+bT concentration.
When growth factor(s) was limiting, the
lactate-producing pathway
was stimulated; when growth factor(s) was not
limiting, the lactate-producing
pathway had reduced activity and the
enzymes involved in other
pathways were stimulated.
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FIG. 2.
Effects of the concentration of YE+bT on
qS, qS-L, and
qS-AEF. Data reported are the averages of data
obtained from batch cultures run in duplicate.
|
|
Effect of the concentration of YE+bT on B. thermoamylovorans energy and cellular yields.
The value of
YATP/s was calculated from end-product
fermentation by considering that one mole of ATP was generated for each mole of lactate or ethanol produced and that two moles of ATP were
generated per mole of acetate produced. When the concentration of YE+bT
was increased from 1.0 to 20.0 g liter1, a switch
from homolactic to heterolactic fermentation was observed. This
metabolic shift to heterolactic fermentation resulted in an increase of
the YATP/s, from 2.24 mol of ATP per mol of
glucose fermented (in a medium limited by growth factor[s]) to 2.70 mol mol1 (in a rich medium) (Table 2).
| |
DISCUSSION |
The effect of the concentration of YE+bT on the growth and the
metabolic profile of a new species, B. thermoamylovorans,
was studied in batch cultures. At an initial glucose concentration of
100 mM, Rx/gf and the growth kinetics showed
that some nutriments present in YE and bT limited the growth of
B. thermoamylovorans at YE+bT concentrations lower than
5 g liter1 (Table 1 and Fig. 1).
When glucose was present in excess, the growth limitation of the
culture by YE+bT led to a marked shift in glucose metabolism from
heterolactic to homolactic fermentation. This metabolic shift resulted
in a decrease of the YATP/s from 2.70 mol of ATP
per mol of glucose fermented in a rich medium to 2.24 mol of ATP per mol of glucose in a medium limited by the growth factor(s) present in
YE+bT. The comparison of B. thermoamylovorans and lactic
bacteria, whose products of fermentations are qualitatively the same,
showed that fermentation patterns in batch culture differed. With
lactic bacteria, grown in a rich medium where growth factors and
glucose were not limiting, lactate was the main end product of glucose fermentation (17). In contrast, under the same culture
conditions, B. thermoamylovorans fermented glucose mainly
into acetate, ethanol, and formate. It is generally acknowledged that
the limitation of lactic bacterial growth by a deficiency in factors
such as vitamins and nitrogenous compounds does not modify their
fermentative metabolism, which remains homolactic. In contrast, with
B. thermoamylovorans, a limitation in growth factor(s)
resulted in a 3.5-fold increase in lactate yield (Table 1). With lactic
bacteria, only the limitation of the growth by glucose or acidic
culture conditions is known to significantly shift the product(s) of
fermentation from lactate to other end products (3, 4, 8-12, 15,
20, 22, 25, 26, 28).
Furthermore, our results indicated that the coupling between cellular
synthesis and energy production (Yx/ATP)
increased with the concentration of growth factors in the medium
relative to that of glucose (Table 2). Such a phenomenon was also
observed with Zymomonas mobilis by Bélaich et al.
(2): increasing pantothenate concentration from 0.05 to
5,000 µg liter1 in a synthetic medium resulted in an
increase of Yx/ATP from 2.5 to 6.5 g of
cells mol of glucose1.
Our results showed that the concentration of some organic compound(s)
present in YE and/or bT relative to that of glucose is a key factor to
optimize lactic acid production. The industrial production of lactic
acid through glucose fermentation by B. thermoamylovorans will probably use organic additives other than yeast or bT, because of
their high cost. Whatever this source (for example, corn steep), the
ratio of glucose to organic compounds will have to be high enough to
provide a medium where glucose is present in excess and the organic
additives are limiting, in order to optimize lactic acid production.
In conclusion, these results supplement our earlier work on the effect
of pH on the growth of B. thermoamylovorans during glucose
fermentation (7). Physiological differences observed between
B. thermoamylovorans and lactic bacteria reported here and
in the earlier report are consistent with the results of the taxonomic
study of B. thermoamylovorans, which was classified as a
member of the genus Bacillus based on the results of 16S rRNA analyses (6). Physiological studies currently being
conducted on B. thermoamylovorans have already shown close
analogies with, but also significant differences from, the fermentative
metabolism of lactic bacteria. Explaining these differences will
require the characterization of the key enzymes thought to be involved in the glucose fermentation pathways of B. thermoamylovorans
and the study of their regulation.
| |
ACKNOWLEDGMENTS |
We thank P. Roger, and we thank J.-L. Garcia (IRD) and B. K. C. Patel (Griffith University) for revising the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Microbiologie IRD, Université de Provence, ESIL Case 925, 163 Ave. de Luminy, 13288 Marseille Cedex 9, France. Phone: (33) 4 91 82 85 76. Fax: (33) 4 91 82 85 70. E-mail:
combet{at}esil.univ-mrs.fr.
| |
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Applied and Environmental Microbiology, October 1999, p. 4582-4585, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
