Previous Article | Next Article 
Applied and Environmental Microbiology, August 1998, p. 2970-2976, Vol. 64, No. 8
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Influence of Carbon Source on Nitrate Removal by Nitrate-Tolerant
Klebsiella oxytoca CECT 4460 in Batch and Chemostat
Cultures
Guadalupe
Piñar,1
Karin
Ková
ová,2
Thomas
Egli,2 and
Juan L.
Ramos1,*
Department of Biochemistry and Molecular and
Cellular Biology of Plants, Estación Experimental del
Zaidín, Consejo Superior de Investigaciones
Científicas, 18008 Granada, Spain,1 and
Department of Microbiology, Swiss Federal Institute for
Environmental Science and Technology (EAWAG), CH-8600 Dübendorf,
Switzerland2
Received 21 January 1998/Accepted 26 May 1998
 |
ABSTRACT |
The nitrate-tolerant organism Klebsiella oxytoca CECT
4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose
or both) on the growth rate and the efficiency of nitrate removal under
laboratory conditions. With sucrose as the sole C source the maximum
specific growth rate was 0.3 h
1, whereas with glycerol it
was 0.45 h1. In batch cultures K. oxytoca
cells grown on sucrose or glycerol were able to immediately use sucrose
as a sole C source, suggesting that sucrose uptake and metabolism were
constitutive. In contrast, glycerol uptake occurred preferentially in
glycerol-grown cells. Independent of the preculture conditions, when
sucrose and glycerol were added simultaneously to batch cultures, the
sucrose was used first, and once the supply of sucrose was exhausted,
the glycerol was consumed. Utilization of nitrate as an N source
occurred without nitrite or ammonium accumulation when glycerol was
used, but nitrite accumulated when sucrose was used. In chemostat
cultures K. oxytoca CECT 4460 efficiently removed nitrate
without accumulation of nitrate or ammonium when sucrose, glycerol, or
mixtures of these two C sources were used. The growth yields and the
efficiencies of C and N utilization were determined at different growth
rates in chemostat cultures. Regardless of the C source, yield carbon (YC) ranged between 1.3 and 1.0 g (dry weight) per g
of sucrose C or glycerol C consumed. Regardless of the specific growth
rate and the C source, yield nitrogen (YN) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to
batch cultures, in continuous cultures glycerol and sucrose were
utilized simultaneously, although the specific rate of sucrose
consumption was higher than the specific rate of glycerol consumption.
In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so
that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h1 the process led to simultaneous and efficient removal
of the C and N sources used. At a growth rate of 0.2 h1
the zone of double limitation was between 8 and 11. This suggests that
the regimen of double limitation is influenced by the C/N ratio and the
growth rate. The results of these experiments were validated by pulse
assays.
| |
INTRODUCTION |
Nitrate is the source of nitrogen
most widely used by aerobic organisms, including plants, fungi, and
bacteria (14, 15, 29). Assimilatory nitrate utilization
involves the sequential action of assimilatory nitrate reductase, which
converts nitrate to nitrite, and nitrite reductase, which converts
nitrite to ammonium. Ammonium is subsequently incorporated into carbon
skeletons, usually via the glutamine synthetase-glutamate synthase
pathway (15, 23).
Although the ability to use nitrate is widespread in nature, nitrate is
considered a ubiquitous pollutant (18) because at high
concentrations it inhibits cell growth (25, 26) and because at low concentrations in drinking water it is toxic (32).
High nitrate loads are commonly found in industrial effluents from wash
tanks in dairy factories and are also produced during the synthesis of
nitroorganic compounds in the pharmaceutical and explosives industries
(4, 20, 25, 30). Reducing high nitrate loads in wastes is of
interest, particularly if the wastes are processed at conventional
treatment plants where futile conversion of nitrate to nitrite can
occur when there are high nitrate loads (19). Wastewaters
from the synthesis of nitroorganic compounds usually contain large
amounts of sulfates and carbonates, which are inhibitors of
denitrification (20, 30). In an attempt to remove high
nitrate loads from this type of industrial waste, we isolated a strain
belonging to the genus Klebsiella that tolerated nitrate at
concentrations up to 1 M and was able to thrive under aerobic
conditions in a culture medium containing up to 150 mM nitrate. This
strain was identified as a Klebsiella oxytoca strain and was
deposited in the Spanish Type Culture Collection (CECT) as strain CECT
4460 (25).
In this paper we describe nitrate removal by this strain under
laboratory conditions in both batch and chemostat cultures containing
sucrose or glycerol or mixtures of these compounds as the sole sources
of C and energy. Glycerol was chosen because it was the C source used
to isolate strain CECT 4460 (25). Several sugars are used by
strain CECT 4460, including sucrose, glucose, lactose, fructose, and
galactose, and sucrose was chosen for use in this study because of its
low cost and because its presence in industrial wastes (e.g., sugar
beet molasses and other wastes) makes it a potentially cheap source of
C for biotreatment of wastes with high nitrate loads. Under optimal
growth conditions nitrate elimination by K. oxytoca CECT
4460 occurred without nitrite or ammonium accumulation. In chemostat
cultures the zone of double nutrient limitation (C limitation and N
limitation) was found to be influenced by the growth rate. At a growth
rate of 0.1 h1, growth was limited by C and N when the
C/N ratio of the influent medium was between 10 and 30, whereas at a
growth rate of 0.2 h1, growth was limited at a C/N ratio
of 8 to 11. Under these conditions both C and N were completely
removed; therefore, these conditions can be considered optimal for
biotreatment.
| |
MATERIALS AND METHODS |
Microorganisms, growth medium, and culture conditions.
K.
oxytoca CECT 4460 was grown on mineral medium M8 supplemented as
suggested by Egli and Fiechter (10). The medium used contained (per liter of deionized water) 2.020 g of KNO3,
5.65 g of KH2PO4, 0.5 g of NaCl,
2.46 g of MgSO4·7H2O, 82 mg of disodium EDTA · 2H2O, 1.25 mg of ZnCl2, 0.75 mg of
MnCl2 · 4H2O, 7.5 mg of
H3BO3, 5 mg of
CoSO4 · 7H2O, 0.25 mg of
CuCl2 · 2H2O, 0.5 mg of
NiCl2 · 6H2O, 0.75 mg of
Na2MoO4·2H2O, and 7 mg of
FeCl3 · 6H2O. Before heat sterilization, the
medium was completely mixed and acidified with concentrated
H2SO4 to pH 3.0. The C source (sucrose or
glycerol or both) was sterilized separately and was added to the
mineral medium after it was cooled so that the final total carbon
concentration was 3.9 g/liter. Continuous cultivation was performed in
bioreactors (working volume, 2.5 liters; MBR, Wetzikon, Switzerland).
Before cells were inoculated, the pH of the medium was adjusted to 7.0, and the pH was maintained at 7.0 ± 0.1 by automatic addition of 1 M NaOH-KOH. The temperature was set at 30 ± 0.1°C. Cultures
were operated in batch or continuous mode as indicated below. The
aeration rate was 1 ± 0.1 volume of air per volume of culture
liquid per min, and the impeller speed was 1,000 rpm.
Analytical methods.
Compounds present in the culture medium
were analyzed after the cells were removed by centrifugation with a
Sorvall model R5C centrifuge at 12,000 × g for 15 min.
The nitrite content was determined by the method of Snell and Snell
(28). The nitrate content was determined by using a
Spectroquant 14773 kit from Merck (Darmstadt, Germany) under the
conditions recommended by the supplier or by using a specific nitrate
electrode and a potentiometer (MicropH 2002; Crison, Barcelona, Spain).
The detection limits were 10 mg/liter for nitrate and 0.05 mg/liter for
nitrite. The dissolved total organic carbon content was measured with a
TOC-UNOR gas analyzer (H. Maihak AG, Hamburg, Germany) for values
ranging from 0 to 100 mg/liter. The glycerol, glucose, and sucrose
contents were determined enzymatically with commercial kits obtained
from Boehringer (Mannheim, Germany). The acetate concentration was measured by high-pressure ion exclusion chromatography as described by
Bally (3), and the detection limit was 1 mg of acetate C per
liter. The C and N contents of the biomass were determined with an
elemental carbon, hydrogen, nitrogen, and sulfur analyzer (model EA
1108; Carlo Erba, Milan, Italy).
Biomass determination.
The biomass was determined as cell
dry weight by filtration through a polycarbonate membrane filter (pore
size, 0.45 µm; diameter, 47 mm; Millipore Corp., Bedford, Mass.).
Cells collected on filters were dried at 105°C to constant weight.
| |
RESULTS AND DISCUSSION |
Determination of the µmax in batch cultures of
K. oxytoca CECT 4460.
To establish the dilution rate
limits for continuous cultivation of K. oxytoca CECT 4460, the maximum specific growth rate (µmax) in batch cultures
was determined. To do this, bacterial cells were grown in minimal
medium containing 0.5 to 4.0 g of C from glycerol or from sucrose
per liter and 20 mM NO3. Cells were
maintained for 35 generations in the exponential growth phase, and then
the µmax was determined. Cells growing with glycerol as
the sole C source had a µmax of 0.45 ± 0.01 h1, whereas cultures growing with sucrose as the sole C
source had a µmax of 0.3 ± 0.01 h1.
Growth of K. oxytoca CECT 4460 in batch cultures with
sucrose and glycerol.
For a number of microorganisms, culture
conditions noticeably influence the pattern of C utilization,
particularly when mixtures of C sources are used. In these cases
catabolite repression is one of the main mechanisms responsible for
preferential use of a given C source (7, 9, 22). We
therefore studied the response of K. oxytoca CECT 4460 to
mixtures of sucrose and glycerol in detail. K. oxytoca CECT
4460 was precultured in a chemostat (dilution rate, 0.2 h1) in which either glycerol or sucrose was the sole C
source. The resulting cells were used as the inocula for batch assays
performed in minimal medium containing a 1:1 mixture of sucrose and
glycerol (Fig. 1 and
2). Regardless of the C source used to
cultivate the inoculum, cells exposed to a mixture of glycerol and
sucrose exhibited a diauxic growth curve. The diauxic phase was shorter
when the inoculum was from glycerol precultures than when the inoculum was from sucrose precultures (Fig. 1A and 2A). In batch cultures both C
sources were used simultaneously from the beginning of the assay,
although there were considerable differences in the specific uptake
rates for glycerol and sucrose utilization (Fig. 1B and 2B). When cells
were precultured with glycerol, both C sources were used simultaneously
during the first 2 h of growth (Fig. 1B). The specific rate of
sucrose consumption was 1.35 ± 0.02 g per g (dry weight) per
h, and the specific rate of glycerol consumption was 1.04 ± 0.03 g per g (dry weight) per h. After 2 h the glycerol
uptake rate was markedly reduced (to 0.2 ± 0.01 g per g
[dry weight] per h), whereas sucrose uptake continued at
approximately the same rate. These results suggest that sucrose repressed glycerol utilization. The supply of sucrose was fully exhausted after 5 to 6 h (Fig. 1A); after this the glycerol
remaining in the culture medium was used as the sole substrate for
growth, and its specific rate of consumption increased to 2.25 ± 0.02 g per g (dry weight) per h.
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 1.
Growth of K. oxytoca CECT 4460 in batch
cultures with a 1:1 mixture of sucrose and glycerol. K. oxytoca CECT 4460 was pregrown in a C-limited chemostat with
glycerol as the C source (dilution rate, 0.2 h1) and was
transferred to a culture medium containing sucrose and glycerol. (A)
Growth ( ) and the concentrations of sucrose ( ) and glycerol ( )
were determined at different times. (B) Specific consumption rates were
determined for glycerol (qGly) ( ) and sucrose
(qSuc) ( ). DW, dry weight; A660nm,
absorbance at 660 nm.
|
|
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 2.
Growth of K. oxytoca CECT 4460 in batch
cultures with a 1:1 mixture of sucrose and glycerol. The conditions
were the same as those described in the legend to Fig. 1 except that
the cells were pregrown in a chemostat with sucrose (dilution rate, 0.2 h1).
|
|
This pattern of utilization of C sources was also reflected in the
specific growth rate. Simultaneous utilization of sucrose
and glycerol
during the first 2 h of culture resulted in a specific
growth rate
(0.49 ± 0.02 h
1) that was higher than the specific
growth rate when either sucrose
(0.3 ± 0.01 h
1) or
glycerol (0.45 ± 0.01 h
1) was the sole C source.
The glycerol which remained in the culture
medium after the supply of
sucrose was exhausted supported a specific
growth rate of 0.21 ± 0.04 h
1 in the second phase of growth (this specific
growth rate was
considerably lower than the µ
max observed
with glycerol-precultured
cells). A synergistic effect due to
simultaneous utilization of
more than one C source by different
microorganisms has been observed
previously when there was an excess of
substrate (
2,
9,
12).
Cells that were pregrown on sucrose and transferred to medium
containing sucrose plus glycerol also utilized sucrose preferentially
(Fig.
2A). During the first 2 h both C sources were used at a
specific rate of 3 ± 0.15 g per g (dry weight) per h (Fig.
2B),
but after 2 h the consumption of sucrose increased to
5.6 ± 0.1
g per g (dry weight) per h and the rate of
consumption of glycerol
decreased until glycerol consumption was
totally repressed. After
5 to 6 h, when the sucrose concentration
fell below our detection
limit, glycerol was again consumed until it
became undetectable
in the culture medium (Fig.
2B). These results
suggest that cells
pregrown in a chemostat with sucrose as the limiting
growth factor
exhibit constitutive utilization of sucrose and glycerol
but that
upon transfer to batch cultures without C source limitation
sucrose
is preferentially used instead of glycerol.
In diauxic cultures the substrate that supports the highest growth rate
is generally utilized first, while consumption of
the second substrate
is repressed (
16). However, with
K. oxytoca CECT
4460 we observed the opposite behavior; the first substrate
to be used
was sucrose, which supported a lower specific growth
rate than glycerol
supported. When the supply of sucrose was exhausted,
growth stopped,
but it later resumed. This may have reflected
the time needed for full
induction of the glycerol utilization
system. These results support the
hypothesis that sucrose uptake
is constitutive, whereas glycerol uptake
can be repressed by sucrose.
Growth of K. oxytoca CECT 4460 in batch cultures with
glucose and glycerol.
To determine whether glucose was responsible
for the inhibition of glycerol utilization, K. oxytoca CECT
4460 cells were grown for 35 generations in minimal medium containing
glycerol or glucose (0.5 g of C per liter) and 20 mM
NO3. Cells in the exponential growth phase
were transferred to minimal medium containing a 1:1 mixture of glucose
and glycerol (Fig. 3). Regardless of the
C source used to grow the inoculum, cells exposed to a mixture of
glycerol and glucose exhibited a diauxic growth curve. In both cases
glucose was used first, and the supply of glucose was completely
exhausted within 6 to 8 h; after this the glycerol remaining in
the culture medium was used as the sole substrate for growth (Fig. 3A
and B). During the first 6 h glycerol consumption was negligible.
Thereafter and when the glucose concentration fell below our detection
limit, glycerol consumption started and continued until the supply of
this C source was exhausted (Fig. 3A and B). These results support the
hypothesis that once sucrose is hydrolyzed to glucose plus fructose,
glycerol utilization is repressed by glucose metabolism.
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 3.
Growth of K. oxytoca CECT 4460 in batch
cultures with a 1:1 mixture of glucose and glycerol. (A) K. oxytoca CECT 4460 was pregrown on glycerol and transferred to a
culture medium containing glucose and glycerol. Growth () and the
concentrations of glucose () and glycerol () were determined at
different times. (B) Same as panel A except that the cells were
pregrown on glucose. A660nm, absorbance at 660 nm.
|
|
Growth kinetics of K. oxytoca CECT 4460 in continuous
cultures with glycerol as the sole C source.
Continuous cultures
of K. oxytoca CECT 4460 with glycerol as the sole C source
were set up at dilution rates ranging from 0.05 to 0.3 h1. The relationships among dilution rate, dry weight of
cells, culture yield, and residual concentrations of glycerol, nitrate, and nitrite were determined under steady-state conditions (Table 1). In general, the higher the growth
rate, the lower the dry weight of the culture and, consequently, the
lower the yield with respect to the amount of C or N consumed. The
residual glycerol concentration was negligible at dilution rates
between 0.05 and 0.15 h1, and the residual concentration
of nitrate was about 20 mg/liter. At a dilution rate equal to or higher
than 0.2 h1 the residual concentration of glycerol was
about 0.5 g/liter, while the residual concentration of nitrate
decreased to approximately 10 mg/liter. These results suggest that
K. oxytoca CECT 4460 was carbon limited at growth rates
equal to or lower than 0.15 h1, whereas it was nitrogen
limited at growth rates equal to or higher than 0.2 h1.
To ensure that N-limited conditions were used in subsequent assays,
chemostats were run at a dilution rate of 0.2 h1. The
relationship between steady-state regimens and growth rates was similar
to that described by Cocaing-Bousquet et al. (5) for
chemostat cultures of Corynebacterium glutamicum; namely, the cultures were C limited at low dilution rates and N limited at high
growth rates.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Relationship between growth rate and culture parameters
for K. oxytoca growing with glycerol
and nitratea
|
|
Growth kinetics of K. oxytoca CECT 4460 in continuous
cultures with sucrose as the sole C source.
In batch cultures the
elimination of nitrate when sucrose was the sole C source was as
efficient as the elimination of nitrate when glycerol was the sole C
source, although some nitrite did accumulate. The accumulation of
nitrite was more evident under anaerobic conditions than under aerobic
conditions (data not shown). Because nitrite accumulation is
detrimental for the operation of a nitrate treatment plant, we tested
whether this was also the case in chemostat cultures under aerobic
conditions. K. oxytoca CECT 4460 was grown aerobically in
chemostat cultures at growth rates between 0.05 and 0.20 h1 (Table 2). When there
was an increase in the growth rate, there was a slight decrease in the
dry weight of the culture, although this decrease hardly influenced
yield nitrogen (YN) and yield carbon (YC)
(Table 2). However, it should be noted that the yields under aerobic
conditions were 1.5- to 2-fold higher than the yields under anaerobic
conditions (data not shown). For growth rates tested, the residual
concentration of nitrate was between 13 and 22 mg/liter, and no nitrite
or ammonium accumulated in the culture medium. The residual sucrose
concentration was negligible at dilution rates between 0.05 and 0.15 h1 (Table 2). At a dilution rate of 0.2 h1
the residual concentration of sucrose was 0.47 ± 0.03 g/liter. These results suggest that when sucrose was the sole C source, K. oxytoca CECT 4460 also became carbon limited at growth rates equal
to or lower than 0.15 h1, as we observed when glycerol
was used as the sole C source. When the dilution rate was increased to
more than 0.2 h1, the culture was washed out (data not
shown), probably because this rate approached the µmax of
the culture for sucrose (0.3 h1).
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Relationship between growth rate and culture parameters
for K. oxytoca growing with sucrose
and nitratea
|
|
Growth of K. oxytoca CECT 4460 in continuous cultures
with mixtures of glycerol and sucrose as C sources.
K.
oxytoca CECT 4460 preferentially used sucrose in batch cultures
when glycerol and sucrose were present simultaneously. As mentioned
above, this was unexpected; usually, when more than one C source is
supplied, the one used preferentially is the one that supports the
higher growth rate, which was not the case for sucrose and glycerol
utilization by K. oxytoca CECT 4460. We therefore tested
whether in a chemostat culture at a fixed growth rate (dilution rate,
0.2 h1) the presence of the two C sources in the medium
also resulted in preferential utilization of sucrose. For this study a
chemostat culture of K. oxytoca CECT 4460 under
steady-steady conditions was fed increasing concentrations of sucrose,
so that the total amount of carbon in the feed was 3.9 g/liter (Table
3).
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Effects of different proportions of glycerol and sucrose
in the continuous culture feed at a constant dilution rate of 0.2 h1 on dry weight and utilization of C and N
|
|
For each glycerol/sucrose ratio, time was allowed for steady-state
conditions to become established, and then different parameters
(dry
weight and residual concentrations of C an N) were measured.
The dry
weight (biomass) of the culture decreased with increasing
amounts of
sucrose in the mixtures. Accordingly, Y
N and Y
C
also
decreased with increasing sucrose concentrations. This suggested
that
K. oxytoca CECT 4460 used sucrose less efficiently than
it
used glycerol, as was expected from the results obtained with
a
single C source (Tables
1 and
2). At the same growth rate,
the
Y
C for glycerol was slightly higher than the Y
C
for sucrose.
The residual nitrate concentration in the medium was on the order of 10 to 20 mg/liter regardless of the sucrose concentration
in the feed. No
accumulation of nitrite was observed during growth
with any of the
mixtures.
The residual concentrations of glycerol and sucrose were also
determined. When the ratio of sucrose to glycerol in the mixture
was 1, the level of sucrose was negligible, whereas the glycerol
concentration
was about 470 ± 30 mg/liter. This suggested that
there was
preferential use of sucrose but at the same time indicated
that there
was simultaneous use of both C sources, because otherwise
the glycerol
concentration would have been 5.0 g/liter. Furthermore,
a decrease in
the initial proportion of glycerol in the sucrose-glycerol
mixture
resulted in lower (indeed negligible) glycerol concentrations.
The
residual concentration of either sucrose or glycerol during
growth with
the mixtures at a dilution rate of 0.2 h
1 was lower than
the residual concentration during growth with
the corresponding
concentration of glycerol or sucrose alone in
cultures (Tables
1
through
3). Similar results have been observed
with other
microorganisms (
11,
21) growing in chemostat cultures
with
different mixtures. This was true for a methylotrophic yeast
culture
growing with glucose and methanol as the C sources and
for
Escherichia coli growing with mixtures of glucose and
galactose.
This finding probably reflects the fact that, in nature,
microbes
must cope with multiple C sources at low concentrations and
suggests
that under these conditions C sources are used more
efficiently
than when a single carbon source is present.
Consumption kinetics under transient-state conditions in continuous
cultures with glycerol.
K. oxytoca CECT 4460 was able to
simultaneously consume sucrose and glycerol under steady-state
conditions in continuous cultures (Table 3). In batch cultures the rate
at which these C sources were utilized was noticeably influenced by the
growth conditions of the inoculum, and sucrose was used in preference
to glycerol. To determine how the addition of one of the C sources
influenced utilization of the other C source, pulses of one of the C
sources were added to a steady-state culture of K. oxytoca that had been established with the other C source (Fig.
4).
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 4.
Simultaneous utilization of sucrose and glycerol after a
pulse of sucrose was added to K. oxytoca cells growing in a
continuous culture with glycerol as the sole C source. An N-limited
steady-state culture (dilution rate, 0.3 h1) of K. oxytoca CECT 4460 with glycerol as the sole C source was pulsed at
zero time with 7.5 g of sucrose per liter. At different times the
concentrations of sucrose () and glycerol ( ) (A) and the specific
glycerol consumption rates (qgly) () (B) were
determined. The initial concentration of glycerol was 10 g/liter. DW,
dry weight.
|
|
A steady-state culture of
K. oxytoca CECT 4460 growing
at a dilution rate of 0.3 h
1 with glycerol as the sole C
source was established. Under steady-state
conditions the dry weight of
the culture was 4.3 ± 0.1 g/liter.
The cells used glycerol at a
rate of 0.58 ± 0.02 g per g (dry
weight) per h (Fig.
4B);
the residual concentration of glycerol
was 1.7 ± 0.1 g/liter
(Fig.
4A), and nitrate or nitrite was undetectable
(data not shown).
This culture was pulsed with 7.5 g of sucrose
per liter, and we
recorded the sucrose, glycerol, nitrate, and
nitrite contents and
growth as a function of time. Nitrate and
nitrite remained practically
undetectable (data not shown) and
the dry weight of the culture
decreased from 4.35 ± 0.01 to 4.1
± 0.02 g/liter during the
time required for consumption of all
of the sucrose (data not shown).
Cells were able to utilize sucrose
and glycerol simultaneously from the
beginning of the assay (Fig.
4A). As sucrose was consumed, the specific
rate of glycerol utilization
decreased from 0.58 ± 0.02 g
per g (dry weight) per h in the absence
of sucrose to 0.44 ± 0.02 g per g (dry weight) per h in the presence
of sucrose (Fig.
4B). This represented a decrease in the initial
glycerol uptake value
of about 25%. As a consequence, glycerol
accumulated in the culture,
reaching a concentration of 3.9 ±
0.1 g/liter (Fig.
4A). Once
sucrose was consumed, the rate of
glycerol utilization increased, and
the residual glycerol concentration
decreased accordingly.
The rate of sucrose consumption was determined by calculating the
difference between the theoretical washout and the sucrose
concentration present in the culture medium at a given time. The
specific sucrose consumption rate was estimated to be approximately
0.16 ± 0.02 g per g (dry weight) per h, and the sucrose
pulse
was completely consumed within 4 h. In the glycerol
steady-state
chemostat the amount of C from sucrose used by the
Klebsiella strain after the pulse was equivalent to the
amount of C from
glycerol that the microbe did not use. These results
suggest that
simultaneous utilization of two C sources was adjusted to
satisfy
the organism's C requirements. These results also confirm that
sucrose utilization is constitutive in cultures of
K. oxytoca CECT 4460 growing on glycerol.
A similar assay was performed in which pulses of 10 g of glycerol
per liter were added to a continuous culture containing
sucrose as the
sole C source at a dilution rate of 0.2 h
1. No
consumption of glycerol was observed, and glycerol was removed
by
washout (data not shown).
Double substrate limitation in cultures grown with sucrose and
nitrate as the sole sources of carbon and nitrogen, respectively.
To determine the C/N ratio that resulted in complete elimination of the
C source supplied and the N source supplied, a series of assays were
performed with different C/N ratios. Tables 1 and 2 show that chemostat
cultures of K. oxytoca CECT 4460 became C or N limited
depending on the growth rate. As zones of double nutrient limitation
have been described for C and N utilization by Egli (8), it
was thought that such a situation can also occur with K. oxytoca depending on the C/N ratio of the culture medium and the
dilution rate. We determined the zone of double nutrient limitation (C
limitation and N limitation) for K. oxytoca CECT 4460 at
dilution rates of 0.1 and 0.2 h1 by using different C/N
ratios.
The residual concentrations of sucrose and nitrate under steady-state
conditions were used to determine whether a culture
was sucrose
limited, nitrate limited, or sucrose and nitrate limited.
Figure
5 shows the results obtained at a
dilution rate of 0.1
h
1. The following three distinct
growth regimens were recognized
(Fig.
5A): (i) sucrose limitation, in
which there was excess nitrate
(this was observed at C/N ratios of
<10); (ii) nitrate limitation,
in which there was excess sucrose (this
was observed at C/N ratios
of >30); and (iii) an intermediate regimen,
in which the concentrations
of both sucrose and nitrate were below our
detection limits (i.e.,
when the C/N ratio was between 10 and 30). In
previous studies
performed with other microorganisms (
1,
6,
8,
13,
24,
27) researchers have described a transitional double
substrate-limited
growth regimen between two distinct
single-nutrient-limited zones.
We observed an increase in the total
organic carbon concentration
in the culture medium with the transition
regimen (Fig.
5B). This
increase in the total organic carbon
concentration corresponded
to excretion of acetate into the culture
medium by
K. oxytoca CECT 4460 during the
double-nutrient-limited growth phase. This
behavior was also observed
in batch cultures of
Klebsiella pneumoniae,
in which
approximately 5 to 10% of the metabolized C was excreted
in the form
of acetate (
31). To determine whether
K. oxytoca CECT 4460 accumulated reserve polymers in any of the three distinct
growth regimens, we measured the C and N contents of the biomass
at
each C/N ratio (data not shown). The proportions of C and N
in the
biomass were constant at all C/N ratios, indicating that
no reserve
polymers accumulated.
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 5.
Effect of the C/N ratio on sucrose limitation, nitrate
limitation, and sucrose-nitrate limitation in a chemostat culture of
K. oxytoca CECT 4460 at a dilution rate of 0.1 h1. K. oxytoca CECT 4460 was grown with
nitrate and sucrose at different C/N ratios. Steady-state residual
concentrations of nitrate N () and sucrose C () were determined
(A), and the concentrations of acetate () and total organic carbon
(DOC) () in the culture medium were determined at the different C/N
ratios (B).
|
|
At a dilution rate of 0.2 h
1 the double-nutrient-limited
zone became narrower and shifted towards a lower C/N ratio; it was
sucrose and nitrate limited when the C/N ratio was between 8.2
and 11 (data not shown). This behavior has been observed previously
with other
microorganisms (
13,
17,
24).
To validate these results, we determined the overcapacity of the
nitrate elimination system in continuous cultures containing
sucrose. A
nitrate-limited chemostat culture of
K. oxytoca CECT
4460 growing at a dilution rate of 0.2 h
1 with sucrose as the
sole C source was established. Once steady-state
conditions were
reached, the culture was pulsed with 2.8 g of
NO
3 per liter (Fig.
6). Six hours later the residual sucrose
concentration
had decreased from about 1,900 to 440 mg/liter, and the
biomass
had increased from 4.4 ± 0.1 to 5.7 ± 0.1 g/liter
(Fig.
6A). Nitrate
consumption was determined by calculating the
difference between
the theoretical washout and the nitrate
concentration present
in the culture medium at a given time (Fig.
6B).
In this assay
the specific rate of C utilization was 0.05 ± 0.005 g per g (dry
weight) per h, and the specific rate of N utilization was
0.0062
± 0.0001 g per g (dry weight) per h, which gave a C/N
ratio of
8.1. These results are in agreement with the zone in which
sucrose
and nitrate simultaneously became limiting growth factors at a
dilution rate of 0.2 h
1.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 6.
Overcapacity of the nitrate elimination system in a
continuous culture with sucrose. A nitrate-limited steady-state culture
(dilution rate, 0.2 h1) of K. oxytoca CECT
4460 with sucrose as the sole C source was pulsed at zero time with
2.8 g of KNO3 per liter. (A) Evolution of biomass
() and concentration of sucrose measured as total organic carbon
(DOC) (). (B) Theoretical washout for nitrate (--), true
concentration of nitrate in the medium (), and consumption of
nitrate (), which was determined by calculating the difference
between the washout of nitrate and the actual nitrate concentration in
the culture medium.
|
|
A similar assay was performed with a nitrate-limited chemostat culture
of
K. oxytoca CECT 4460 growing at a dilution rate
of 0.25 h
1 with glycerol as the sole C source. Once steady-state
conditions
were reached, the residual glycerol concentration was about
500
mg/liter. The culture was pulsed with 0.8 g of
NO
3 per liter, and the specific rates of C
and N utilization were
determined. Two hours later the residual
glycerol concentration
was about 200 mg/liter, and this concentration
remained constant
with time. This was accompanied by an increase in the
culture
biomass from 4.6 ± 0.05 to 4.85 ± 0.05 g/liter. The
specific rate
of C utilization was 0.0165 ± 0.0005 g per g (dry
weight) per
h, and the specific rate of N utilization was 0.002 ± 0.0005 g
per g (dry weight) per h, which gave a C/N ratio of 8.1. These
results are also in agreement with the data obtained when sucrose
was
the sole C source.
Conclusions.
We found that in chemostat cultures
containing sucrose or glycerol or mixtures of these C sources,
K. oxytoca CECT 4460 efficiently removed high
nitrate loads without accumulating nitrite or ammonium. In chemostat
cultures containing sucrose and glycerol, K. oxytoca CECT
4460 simultaneously consumed both C sources, whereas in batch cultures
consumption was sequential, with sucrose used preferentially before
glycerol.
In chemostat cultures of
K. oxytoca CECT 4460 at a dilution
rate of 0.1 h
1, when the C/N ratio of the influent medium
was in the range from
10 to 30, the culture was simultaneously sucrose
and nitrate limited.
At a higher growth rate (e.g., at a dilution rate
of 0.2 h
1) the zone of double nutrient limitation was
narrower and shifted
towards lower C/N ratios (C/N ratios between 8 and
11). These
results suggest that both the N source and the C source were
removed
so that their concentrations were negligible. Therefore, these
conditions can be considered optimal for biotreatment of the industrial
wastewater tested here with regard to simultaneous elimination
of C and
N.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from Unión
Española de Explosivos and by grant PETRI 93-084 from CICYT.
G.P. thanks the MAPFRE Foundation for a fellowship and EAWAG for the
use of facilities in Dübendorf and for partial financial support.
We thank H. P. Füchslin for excellent technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: EEZ-CSIC,
Apdo. Correos 419, E-18008 Granada, Spain. Phone: 34-958-121011. Fax:
34-958-129600. E-mail: jlramos{at}eez.csic.es.
| |
REFERENCES |
| 1.
|
Al-Awadhi, N.,
T. Egli,
G. Hamer, and C. A. Mason.
1990.
The process utility of thermotolerant methylotrophic bacteria. I. An evaluation in chemostat culture.
Biotechnol. Bioeng.
36:816-820.
|
| 2.
|
Babel, W.
1982.
Energetische und biochemische Aspekte der Mischsubstrat-Fermentation, p. 183-188.
In
M. Ringpfeil (ed.), Biotechnologie. Akademie Verlag, Berlin, Germany.
|
| 3.
|
Bally, M.
1994.
Physiology and ecology of nitriloacetate degrading bacteria in pure culture, activated sludge and surface waters. Ph.D. dissertation.
Swiss Federal Institute of Technology, Zürich, Switzerland.
|
| 4.
|
Clarkson, W. W.,
B. J. B. Ross, and S. Krishnamachari.
1991.
Denitrification of high-strength industrial wastewater, p. 347-357.
In
45th Purdue Industrial Waste Conference Proceedings. Lewis Publishers, Chelsea, Mich.
|
| 5.
|
Cocaing-Bousquet, M.,
A. Guyonvarch, and N. D. Lindley.
1996.
Growth rate-dependent modulation of carbon flux through central metabolism and the kinetic consequences for glucose-limited chemostat cultures of Corynebacterium glutamicum.
Appl. Environ. Microbiol.
62:429-436[Abstract].
|
| 6.
|
Duchars, M. G., and M. M. Attwood.
1989.
The influence of the carbon:nitrogen ratio of the growth medium on the cellular composition and regulation of enzyme activity in Hyphomicrobium sp.
J. Gen. Microbiol.
135:787-793.
|
| 7.
|
Duetz, W. A.,
S. Marqués,
C. De Jong,
J. L. Ramos, and J. G. Van Andel.
1994.
Inducibility of the TOL catabolic pathway in Pseudomonas putida (pWW0) growing on succinate in continuous culture: evidence of carbon catabolite repression control.
J. Bacteriol.
176:2354-2361[Abstract/Free Full Text].
|
| 8.
|
Egli, T.
1991.
On multiple-nutrient-limited growth of microorganisms, with special reference to dual limitation by carbon and nitrogen substrates.
Antonie Leeuwenhoek
60:225-234.
|
| 9.
|
Egli, T.
1995.
The ecological and physiological significance of the growth of heterotrophic microorganisms with mixtures of substrates.
Adv. Microb. Ecol.
14:305-386.
|
| 10.
|
Egli, T., and A. Fiechter.
1981.
Theoretical analysis of media used in the growth of yeasts on methanol.
J. Gen. Microbiol.
123:365-369.
|
| 11.
|
Egli, T.,
U. Lendenmann, and M. Snozzi.
1993.
Kinetics of microbial growth with mixtures of carbon sources.
Antonie Leeuwenhoek
63:289-298[Medline].
|
| 12.
|
Egli, T.,
N. D. Lindley, and J. R. Quayle.
1983.
Regulation of enzyme synthesis and variation of residual methanol concentration during carbon-limited growth of Kloeckera sp. 2201 on mixtures of methanol and glucose.
J. Gen. Microbiol.
129:1269-1281.
|
| 13.
|
Egli, T., and J. R. Quayle.
1986.
Influence of the carbon:nitrogen ratio of the growth medium on the cellular composition and the ability of the methylotrophic yeast Hansenula polymorpha to utilize mixed carbon sources.
J. Gen. Microbiol.
132:1779-1788.
|
| 14.
|
Goldman, B. S.,
J. T. Lin, and V. Stewart.
1994.
Identification and structure of the nasR gene encoding a nitrate- and nitrite-responsive positive regulator of nasFEDCBA (nitrate assimilation) operon expression in Klebsiella pneumoniae MSal.
J. Bacteriol.
176:5077-5085[Abstract/Free Full Text].
|
| 15.
|
Guerrero, M. G.,
J. M. Vega, and M. Losada.
1981.
The assimilatory nitrate-reducing system and its regulation.
Annu. Rev. Plant Physiol.
32:169-204.
|
| 16.
|
Harder, W., and L. Dijkhuizen.
1982.
Strategies of mixed substrate utilization in microorganisms.
Phil. Trans. R. Soc. London B
297:459-480[Medline].
|
| 17.
|
Herbert, D.
1976.
Stoichiometric aspects of microbial growth, p. 1-30.
In
A. C. R. Dean, D. C. Ellwood, C. G. T. Evans, and J. Melling (ed.), Continuous culture 6: applications and new fields. Ellis Hordwood, Chichester, United Kingdom.
|
| 18.
|
Keith, L. H., and W. A. Telliard.
1979.
Priority pollutants. I. A perspective view.
Environ. Sci. Technol.
13:416-423.
|
| 19.
|
Krishnamachari, S., and W. W. Clarkson.
1993.
Nitrite accumulation in the effluents from high-strength denitrification of industrial wastewater, p. 383-392.
In
47th Purdue Industrial Waste Conference Proceedings. Lewis Publishers, Chelsea, Mich.
|
| 20.
|
Lawson, C. T.
1981.
Development of a biological denitrification process for a high-strength industrial waste, p. 882-888.
In
35th Purdue Industrial Waste Conference Proceedings. Lewis Publishers, Chelsea, Mich.
|
| 21.
|
Lendenmann, U.,
M. Snozzi, and T. Egli.
1996.
Kinetics of the simultaneous utilization of sugar mixtures by Escherichia coli in continuous culture.
Appl. Environ. Microbiol.
62:1493-1499[Abstract].
|
| 22.
|
Magasanik, B.
1961.
Catabolite repression.
Cold Spring Harbor Symp. Quant. Biol.
26:249-262[Abstract/Free Full Text].
|
| 23.
|
Magasanik, B., and F. C. Neidhardt.
1996.
Regulation of carbon and nitrogen utilization, p. 1318-1325.
In
F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C.
|
| 24.
|
Minkevich, I. G.,
A. Y. Krynitskaya, and V. K. Eroshin.
1988.
A double substrate limitation zone of continuous microbial growth, p. 171-189.
In
P. Kyslik, E. A. Dawes, V. Krumphanzl, and M. Novak (ed.), Continuous culture. Academic Press, London, United Kingdom.
|
| 25.
|
Piñar, G.,
E. Duque,
A. Haïdour,
J. M. Oliva,
L. Sánchez-Barbero,
V. Calvo, and J. L. Ramos.
1997.
Removal of high concentrations of nitrate from industrial wastewater by bacteria.
Appl. Environ. Microbiol.
63:2071-2073[Abstract].
|
| 26.
|
Ramos, J. L.,
A. Haïdour,
E. Duque,
G. Piñar,
V. Calvo, and J. M. Oliva.
1996.
Metabolism of nitrate esters by a consortium of two bacteria.
Nat. Biotechnol.
14:320-322[Medline].
|
| 27.
|
Rutgers, M.,
P. A. Balk, and K. Van Dam.
1990.
Quantification of multiple-substrate controlled growth. Simultaneous ammonium and glucose limitation in chemostat cultures of Klebsiella pneumoniae.
Arch. Microbiol.
153:478-484[Medline].
|
| 28.
|
Snell, F. D., and C. T. Snell.
1949.
Colorimetric methods of analysis, vol. 2. , p. 802-807.
Van Nostrand, New York, N.Y.
|
| 29.
|
Stewart, V.
1988.
Nitrate respiration in relation to facultative metabolism in enterobacteria.
Microbiol. Rev.
52:190-232[Free Full Text].
|
| 30.
|
Walker, J. F., Jr.,
M. V. Helfrich, and T. L. Donaldson.
1989.
Biodenitrification of uranium refinery wastewaters.
Environ. Prog.
8:97-101.
|
| 31.
|
Wanner, U., and T. Egli.
1990.
Dynamics of microbial growth and cell composition in batch culture.
FEMS Microbiol. Rev.
75:19-44.
|
| 32.
|
World Heath Organization.
1984.
Guidelines for drinking-water quality.
World Health Organization, Geneva, Switzerland.
|
Applied and Environmental Microbiology, August 1998, p. 2970-2976, Vol. 64, No. 8
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
del Castillo, T., Ramos, J. L.
(2007). Simultaneous Catabolite Repression between Glucose and Toluene Metabolism in Pseudomonas putida Is Channeled through Different Signaling Pathways. J. Bacteriol.
189: 6602-6610
[Abstract]
[Full Text]
-
Piñar, G., Ramos, J. L.
(1998). Recombinant Klebsiella oxytoca Strains with Improved Efficiency in Removal of High Nitrate Loads. Appl. Environ. Microbiol.
64: 5016-5019
[Abstract]
[Full Text]
Top
Abstract
Introduction
