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Applied and Environmental Microbiology, September 1999, p. 4008-4013, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Nitrification and Autotrophic Nitrifying Bacteria
in a Hydrocarbon-Polluted Soil
Jamal
Deni and
Michel J.
Penninckx*
Laboratoire de Physiologie et Ecologie
Microbiennes, Section Interfacultaire d'Agronomie,
Université Libre de Bruxelles c/o Institut Pasteur, B-1180,
Brussels, Belgium
Received 22 March 1999/Accepted 24 June 1999
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ABSTRACT |
In vitro ammonia-oxidizing bacteria are capable of oxidizing
hydrocarbons incompletely. This transformation is accompanied by
competitive inhibition of ammonia monooxygenase, the first key enzyme
in nitrification. The effect of hydrocarbon pollution on soil
nitrification was examined in situ. In a microcosm study, adding diesel
fuel hydrocarbon to an uncontaminated soil (agricultural unfertilized
soil) treated with ammonium sulfate dramatically reduced the amount of
KCl-extractable nitrate but stimulated ammonium consumption. In a soil
with long history of pollution that was treated with ammonium sulfate,
90% of the ammonium was transformed into nitrate after 3 weeks of
incubation. Nitrate production was twofold higher in the contaminated
soil than in the agricultural soil to which hydrocarbon was not added.
To assess if ammonia-oxidizing bacteria acquired resistance to
inhibition by hydrocarbon, the contaminated soil was reexposed to
diesel fuel. Ammonium consumption was not affected, but nitrate
production was 30% lower than nitrate production in the absence of
hydrocarbon. The apparent reduction in nitrification resulted from
immobilization of ammonium by hydrocarbon-stimulated microbial
activity. These results indicated that the hydrocarbon inhibited
nitrification in the noncontaminated soil (agricultural soil) and that
ammonia-oxidizing bacteria in the polluted soil acquired resistance to
inhibition by the hydrocarbon, possibly by increasing the affinity of
nitrifying bacteria for ammonium in the soil.
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INTRODUCTION |
Bioremediation is the most recent
technology used for cleaning areas contaminated with hydrocarbon
derivatives. The approach that has been exploited most consists of
stimulation of the soil endogenous microflora by adding an electron
acceptor and/or nutriments, in particular nitrogen in the form of
ammonium salts (2, 43, 44). This nitrogen source is
exploited mainly by microbial biomass for growth and production of
degradative enzymes. Some of the ammonium may be transformed into
nitrite and nitrate by the nitrification pathway (26). A
number of in vitro studies have shown that pure cultures of
Nitrosomonas europaea oxidize a wide variety of hydrocarbon substrates through the action of ammonia monooxygenase, the first key
enzyme in the autotrophic nitrification process. In contrat, in situ
studies of the effects of hydrocarbon soil pollution on nitrification
apparently have not been performed previously. The common hydrocarbon
substrates include alkanes, alkenes, and aromatic and chlorinated
aliphatic compounds (13, 15, 27, 40). Transformation of
hydrocarbons via the ammonia monooxygenase pathway may be considered
competitive cooxidation which reduces the rate and extent of ammonia
oxidation. The oxidation products obtained from alternative hydrocarbon
substrates are not assimilated by N. europaea and accumulate
in the culture medium. It is thought that in this case the nitrifying
bacteria present in a polluted environment initiate a syntrophic
pathway that provides intermediates for heterotrophic bacteria. Thus,
nitrifying bacteria appear to be excellent candidates for hydrocarbon
remediation because it may be possible to enhance the biodegradative
capacity of these ubiquitous soil bacteria by adding ammonia and oxygen
in order to support hydrocarbon cometabolism.
However, if bacteria are to be used effectively in bioremediation
schemes, it is important to obtain information concerning the
nitrification process that occurs in the presence of hydrocarbon in a
natural soil medium; studies performed with pure cultures ignore
interactions of bacteria and environmental components and bacterial
diversity (36, 37).
The objective of the present study was to determine the effect of
adding a hydrocarbon fuel on nitrification and nitrifying bacteria in
an uncontaminated agricultural soil and in a soil with a long history
of pollution. We present evidence that nitrifying bacteria in polluted
soil were characterized by a lower affinity for hydrocarbons and that
the apparent inhibition of nitrification observed in the presence of
hydrocarbons resulted not from a competitive effect but from
immobilization of nitrogen in the microbial biomass.
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MATERIALS AND METHODS |
Soil samples.
Two loamy soils were used in this study. A
soil that was contaminated with diesel fuel was obtained from an
abandoned area of a petroleum refinery. This soil was characterized by
absence of vegetation and a management program. The operation at the
refinery ceased 12 years ago. The other soil was obtained from a
unfertilized agricultural plot that had been planted with ryegrass for
at least 3 years. All of the inorganic nitrogen in this soil was
derived from mineralization of organic nitrogen. Soil samples were
collected aseptically from the upper 20 cm and were stored at 4°C.
Before use, they were sieved (mesh size, 2 mm) and kept at room
temperature for 24 h. Characteristics of the soils are summarized
in Table 1.
Incubation experiment.
Laboratory microcosms were used for
the incubation experiment. Each sample was separated into 500-g (dry
weight) portions, and each portion was transferred to a sterile
2,000-ml Mason jar capped with a lid fitted with a rubber septum.
Triplicate jars were prepared for each treatment. The microcosms were
incubated for 4 weeks at 28°C in the dark and were aerated weekly to
maintain aerobic conditions. The mineral N content was determined each week. Total mineralized carbon contents were determined by using sterile 500-ml Mason jars. Triplicate jars (50 g [dry weight] of
soil/jar) were used for each soil treatment. Total mineralized carbon
contents were measured weekly by titrating the CO2 trapped during incubation in NaOH (25). All water-soluble substrates were dissolved in a volume of water sufficient to adjust the water content of the soil to 20% on the basis of wet weight. Substrates were
neutralized with NaOH, if necessary, before they were added. All
substrates were added to samples by spraying them with a 2-ml syringe.
Ammonium was added in the form of
(NH4)2SO4, and 150 µg of P/g (dry
weight) of soil was added as KH2PO4 to prevent any limitation of activity by nutriment imbalance. Diesel fuel (density
at 15°C, 0.83 kg/liter) was purchased from Petro-FINA Belgium.
Nitrapyrin (90% pure) was obtained from Sigma Chemical, was dissolved
in 100 mM dimethyl sulfoxide at a concentration of 0.5% (wt/wt), and
was added at a concentration of 10 µg/g of soil. To obtain maximal
inhibition of nitrification, the nitrapyrin solution was mixed with the
ammonium salt solution before it was added to the soil.
Analytical procedures.
Exchangeable ammonium, nitrite, and
nitrate contents were determined after soil samples were extracted with
1 M KCl (1:5, wt/vol) for 2 h by using a Tecator Aquatec model
5400 autoanalyzer with a detection level of 0.1 ppm of N for all three
compounds. Mineral nitrogen was quantified colorimetrically by the
indophenol (ammonium) and cadmium reduction (nitrate) methods
(16).
Petroleum hydrocarbon in the polluted soil was extracted with carbon
tetrachloride and was analyzed quantitatively by infrared
spectrophotometry and qualitatively with a gas chromatograph (GC)
equipped with a flame ionization detector (FID) (
29).
Enumeration of nitrifying bacteria.
Ammonia- and
nitrite-oxidizing bacteria were enumerated by a most-probable-number
(MPN) procedure (30). Suspensions of 5.0 g of moist
soil and 45 ml of sterile phosphate buffer containing 139 mg of
K2HPO4 per liter and 27 mg of
KH2PO4 per liter (pH 7.0) were shaken at 100 rpm for 2 h. Subsamples of the suspensions were diluted in sterile
microtiter plates containing the appropriate medium for the ammonium-
and nitrite-oxidizing bacteria (42). Twelve replicates were
made per dilution. Samples were incubated for a maximum of 3 months at
28°C in the dark. The number of nitrifying bacteria was determined
with Cochran's tables (10) after detection of
NO2
with the Griess reagent (33).
DNA extraction.
DNA was extracted from 0.5-g portions of
soil in 2-ml microcentrifuge tubes containing 0.5 ml of 100 mM
phosphate buffer (pH 8), 0.5 ml of a 10% sodium dodecyl sulfate
solution (100 mM NaCl, 500 mM Tris [pH 8], 10% sodium dodecyl
sulfate), and 2.5 g of 0.1-mm-diameter zirconia beads. The tubes
were shaken at 2,000 rpm for 20 min in a bead mill homogenizer. The
supernatant was recovered by centrifugation for 5 min at
12,000 × g and was extracted with a phenol-chloroform
mixture (3). After ethanol precipitation, the DNA was
resuspended in 100 µl of TE (10 mM Tris, 0.1 mM EDTA).
PCR amplification.
Crude DNA was purified by gel filtration
on Sephadex G-200 (39). PCR amplification of 16S ribosomal
DNA fragments was carried out by using the CTO primers specific for
ammonia oxidizers belonging to the 
subgroup of the class
Proteobacteria (19) and primer FGPS specific for
the genus Nitrobacter (12). Each reaction mixture
(total volume, 50 µl) was prepared as recommended by the manufacturer
by using 2.5 U of Expand High Fidelity polymerase (Boehringer
Mannheim). To minimize amplification inhibition in the PCR, 400 ng of
bovine serum albumin per µl was added to the PCR mixture
(20). The thermal cycle included an initial denaturation step consisting of 94°C for 120 s, followed by 35 cycles
consisting of denaturation at 94°C for 30 s, annealing at 57°C
with the CTO primers and at 50°C with FGPS for 30 s, and
elongation at 68°C for 60 s. The cycle was completed by a final
elongation step consisting of 72°C for 5 min.
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RESULTS AND DISCUSSION |
Hydrocarbon in the polluted soil.
The concentrations of total
petroleum hydrocarbons, as estimated by infrared spectroscopy
analysis, ranged from 2,500 to 4,000 µg/g of soil (Table 1).
The large differences in the total petroleum hydrocarbon concentrations
in samples resulted from the heterogeneous distribution of petroleum
hydrocarbons in the soil. A GC-FID analysis revealed that polyaromatic
hydrocarbons and aliphatic hydrocarbons (C11 to
C19) were the dominant pollutants (data not shown).
Several reports have documented that mineralization (conversion to
CO
2) of the hydrocarbons in polluted soils is enhanced
by
adding mineral N (
2,
43,
44). With our sample of polluted
soil this effect was observed in the presence of an extra dose
of
diesel fuel but not in the initial soil sample (Fig.
1). The
lack of enhancement of
mineralization of the indigenous hydrocarbons
could have been due
to restricted access of microorganisms to
hydrocarbons that were
adsorbed or trapped in microaggregates
and/or were not easy to
mineralize (
18).
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FIG. 1.
Cumulative CO2 evolution during incubation
of polluted soil amended with 300 µg of
NH4+ N/g (dry weight) of soil ( ), 4,000 µg
of diesel fuel/g (dry weight) of soil ( ), or 300 µg of
NH4+ N/g (dry weight) of soil plus 4,000 µg
of diesel fuel/g (dry weight) of soil ( ) and unamended polluted soil
( ). Data are averages based on three replicates; the error bars are
smaller than the symbols.
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Nitrogen in polluted soil.
The levels of
NH4+ N and NO3 N
extracted with KCl were below the limit of detection (0.5 µg/g) in
the polluted soil (Table 1). This situation did not evolve during the
4-week incubation period (data not shown). The total-N content of this
soil was also very low (0.05%) (Table 1). The low N content was
probably due to the absence of any N management of the abandoned area
which was the source of the soil.
In the agricultural soil, the concentration of nitrate increased from
6.5 to 15 µg of NO
3 N/g (dry weight) of
soil after 4 weeks of incubation. However,
the concentration of
ammonium did not change. This showed that
nitrification in this soil
depended on mineralization of organic
nitrogen. Generally, the
populations and in situ activities of
nitrifiers may be limited by the
rate of production of ammonium
during mineralization of organic
nitrogen. This can easily be
demonstrated in many soils by adding
ammonium and observing stimulation
of growth of the nitrifier
population (
5). In contrast, the
contaminated soil was
characterized by low total-N contents and
no accumulation of any form
of inorganic nitrogen. This probably
resulted from the absence of
organic nitrogen available for
mineralization.
Nitrifiers in polluted soil.
Ammonia-oxidizing bacteria
belonging to the subgroup of the Proteobacteria and
nitrite-oxidizing bacteria belonging to the genus
Nitrobacter are the principal autotrophic nitrifiers that have been found in soils (7, 36). Use of PCR amplification primers CTO and FGPS, which are specific for the 16S rRNA genes of
ammonia-oxidizing bacteria (19) and nitrite-oxidizing
bacteria (12), respectively, with DNA extracted from the
polluted and agricultural soils produced the expected amplification
products (465-bp product for ammonia-oxidizing bacteria and 397-bp
product for nitrite-oxidizing bacteria) (Fig.
2, lanes 1 and 3). A PCR analysis of
serial dilutions of DNA extracted from both soils revealed that the
lower detection limit for ammonia-oxidizing bacteria and
nitrite-oxidizing bacteria was approximately 10 times higher in the
polluted soil than in the agricultural soil (Fig. 2). This result
supports the finding that there was a clear quantitative distinction
between the MPN values for nitrifying bacteria in polluted and
agricultural soils (Fig. 3). The lower
value obtained by the MPN procedure showed that the PCR product
obtained with DNA extracted from polluted soil was derived from living
nitrifiers and not from dead cells or DNA adsorbed to soil particles
(1, 21). Moreover, it demonstrated that this group of
bacteria persisted in hydrocarbon-polluted soil for several years in
the absence of an energy supply (ammonium and nitrite) and in the
presence of the polluting hydrocarbons.
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FIG. 2.
Agarose gel analysis of 16S ribosomal DNA from
agricultural and polluted soils amplified with primers CTO (A) and FGPS
(B). Lanes 1 through 3, DNA from agricultural soil; lanes 4 through 6, DNA from polluted soil; lane 7, positive control (N. europaea [A] or Nitrobacter winogradskyi [B]).
Lanes 1 and 4 contained undiluted soil DNA; lanes 2 and 5 contained
soil DNA diluted 1:10; lanes 3 and 6 contained soil DNA diluted 1:100;
and lane M contained a 1-kb DNA ladder size standard (Gibco BRL).
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FIG. 3.
MPN of ammonia-oxidizing bacteria (AOB) and
nitrite-oxidizing bacteria (NOB) in agricultural soil (open bars) and
polluted soil (cross-hatched bars). The error bars indicate the 95%
confidence limits.
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Effect of ammonium addition.
Addition of 300 µg of
NH4+ N/g (dry weight) of soil to the polluted
soil was followed by a large and rapid increase in the mineral nitrate
content of the soil, which was about 240 µg of NO3 N/g (dry weight) of soil after 3 weeks of
incubation (Fig. 4A). The concomitant
decrease in the ammonium concentration attained 95% of the initial
value. In contrast, the rate of nitrification of 300 µg of
NH4+ N/g (dry weight) of soil added to an
agricultural soil was apparently slower (Fig.
5). Previous laboratory studies have
shown that the rate of nitrification in agricultural soils after
ammonium sulfate is added depends on the soil properties and that the
inhibitory effect of ammonium on nitrification occurs at concentrations
of >300 µg of N/g (23, 24, 38). The dominant factors that
control the rates of nitrification in many soils are (i) the supply of NH4+ substrate, (ii) the acidity, (iii) the
water content, and (iv) the temperature (8, 28). These
factors were equivalent or optimal for nitrification in the two soils
used in this investigation. This suggests that the observed difference
in the rates of nitrification for the two soils resulted from a
qualitative difference in the populations of nitrifying bacteria.
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FIG. 4.
Changes in the concentrations of ammonium (), nitrate
(), and nitrite ( ) during incubation of polluted soil amended
with 300 µg of NH4+ N/g (dry weight) of soil
(A) or 4,000 µg of diesel fuel/g (dry weight) of soil plus 300 µg
of NH4+ N/g (dry weight) of soil (B). dw, dry
weight.
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FIG. 5.
Changes in nitrate ( and ) and ammonium ( and
) concentrations in agricultural soil amended with 300 µg of
NH4+ N/g (dry weight) of soil in presence ( and ) and in the absence ( and ) of 4,000 µg of diesel
fuel/g (dry weight) of soil. The error bars are smaller than the
symbols. dw, dry weight.
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In the agricultural soil, the disappearance of ammonium reflected the
appearance of nitrate (Fig.
5). In the polluted soil,
the ammonium
concentration was reduced to 30% of its initial value
during the first
week, but the nitrate concentration represented
only 30% of the
observed value after 3 weeks of incubation (Fig.
4A). During the first
week, most of the ammonium consumed was
apparently in the form of
nitrification intermediates other than
nitrite, and the transitory
amount of nitrite which accumulated
was very small (Fig.
4A). The high
rate of ammonium consumption
in the first week suggested that the first
key enzyme in nitrification,
ammonia monooxygenase, is not a limiting
factor. Thus, the ammonia-oxidizing
bacteria in polluted soil seem to
be dominated by a small number
of bacteria with a high affinity for
ammonium.
Effect of a nitrification inhibitor.
Previous laboratory
studies showed that nitrapyrin was an effective nitrification inhibitor
and that the degrees of inhibition were different in different soils
(4, 9) and varied with the genus and strain of
ammonia-oxidizing bacteria (6). In the agricultural soil,
production of nitrate and disappearance of ammonium were not observed
even after 50 days of incubation (Fig. 6)
in the presence of the nitrification inhibitor nitrapyrin (10 µg/g
[dry weight]) (9). In contrast, in the polluted soil, nitrification and ammonium consumption started after a lag period of
about 3 weeks. The nitrapyrin was completely consumed after 3 weeks in
the polluted soil, as shown by the GC-FID analysis (data not shown).
Degradation of nitrapyrin and degradation of the intermediates (also
inhibitors of nitrification [4]) in polluted soil
could have been due to the action of the nitrifying bacteria, as
observed in vitro (41). The rapid degradation of nitrapyrin
in the polluted soil but not in the agricultural soil could have been
due to the high specific activity of nitrifying bacteria in the
polluted soil.
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FIG. 6.
Effects of nitrapyrin (10 µg/g [dry weight] of soil)
on the concentrations of nitrate ( and ) and ammonium ( and
) in agricultural soil ( and ) and polluted soil ( and )
amended with 300 µg of NH4+ N/g (dry weight)
of soil. dw, dry weight.
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The complete inhibition of nitrification by nitrapyrin in the two soils
examined during the first few weeks of incubation
suggested that the
contribution of heterotrophic nitrification
to nitrate production was
insignificant. In fact, nitrapyrin is
a specific inhibitor of
chemoautotrophic ammonia oxidation but
not heterotrophic ammonia
oxidation (
11,
34) and has been
used as a differential
inhibitor in environmental studies (
32).
Moreover, the soils
which we used had neutral pH values, and contributions
to nitrate
production by organotrophic microorganisms have been
observed only in
acid soils that are rich in organic matter (
17,
31,
32).
Effect of ammonium and diesel fuel.
In order to compare the
effects of a hydrocarbon on nitrification in the contaminated and
uncontaminated soils, both soils were amended with 4,000 µg of diesel
fuel per g and 300 µg of NH4+ N/g (dry
weight) of soil. This was done because the hydrocarbons present in the
contaminated soil were apparently not accessible to microorganisms and
had no effect on nitrification.
Addition of a hydrocarbon to the agricultural soil resulted in a lower
increase in nitrate production compared to the same
soil when diesel
fuel was not added; the evolution of ammonium
was apparently not
affected by the diesel fuel addition (Fig.
5). The decrease in the
ammonium concentration in the presence
of the hydrocarbon resulted in
large part from immobilization
by incorporation into organic components
(i.e., microbially synthesized
amino acids and proteins)
(
14). Consequently, nitrification
was directly affected by
the hydrocarbon addition, so that decreases
in denitrification were not
expected during this incubation
period.
When the previously polluted soil was supplemented with an extra dose
of diesel fuel (Fig.
4B), the ammonium concentration
was apparently not
affected. Nevertheless, the plateau concentration
of nitrate was 30%
lower than the concentration observed in the
absence of extra added
hydrocarbon (Fig.
4A). The total amount
of ammonium consumed after 4 weeks suggested that the low level
of nitrate production in the
polluted soil and in the presence
of an extra dose of diesel fuel was
mainly due to immobilization
of nitrogen by heterotrophic bacteria
rather than to inhibition
of nitrifying bacteria by hydrocarbons. Under
the conditions which
we used, the availability of ammonium to
nitrifying bacteria appeared
to be a limiting factor for
nitrification.
In order to check that the apparent inhibition of nitrification
observed when polluted soil was exposed to diesel fuel was
the result
of immobilization of NH
4+ N by
hydrocarbon-stimulated microbial activity, we compared the
effects of
hydrocarbons and glucose on the amounts of ammonium
and nitrate
detected after incubation of soil treated with 300
µg of
NH
4+ N/g (Fig.
7). After incubation, the KCl-extracted
ammonium values
for all treatments were comparable to the value
observed in the
presence of ammonium alone. This result showed that
hydrocarbons
were as effective as glucose for inducing immobilization
of NH
4+ N. The conclusion that nitrogen was
immobilized by heterotrophic
bacteria stimulated by addition of a
carbon source was confirmed
by the amounts of DNA extracted from the
polluted soil amended
with both ammonium and diesel fuel, which were
higher than the
amounts extracted from the polluted soil amended with
diesel fuel
alone or with ammonium alone (Fig.
8). In fact, several studies
have shown
that changes in the DNA contents of environmental samples
corresponded
to changes in the population densities determined
by using direct count
epifluorescence microscopy (
22,
35).
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FIG. 7.
Effects of different hydrocarbons and glucose on the
amounts of NO3 N after 4 weeks of incubation
of polluted soil treated with 300 µg of NH4+
N/g (dry weight) of soil.
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FIG. 8.
Agarose gel electrophoresis of DNA extracted from
polluted soil after 4 weeks of incubation. Lane 1, untreated soil; lane
2, soil treated with 300 µg of NH4+ N/g (dry
weight) of soil; lane 3, soil treated with 4,000 µg of diesel fuel/g
(dry weight) of soil; lane 4, soil treated with 300 µg of
NH4+ N/g (dry weight) of soil and 4,000 µg of
diesel fuel/g (dry weight) of soil; lane M, DNA molecular weight marker
II (Boehringer Mannheim).
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The observation that a hydrocarbon did not have a direct effect on the
activity of nitrifying bacteria could be explained
by the high affinity
of ammonia-oxidizing bacteria for ammonium
in the polluted soil. This
apparently did not occur in the agricultural
soil, in which a
significant amount of ammonium was always present
(Fig.
5).
Conclusions.
Addition of hydrocarbon to an uncontaminated soil
stimulated immobilization of nitrogen and reduced nitrification. In the absence of a bioremediation program (nutriment addition), the N
immobilized in situ was derived from mineralization of the available organic nitrogen. In the case of long-term pollution and in the absence
of a bioremediation program, the soil was excessively poor in nitrogen.
Perhaps the same situation occurred in the contaminated soil used in
this study, which had a long history of pollution, because initially
this soil had a low total-N content and a small population of
nitrifying bacteria. Apparently, nitrifying bacteria persisted in
contaminated soil containing a limited amount of ammonium for several
years. These conditions reduced the number of nitrifying bacteria but
probably selected an ammonia-oxidizing community with a higher ammonium
affinity and a higher activity than the community in the agricultural
soil. The ammonia-oxidizing bacterial community in contaminated soil
was not directly affected by a hydrocarbon addition, possibly because
of its high affinity for ammonium.
A contaminated soil is a nitrogen-limited environment, and the
adaptations which we observed may be very important for nitrifier
survival in a ammonium-limited soil. Whether these adaptations
are due
to physiological plasticity or to the presence of strains
specialized
for living in an ammonium-limited and hydrocarbon-polluted
soil will be
examined by using the new molecular techniques for
studying the
diversity of these organisms (
19).
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ACKNOWLEDGMENTS |
J.D. thanks the David & Alice Van Buuren Foundation and M.J.P.
thanks the Federal Ministry of Agriculture of Belgium for financial support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Physiologie et Ecologie Microbiennes, Section Interfacultaire
d'Agronomie, Université Libre de Bruxelles c/o Institute
Pasteur, Rue Engeland 642, B-1180, Brussels, Belgium. Phone: 32 2 373 33 03. Fax: 32 2 3733309. E-mail:
upemulb{at}resulb.ulb.ac.be.
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Applied and Environmental Microbiology, September 1999, p. 4008-4013, Vol. 65, No. 9
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Abstract
Introduction
