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Previous Article | Next Article 
Applied and Environmental Microbiology, August 2001, p. 3650-3654, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3650-3654.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
L-Pyroglutamate Spontaneously Formed
from L-Glutamate Inhibits Growth of the Hyperthermophilic
Archaeon Sulfolobus solfataricus
Chan B.
Park,1,
Sun Bok
Lee,1,* and
Dewey D. Y.
Ryu2
Department of Chemical Engineering, Pohang
University of Science and Technology, San 31, Hyoja-Dong, Pohang
790-784, Korea,1 and Department of
Chemical Engineering and Materials Science, University of
California, Davis, California 956162
Received 7 February 2001/Accepted 25 May 2001
 |
ABSTRACT |
Identification of physiological and environmental factors that
limit efficient growth of hyperthermophiles is important for practical
application of these organisms to the production of useful enzymes or
metabolites. During fed-batch cultivation of Sulfolobus
solfataricus in medium containing L-glutamate, we
observed formation of L-pyroglutamic acid (PGA). PGA formed
spontaneously from L-glutamate under culture conditions
(78°C and pH 3.0), and the PGA formation rate was much higher at an
acidic or alkaline pH than at neutral pH. It was also found that PGA is
a potent inhibitor of S. solfataricus growth. The cell
growth rate was reduced by one-half by the presence of 5.1 mM PGA, and
no growth was observed in the presence of 15.5 mM PGA. On the other
hand, the inhibitory effect of PGA on cell growth was alleviated by addition of L-glutamate or L-aspartate to the
medium. PGA was also produced from the L-glutamate in yeast
extract; the PGA content increased to 8.5% (wt/wt) after 80 h of
incubation of a yeast extract solution at 78°C and pH 3.0. In medium
supplemented with yeast extract, cell growth was optimal in the
presence of 3.0 g of yeast extract per liter, and higher yeast
extract concentrations resulted in reduced cell yields. The extents of
cell growth inhibition at yeast extract concentrations above the
optimal concentration were correlated with the PGA concentration in the
culture broth. Although other structural analogues of
L-glutamate, such as L-methionine sulfoxide,
glutaric acid, succinic acid, and L-glutamic acid
-methyl ester, also inhibited the growth of S. solfataricus, the greatest cell growth inhibition was observed
with PGA. We also observed that unlike other glutamate analogues,
N-acetyl-L-glutamate enhanced the growth
of S. solfataricus. This compound was stable under cell
culture conditions, and replacement of L-glutamate
with N-acetyl-L-glutamate in the medium
resulted in increased cell density.
| |
INTRODUCTION |
Recently, hyperthermophiles have
attracted the attention of many workers in the biotechnology research
community because of the potential industrial applications of these
organisms and because they may provide a better understanding of how
cell components are stabilized when they are heated (1, 13, 18,
28, 29). Despite the biotechnological potential of
hyperthermophiles, thus far the uses of these organisms have been
limited because of the low cell yields when they are cultivated, which
are attributed mainly to a lack of knowledge concerning the
physiological characteristics and high-temperature cultivation
techniques (5, 17). Therefore, identification of
physiological and environmental factors that limit efficient growth of
hyperthermophiles and development of strategies to obtain high cell
densities under high-temperature conditions are particularly important
for increasing the biomass yields of these microorganisms in
environments different from their natural habitats.
Sulfolobus solfataricus P2 is a hyperthermophilic archaeon
which normally grows at 75 to 85°C and pH 2.0 to 4.0 (4, 7, 32). Since physiological and genomic studies of this species have been carried out most intensively among various members of the
order Sulfolobales (11, 15, 20, 21, 27), we
selected S. solfataricus as a model archaeon and have been
investigating the factors that affect the growth of this
microorganism in a laboratory-scale fermentor (22-25).
Unlike most other hyperthermophiles, S. solfataricus grows
under aerobic conditions, and thus it is anticipated that this archeon
can be cultivated as efficiently as other aerobes, such as
Escherichia coli and yeast cells, if physiological and
environmental factors unfavorable for cell growth are identified and eliminated.
Recently, we studied the effects of low-molecular-weight solutes, such
as compatible solutes and L amino acids, on the growth of
S. solfataricus (25). Of the
low-molecular-weight solutes tested, L-glutamate was found
to be the best growth enhancer for S. solfataricus. When cells were grown in medium containing
both glucose and L-glutamate, S. solfataricus
preferentially utilized L-glutamate instead of glucose. In
this regard, it is noteworthy that complex nutrients, such as
hydrolyzed peptone and yeast extract, which are essential for efficient
growth of most heterotrophic hyperthermophiles (14),
contain L-glutamate as a major component. Growth
stimulation and reduction of the lag time by yeast extract in the
medium are attributable to the role of L-glutamate as a compatible solute that facilitates adaptation of cells to stressful environmental factors, such as high temperatures and extremely acidic conditions.
The finding that exogenously supplied L-glutamate can
promote the growth of S. solfataricus in batch cultures
prompted us to investigate whether cell densities can be further
increased by continuous addition of L-glutamate to the
medium in a fed-batch operation. In the present study, we found that
prolonged incubation of L-glutamate under culture
conditions (high temperature and low pH) resulted in conversion of
L-glutamate to L-pyroglutamic acid
(2-pyrrolidone-5-carboxylic acid) (PGA) and that PGA is a potent
inhibitor of growth of S. solfataricus. Although it has been
known for decades that thermal conversion of L-glutamate to
PGA occurs (31) and although hyperthermophiles require
temperatures that produce PGA, the effect of PGA production on the
growth of hyperthermophiles has not been investigated previously. Our
data indicate that thermal decomposition of a medium component at an elevated temperature can be one of the major factors that limit efficient growth of hyperthermophiles.
| |
MATERIALS AND METHODS |
Microorganism.
S. solfataricus P2 (= DSM 1617),
which was isolated from a volcanic hot spring in Italy, was obtained
from the Deutsche Sammlung von Mikroorganismen und Zellkulturen
(Braunschweig, Germany).
Culture methods.
Batch cultivation was carried out in
screw-cap flasks (working volume, 50 ml) at 78°C in a shaking water
bath with agitation at 100 oscillations per min. The glucose minimal
medium (GM medium) used for seed culture and control experiments was
composed of glucose (3.0 g/liter) and modified Allen's basal salt
solution. Modified Allen's basal salt solution contained (per liter of
distilled water) 1.3 g of
(NH4)2SO4, 0.28 g of
KH2PO4, 0.25 g of Mg SO4 · 7H2O, 70 mg of CaCl2 · 2H2O, 20 mg of FeCl3 · 6H2O,
4.5 mg of Na2B4O7 · 10H2O, 1.8 mg of MnCl2 · 4H2O, 0.05 mg of ZnSO4 · 7H2O, 0.05 mg of CuCl2 · 2H2O, 0.04 mg of VOSO45H2O, 0.03 mg
of Na2MoO4 · 5H2O, and 0.01 mg of CoSO4 · 7H2O (15).
GYM medium was prepared by adding 3.0 g of yeast extract per liter to
GM medium. The pH of the culture medium was adjusted to 3.0 with
sulfuric acid. Yeast extract was obtained from Difco Laboratories
(Detroit, Mich.), and all other chemicals used in this work were
obtained from Sigma (St. Louis, Mo.).
Fed-batch cultivation was conducted in a bench top fermentor with a
working volume of 2.3 liters (KLF2000; Bioengineering AG, Wald,
Switzerland) as described previously (22, 23). The pH of
the culture broth was automatically adjusted to 3.0, and the dissolved
oxygen level was maintained at more than 30% of air saturation. GM
medium supplemented with L-glutamate at a specified concentration was used as a base medium. The feed rate was controlled to maintain constant residual concentrations of L-glutamate
and glucose by using the constant-volume fed-batch protocol
(22).
Analytical methods.
The cell densities of S. solfataricus cultures were determined by measuring turbidity at
540 nm and were correlated to cell dry weights. To determine cell dry
weight, cells were washed twice with distilled water and dried for
48 h at 110°C. The concentrations of PGA,
L-glutamate, and
N-acetyl-L-glutamate were determined by using a
high-performance liquid chromatograph (HPLC) (Knauer, Berlin, Germany)
equipped with a UV detector (210 nm) and a reverse-phase C18 column (Waters, Milford, Mass.). Deionized water
adjusted to pH 2.1 with phosphoric acid was used as the eluent (flow
rate, 1.0 ml/min). The specific growth rate was calculated from the slope of a straight line on a semilog plot in which the logarithm of
cell concentration was plotted versus the culture time. The rate
constant for decomposition of L-glutamate was determined by
assuming that first-order reaction kinetics were operating. The
I50 of PGA was the concentration of PGA at which the growth rate of S. solfataricus was one-half the growth rate in
medium without PGA.
| |
RESULTS |
PGA formation from L-glutamate under culture
conditions.
Based on our previous finding that
L-glutamate enhances the growth of S. solfataricus in batch cultures (25), we attempted fed-batch cultivation of S. solfataricus by continuously
adding L-glutamate to the culture medium. Since the cell
density and growth rate were maximal at an L-glutamate
concentration of 1.0 g/liter (25), the initial fed-batch
experiments were performed by controlling the residual concentration of
L-glutamate so that it was at this level. In contrast to
our expectation, however, continuous addition of
L-glutamate resulted in a very marginal increase in the
cell density. Moreover, growth of S. solfataricus in
fed-batch cultures was suppressed as the residual glutamate concentration was increased to 3.0 g/liter, and no cell growth was
observed in the presence of 5.0 g of L-glutamate per liter. During our study of fed-batch cultivation of S. solfataricus
in glutamate-supplemented media, we also observed that the
concentrations of L-glutamate in culture media gradually
decreased even in the absence of cell growth.
It has been determined previously that L-glutamate can be
converted to PGA at a high temperature (31). To explore
the possibility that L-glutamate is spontaneously converted
to PGA, GM medium containing L-glutamate was maintained
under the conditions employed for the fed-batch experiments. Samples
were withdrawn at appropriate times and analyzed by using a
reverse-phase HPLC. From HPLC chromatograms, we confirmed that
L-glutamate in GM medium was dehydrated to PGA under the
cultivation conditions used; the peak corresponding to
L-glutamate (2.21 min) decreased gradually with incubation time, whereas a new peak (7.74 min), whose retention time was identical
to that of authentic PGA, increased simultaneously. The stoichiometry
of the conversion was as follows: 1 mol of L-glutamate 
1 mol of PGA + 1 mol of H2O. To quantify dehydration
of L-glutamate in GM medium, the rates of PGA formation
were determined at different temperatures and pHs. It was found that
the first-order dehydration rate of L-glutamate was
markedly influenced by pH and temperature. Glutamate dehydration was
rather modest at pH 7.0, but the dehydration rates were significantly
higher at pH 3.0 and 10.0. At 78°C, for example, the glutamate
dehydration rate at pH 3.0 (1.08 × 10
2
h1) was 36 times higher than that at pH 7.0 (0.03 × 102h1). On the other hand, the dehydration
rate of L-glutamate was almost unaffected by the presence
of the modified Allen's salts and glucose in GM medium (data not shown).
Effect of PGA on growth of S. solfataricus.
When
the PGA concentrations in samples from fed-batch experiments were
analyzed by the HPLC method, the PGA level in the culture medium
increased with the L-glutamate concentration. In addition, there was an inverse relationship between the maximal cell densities and the PGA concentrations in the media (data not shown). These results
suggest that cell growth is adversely affected by accumulation of PGA
in fed-batch cultures with L-glutamate.
In order to investigate the effect of PGA on growth of S. solfataricus, various amounts of PGA were added to two media: a glucose minimal medium (GM medium) and a complex medium (GYM medium). For comparison, growth of S. solfataricus was also monitored
after L-glutamate was added to the same medium. As shown in
Fig. 1, PGA significantly inhibited the
growth of S. solfataricus, whereas L-glutamate
enhanced cell growth. In GYM medium, reduced inhibitory effects of PGA
were observed compared to the results obtained with GM medium.
Irrespective of the medium composition, however, S. solfataricus did not grow in the presence of 15.5 mM PGA.
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FIG. 1.
Effects of L-glutamate and PGA on growth of
S. solfataricus. L-Glutamate (Glu) or PGA was
added to GM medium and GYM medium. The following amounts of glutamate
were added: none ( ), 3.4 mM ( ), 6.8 mM ( ), and 20.4 mM ( ).
The following amounts of PGA were added: none (), 3.9 mM (), 7.7 mM (), and 15.5 mM ().
|
|
Since both L-glutamate and L-aspartate enhance
the growth of S. solfataricus (25), we
investigated the effects of these two amino acids on the inhibitory
effect of PGA by measuring the growth rate in GM medium supplemented
with 10 mM L-glutamate or 10 mM L-aspartate in
the presence of various concentrations of PGA. As shown in Fig.
2, inhibition of S. solfataricus growth by PGA was relieved by the addition of
L-glutamate or L-aspartate. While the
I50 of PGA for cell growth was 5.1 mM in GM medium (with no
amino acid added), the I50s of PGA were 8.4 and 12.1 mM in the presence of L-aspartate and L-glutamate,
respectively. No amino acid other than L-glutamate and
L-aspartate resulted in an appreciable reduction in growth
inhibition by PGA (data not shown).
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FIG. 2.
Effects of PGA on growth of S. solfataricus
in GM medium () and in medium supplemented with 10 mM
L-aspartate () or 10 mM L-glutamate ().
The specific growth rates for each PGA concentration were normalized to
those obtained in the absence of PGA.
|
|
PGA formation in a yeast extract solution and its effect on growth
of S. solfataricus.
L-Glutamate is one of
the most abundant amino acids in yeast extract (14). For
this reason, we investigated whether PGA can be produced from yeast
extract and whether there is any relationship between PGA formation and
inhibition of S. solfataricus growth in the presence of high
yeast extract concentrations.
To examine spontaneous formation of PGA under S. solfataricus culture conditions, we incubated a yeast extract
solution at 78°C and pH 3.0 without cells and analyzed the culture
broth by using a reverse-phase HPLC. From this HPLC analysis, we found that the yeast extract used in this work originally contained 2.2%
(wt/wt) PGA. After 80 h of incubation of the yeast extract solution under cell culture conditions, the PGA content was 8.5% (wt/wt). In order to examine the relationship between growth inhibition by yeast extract and PGA formation, cells were cultivated in GM medium
supplemented with different amounts of yeast extract. The cell
densities and PGA concentrations in culture broth are shown in Fig.
3. As reported previously (4,
15), growth of S. solfataricus was stimulated by
addition of a small amount (3.0 g/liter) of yeast extract to GM medium.
When the yeast extract concentration exceeded 6.0 g/liter (Fig. 3A),
however, significant growth inhibition was observed. From time course
profiles of PGA formation, it was found that accumulation of PGA in the
medium was significant at high yeast extract concentrations (Fig. 3B).
The results described above indicate that the growth inhibition
observed in the presence of high yeast extract concentrations is due at
least in part to accumulation of PGA in the culture broth.
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FIG. 3.
(A) Batch growth of S. solfataricus in GM
medium supplemented with yeast extract. (B) Time course profiles for
residual PGA concentrations in the culture broth. The following amounts
of yeast extract were added: none (), 3.0 g/liter (), 6.0 g/liter
(), 9.0 g/liter (), and 12.0 g/liter ( ).
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|
Effects of structural analogues of L-glutamate on
growth of S. solfataricus.
We examined the effects of
various structural analogues of L-glutamate on growth of
S. solfataricus. In this experiment, cells were cultivated
in GM medium supplemented with each analogue at a concentration of 10 mM, and the specific growth rate was determined from the growth curve.
As shown in Table 1, the growth
inhibition was greatest with PGA among the glutamate analogues tested
in this work. Significant growth inhibition was also observed in the
presence of L-methionine sulfoxide, glutaric acid, succinic acid, and L-glutamic acid -methyl ester. When
N-acetyl-L-glutamic acid was added, on the other
hand, the growth rate of S. solfataricus increased about
1.7-fold compared to the growth rate in GM medium.
We also investigated cell growth in GM medium supplemented with
L-glutamate or N-acetyl-L-glutamate.
As shown in Fig. 4, there was an increase
in the maximal cell density when
N-acetyl-L-glutamate was used in place of
L-glutamate. In addition, the biomass yield was much higher
with N-acetyl-L-glutamate
(Yx/s = 0.59) than with L-glutamate (Yx/s = 0.31). We
also examined the stability of
N-acetyl-L-glutamate under the culture
conditions used (78°C and pH 3.0). In contrast to
L-glutamate, N-acetyl-L-glutamate
was stably maintained and was not spontaneously converted to other
compounds under the test conditions.
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FIG. 4.
Growth of S. solfataricus in GM medium
supplemented with L-glutamate (A) or
N-acetyl-L-glutamate (B). Symbols: ,
L-glutamate or N-acetyl-L-glutamate
concentration in medium without cells; , cell density;  ,
L-glutamate or N-acetyl-L-glutamate
concentration in inoculated culture.
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| |
DISCUSSION |
The thermal lability of nutrients has been known for several
decades, especially as it relates to food processing (12,
16). Many nutrient instability problems are related to
thermochemical modifications of carbohydrates and their effects on the
growth of microorganisms (9, 10). In this study, we showed
that L-glutamate, one of the most abundant amino acids in
yeast extract, can be spontaneously converted to PGA during cultivation
of hyperthermophiles and that PGA significantly suppresses growth of
S. solfataricus. Although thermal conversion of
L-glutamate to PGA was originally described several decades
ago (31) and the role of PGA as an agonist for the
glutamate receptor has been investigated with nerve cells (6,
19), there have been no previous reports concerning the effects
of PGA on the growth of microorganisms.
Formation of PGA from L-glutamate during cultivation can
adversely affect the growth of S. solfataricus in two ways:
the level of the growth enhancer (L-glutamate) can decrease
and the growth inhibitor (PGA) can accumulate. As reported previously
(25), L-glutamate is an important growth
enhancer for S. solfataricus, and hence a loss of
L-glutamate during cultivation should accompany a decrease
in cell growth. Since the rate of dehydration of
L-glutamate was higher at a low pH, acidophilic
hyperthermophiles, such as Sulfolobus sp.,
Metallosphaera sp., Acidianus sp., and
Stygiolobus sp., might be much more vulnerable to growth
suppression by decomposition of L-glutamate than
neutrophilic hyperthermophiles are.
It was observed in this study that PGA was lethal to S. solfataricus. There was no cell growth in the presence of PGA
concentrations greater than 15.5 mM in GM medium. According to a recent
study of a high-density culture of S. solfataricus in which
a microfiltration bioreactor was used (26), toxic
compounds with molecular masses less than 1 kDa were produced during
fermentation and reduced the maximum cell density. In view of the
results presented in this paper, it is very probable that PGA
(molecular weight, 129.1) is one of the toxic compounds that accumulate
in culture broth. It is noteworthy that a complex nutrient, such as
yeast extract, which contains a large amount of
L-glutamate, was added continuously in the study of
Schiraldi et al. (26).
The inhibitory effect of PGA produced from L-glutamate
explains in part the growth inhibition in the presence of high yeast extract concentrations observed with batch cultures of S. solfataricus and Thermoplasma acidophilum (2, 4,
15). Previously, we reported that addition of excess yeast
extract to fed-batch cultures of S. solfataricus results in
low maximum cell densities, and the inhibitory effect was attributed to
accumulation of mineral ions in yeast extract (24).
However, at that time it was difficult to explain the inhibitory effect
of yeast extract in batch cultures in which the concentrations of
mineral ions were not as high as those in fed-batch cultures. From the
results obtained in this study, it is clear that formation of PGA is
one of major reasons for growth inhibition in the presence of high
yeast extract concentrations in batch cultures of thermoacidophiles.
In our study, the inhibition caused by PGA was reduced when acidic
amino acids, such as L-aspartate and
L-glutamate, were added. In many cases, the carrier protein
for L-glutamate transport is similar to that for
L-aspartate transport, and L-aspartate works as
a competitive inhibitor of L-glutamate transport among amino acids (8, 30). Considering this, it is likely that PGA is a competitive inhibitor for transport of L-glutamate
or L-aspartate in S. solfataricus. Although our
results imply that PGA functions as a structural analogue of
L-glutamate, it remains to be seen how PGA causes death of
S. solfataricus upon uptake.
Various structural analogues of L-glutamate were examined,
and inhibitory effects were observed in the presence of
L-glutamic acid -benzyl ester, L-glutamic
acid -methyl ester, glutaric acid, L-methionine
sulfoxide, PGA, and succinic acid. There was not as much correlation
between affinity of the analogues for uptake of L-glutamate
by nerve cells (3) and the extent of inhibition of growth
of S. solfataricus. For example, glutaric acid,
L-methionine sulfoxide, PGA, and succinic acid were
reported to have negligible affinity for L-glutamate
transport in nerve cells. It is interesting that N-acetyl
glutamate did not decay to another form under S. solfataricus culture conditions. The amine group of
N-acetyl-L-glutamate was presumed to be
protected from internal cyclization between amine and carboxylic groups by attachment of an acetyl group moiety. S. solfataricus
utilized N-acetyl-L-glutamate as well as
L-glutamate, and growth was greatly enhanced by addition of
N-acetyl-L-glutamate to GM medium. Furthermore, the cell mass yield was much higher when
N-acetyl-L-glutamate was used instead of
L-glutamate. Considering these results,
N-acetyl-L-glutamate might be substituted for
L-glutamate during cultivation of S. solfataricus.
| |
ACKNOWLEDGMENTS |
This work was supported by the Korean Ministry of Science and
Technology and by in-house grants from the Pohang University of Science
and Technology.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Chemical Engineering, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang 790-784, Korea. Phone: 82-54-279-2268. Fax:
82-54-279-2699. E-mail: sblee{at}postech.ac.kr.
Present address: Department of Chemical Engineering, University of
California, Berkeley, CA 94720.
| |
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Applied and Environmental Microbiology, August 2001, p. 3650-3654, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3650-3654.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
