Previous Article | Next Article 
Applied and Environmental Microbiology, July 2001, p. 2952-2957, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.2952-2957.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Autotrophic Ammonia Oxidation at Low pH through Urea
Hydrolysis
Simon A. Q.
Burton
and
Jim I.
Prosser*
Department of Molecular and Cell Biology,
Institute of Medical Sciences, University of Aberdeen,
Foresterhill, Aberdeen AB25 2ZD, United Kingdom
Received 19 January 2001/Accepted 15 April 2001
 |
ABSTRACT |
Ammonia oxidation in laboratory liquid batch cultures of
autotrophic ammonia oxidizers rarely occurs at pH values less than 7, due to ionization of ammonia and the requirement for ammonium transport
rather than diffusion of ammonia. Nevertheless, there is strong
evidence for autotrophic nitrification in acid soils, which may be
carried out by ammonia oxidizers capable of using urea as a source of
ammonia. To determine the mechanism of urea-linked ammonia oxidation, a
ureolytic autotrophic ammonia oxidizer, Nitrosospira sp.
strain NPAV, was grown in liquid batch culture at a range of pH values
with either ammonium or urea as the sole nitrogen source. Growth and
nitrite production from ammonium did not occur at pH values below 7. Growth on urea occurred at pH values in the range 4 to 7.5 but ceased
when urea hydrolysis was complete, even though ammonia, released during
urea hydrolysis, remained in the medium. The results support a
mechanism whereby urea enters the cells by diffusion and intracellular
urea hydrolysis and ammonia oxidation occur independently of
extracellular pH in the range 4 to 7.5. A proportion of the ammonia
produced during this process diffuses from the cell and is not
subsequently available for growth if the extracellular pH is less than
7. Ureolysis therefore provides a mechanism for nitrification in acid
soils, but a proportion of the ammonium produced is likely to be
released from the cell and may be used by other soil organisms.
| |
INTRODUCTION |
Growth of pure cultures of
autotrophic ammonia-oxidizing bacteria in liquid culture is optimal
within the pH range 7.0 to 8.5 and typically does not occur below a pH
of 6.5 (3). Nitrification has, however, been reported in
acid soils at pH values as low as 3.7 (5, 9, 11, 25, 26, 34, 35,
37). Although this may be due, in some cases, to heterotrophic
nitrification (1, 22) there is strong evidence for
autotrophic nitrification in acid soils (19, 38).
Autotrophic ammonia oxidizers have been isolated from and enumerated in
acid soils (7, 13, 16, 20, 26) and 16S rRNA gene sequences
of ammonia oxidizers are frequently detected (6, 33), with
evidence for changes in relative abundance of different sequence
clusters in soils of different pH (32). However, pure
cultures often fail to nitrify at the bulk pH value of the soil from
which they were isolated (1). Pure cultures of acidophilic
nitrite oxidizers have been isolated (8, 14), but there is
only a single report of slow-growing acidophilic ammonia oxidizers from
fertilized acid tea soils, capable of growing at low pH in mineral
medium containing 1,000 mg of NH4+-N
liter
1 (15). Acid-sensitive ammonia
oxidizers may be responsible for nitrification in acid soils, since
fertilization can lead to an increase in the number of autotrophic
nitrifiers (23). However, this increase in number is often
associated with the addition of compounds that may increase pH, such as
urea or carbonate, and is consistent with the view that autotrophic
nitrification may be restricted to microsites of alkaline pH
(29). The localized pH inhibition of autotrophic
nitrification may also be relieved through the buffering capacity of
the soil in which the oxidizers are found (4), but in
laboratory culture, growth at low pH has only been observed in biofilms
(3, 17) or agglomerates (10).
An alternate explanation for nitrification in acid soils is the ability
of many ammonia oxidizers to hydrolyze urea (1, 21, 36).
Urea has been found to stimulate autotrophic nitrification in Dutch
acid heathland and forest soils (9, 11), and stimulation was shown to be independent of an increase in pH. The isolation of a
ureolytic ammonia oxidizer belonging to the genus
Nitrosospira from this soil, and its subsequent growth in
liquid medium containing urea with an acidophilic nitrite oxidizer,
resulted in autotrophic nitrification at pH 4.5 (8).
Growth of this strain on ammonium sulfate was not observed below pH
5.5.
The mechanism for acidophilic growth of ureolytic ammonia oxidizers is
not clear. A wide range of nonnitrifying bacteria possess urease
activity (24), and ammonia released by such organisms will
provide a substrate for ammonia oxidizers and will also, potentially,
increase local pH. Similarly, ureolytic ammonia oxidizers would have to
compete with heterotrophs for the ammonia released into the
environment. This suggests that ammonia produced from urea must be
retained within the cell to provide an ecological advantage.
The aim of this study was to assess urea hydrolysis followed by ammonia
oxidation as a mechanism for autotrophic nitrification in acid soil.
This was achieved by determining the influence of pH on the kinetics of
urea hydrolysis and ammonia oxidation in liquid culture by a ureolytic
strain of Nitrosospira that is unable to grow on
ammonium at pH values less than 7.
| |
MATERIALS AND METHODS |
Bacterial strains and cultural conditions.
A ureolytic
autotrophic ammonia oxidizer, Nitrosospira sp. strain NPAV,
originally isolated from soil, was supplied by E. Schmidt, University
of Minnesota. Routine growth and maintenance were carried out as
described by Keen and Prosser (18) in Skinner and Walker
(31) (SW) medium as modified by Powell and Prosser (27) and containing 50 µg NH4+-N
ml1 as ammonium sulfate. An acidophilic nitrite oxidizer,
Nitrobacter sp. NHBI, was supplied by W. de Boer, Institute
for Ecological Research, Heteren, The Netherlands. This strain was
subcultured as for Nitrosospira sp. strain NPAV but in
Schmidt and Belser (30) (SB) medium containing 50 µg
NO2-N ml1 and adjusted to pH 7 by addition of 10 M NaOH prior to autoclaving. The absence of
contamination by heterotrophs was checked by plating on nutrient agar
(Oxoid) and incubation at 25°C for at least 21 days. Cultures were
also examined microscopically for the presence of contaminants. Growth
was assessed as the disappearance of ammonia and production of acid and
nitrite (Nitrosospira) and as disappearance of nitrite
(Nitrobacter) using spot tests for ammonia (Nessler's reagent) and nitrite (Griess llosvay's reagents 1 and 2). Acid production was detected by inclusion of the pH indicator phenol red in media.
Influence of pH on growth on ammonia and urea in poorly buffered
medium.
The effect of pH on growth of Nitrosospira sp.
strain NPAV was determined in batch cultures prepared using 250-ml
Erlenmeyer flasks containing 100 ml of SB medium with either 23.3 µg
of NH4+-N ml1 or 23.3 µg of
urea-N ml1. After autoclaving at 121°C for 20 min, the
pH of the medium was adjusted to 4, 5, 6, 7, or 7.5 by aseptic addition
of sterile 1 M NaOH or 1 M H2SO4, as
appropriate. Medium was then inoculated with 3 ml of a stationary-phase
culture of Nitrosospira sp. strain NPAV, grown in the same
medium at pH 7. Cocultures of Nitrosospira sp. strain NPAV
and Nitrobacter sp. strain NHB1 were also prepared by
inoculation of 3 ml of stationary phase cultures of each strain.
Cultures were incubated at 25°C in the dark without shaking, although
a number of control cultures were incubated shaken to check for oxygen
limitation. No difference was observed in growth kinetics of shaken or
static cultures under these conditions. Samples (1 to 2 ml) were
removed for measurement of pH (Russell pH electrode) and assessment of
growth and were preserved at 4°C following addition of 10 µl of a
1% (wt/vol) solution of potassium ethyl xanthate, an inhibitor of both
ammonia- and nitrite-oxidizing bacteria. Growth was assessed by
analysis for urea, ammonia, nitrite, and nitrate, as required, using an
RFA Autoanalyser system (Alpkem Corporation, Clackamas, Oreg.).
Cultures were checked for purity at 1- to 2-day intervals as described above.
Growth on urea and ammonium at constant pH.
Combined growth
of Nitrosospira sp. strain NPAV and Nitrobacter
sp. strain NHB1 under controlled pH conditions was studied in a modular
fermentor (Gallenkamp, Loughborough, United Kingdom) consisting of a
1.3-liter-glass vessel with an operating volume of 1 liter. The pH of
the medium was controlled by automated addition of 0.02 M NaOH or 0.1 M
H2SO4. Silicone rubber tubing was used throughout, and the vessel was covered with aluminium foil to prevent
photoinhibition. The culture vessel and components were autoclaved at
121°C for 20 min with SB medium containing 23.3 µg of
NH4+-N ml1 or nitrogen-free
medium, after which filter-sterilized urea was dispensed by passage
through a 0.2-µm-pore-size Millipore filter to give a final
concentration of 23.3 µg of urea-N ml1. Agitation was
achieved using a magnetic impeller (120 rpm), and the temperature was
maintained at 25°C. Filter-sterilized, humidified air was delivered
by a HyFlo model C air pump (Medcalf Bros. Ltd., Potters Bar, England)
at a rate of 500 ml min1. Medium was inoculated with 27 ml of stationary phase cultures of Nitrosospira sp. strain
NPAV and Nitrobacter sp. strain NHB1, and purity and growth
were assessed in samples as described above.
Growth of
Nitrosospira in medium containing ammonia and urea
was also assessed in SW medium buffered with 50 mM
2(
n-morpholino)ethane
sulphonic acid (MES) (Sigma) and
adjusted to pH 6.2 with 10 M
NaOH and concentrated HCI prior to
autoclaving. Specific rates
of product formation were calculated by
linear regression of linear
regions of semilogarithmic plots of product
concentration versus
time. All experiments and treatments were repeated
at least three
times. Means and standard errors were calculated from
triplicates.
Graphical data are from a single replicate typical of a
particular
treatment.
| |
RESULTS |
Growth on ammonia in poorly buffered medium.
Batch growth of
Nitrosospira sp. strain NPAV occurred in SB medium
containing ammonium at initial pH values of 7 and 7.5 (Fig. 1) but not at pH 6 or lower. SB medium is
poorly buffered, and complete conversion of ammonium to nitrite was
prevented by production of nitrous acid and consequent pH reduction to
<6. Variation in the lag phase was seen between replicates, but when
growth commenced, rates of change in ammonia, nitrite, and pH were
similar in triplicate cultures. Initial ammonium concentration was
lower in medium adjusted to the higher initial pH value, due to greater
volatilization of ammonia prior to inoculation. Ammonium limitation
will not have occurred, however, as concentrations were always
significantly greater than reported saturation constants for ammonia
oxidizers (28). Growth from initial pH values of 7.5 and 7 led to production of similar amounts of nitrite (5.31 and 5.35 µg
NO2-N ml1, respectively [Table
1]) and the final pH values were not
significantly different (Student's t test, P = 0.10). The pH of the medium fell below the minimum pH for growth
and rose slightly following cessation of nitrite oxidation.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 1.
Changes in ammonium and nitrite concentrations and pH
during growth of Nitrosospira sp. strain NPAV in liquid
batch culture on poorly buffered medium containing ammonium, at initial
pH values of 7 (a) and 7.5 (b).
|
|
To determine whether growth was inhibited by accumulation of nitrite,
the above experiments were repeated with a coculture
of
Nitrosospira sp. strain NPAV and
Nitrobacter sp.
strain NHB1,
an acidophilic nitrite oxidizer. Growth of the coculture
occurred
only at initial pH values of 7 and 7.5. At pH 7, no nitrite
was
detected and nitrate was the sole oxidation product. At pH 7.5,
nitrite accumulated during the first 260 h before decreasing below
the level of detection following conversion to nitrate. There
was no
evidence of nitrite toxicity. At pH 7, nitrite produced
by monocultures
of
Nitrosospira was greater than nitrite and nitrate
produced during coculture with
Nitrobacter (Student's
t test,
P = 0.003), while at pH 7.5, final
product concentrations were
not significantly different (
P = 0.42) (Table
1). Despite this,
the final pH values were
significantly lower in cocultures of
Nitrosospira and
Nitrobacter, possibly due to greater ionization
of nitric
acid in the weakly buffered
medium.
Growth on urea in poorly buffered medium.
Growth of
Nitrosospira sp. strain NPAV in SB medium containing urea
occurred at all pH values tested in the range 4 to 7.5. No urea
hydrolysis was detected in uninoculated controls at the same initial pH
values. Lag phases prior to growth were much shorter than those
observed during growth on ammonium and were often not detectable. In
addition, standard errors associated with specific rates of product
formation during growth on urea were much lower than those during
growth on ammonium (Table 1). Following inoculation of medium at an
initial pH value of 4, urea hydrolysis was accompanied by an
exponential increase in ammonium and nitrite concentrations (Fig. 2a),
but no further increases were observed following completion of urea
hydrolysis at approximately 180 h. The pH increased to 4.7 during
initial production of ammonium from urea and decreased to 4.2 as
ammonia oxidation proceeded. Following cessation of urea hydrolysis and
ammonia oxidation, the pH increased to a final value of 5.4. Thus,
ammonia oxidation to nitrite occurred at pH values below 4.7 but ceased
when urea was fully utilized, even though ammonium was present in the
medium. The final ammonium and nitrite concentrations were 16.4 and
13.5 µg of N ml1, respectively, representing conversion
of approximately 45% of supplied urea-N to nitrite-N. The total final
N concentration was greater than that initially supplied due to water
loss during autoclaving and to evaporation during long-term incubation.
At an initial pH of 5, urea hydrolysis was again associated with
initial exponential increases in ammonia and nitrite concentrations
(Fig.
2b). Ammonia oxidation to nitrite
ceased when urea was fully
utilized and the final ammonium
concentration was similar to that
following growth at pH 4 (Table
1).
The pH did not increase during
initial production of ammonium from urea
but decreased during
nitrite formation, as observed above. Following
completion of
urea hydrolysis, the pH increased to a final value of
5.5, and
approximately 49% of urea-N supplied was converted to
nitrite-N.
Similar changes in urea, ammonium, and nitrite
concentrations
and in pH were seen during growth from an initial pH
value of
6 (data not shown), and final nitrite concentrations and pH
values
were similar to those in cultures with initial pH values of 4
and 5 (Table
1).
View larger version (36K):
[in this window]
[in a new window]
|
FIG. 2.
Changes in urea, ammonium, and nitrite concentrations
and pH during growth of Nitrosospira sp. strain NPAV in
liquid batch culture on poorly buffered medium containing urea, at
initial pH values of 4 (a), 5 (b), 7 (c), and 7.5 (d).
|
|
Growth in medium at an initial pH of 7 showed different behavior
because of the ability of
Nitrosospira to oxidize ammonium
directly at this pH. Urea hydrolysis was accompanied, as at pH
values 4 to 6, by increases in ammonium and nitrite concentrations
(Fig.
2c).
Ammonia concentration decreased, however, for a short
period after
exhaustion of urea and led to further increase in
nitrite
concentration. This was accompanied by a reduction in
pH, which
eventually inhibited ammonia oxidation and nitrite production.
The
final ammonium concentration was lower than that at acid pH
values. The
final nitrite concentration, although greater than
those at initial pH
values of 4 and 6, was not significantly different
from that at pH 5 (Table
1), and approximately 62% of urea-N
was converted to nitrite-N.
The final pH was similar to those
in cultures grown from lower initial
pH
values.
Growth from an initial pH of 7.5 led to ammonia production at the same
rate as at lower initial pH values, but subsequent
conversion to
nitrite was slower (Fig.
2d). Significant ammonia
oxidation occurred
after exhaustion of urea, and final ammonia
and nitrite concentrations
were lower and higher, respectively,
than those at acidic initial pH
values. Reduction in pH eventually
inhibited ammonia oxidation, and the
final pH of 5.67 was not
significantly different from that at lower
initial pH values (Table
1).
Despite variation in pH during growth, nitrite concentration increased
exponentially over significant periods during growth
on urea, enabling
calculation of specific rates of nitrite production
at different
initial pH values (Table
1). Under these conditions,
the specific rate
of nitrite production is equivalent to specific
growth rate during
exponential growth (
27). Specific rates of
nitrite
production from urea showed little variation with initial
pH values in
the range 4 to 6, reflecting the fact that most nitrite
production
occurred when the pH was approximately 5.5 in these
cultures. The rate
increased in cultures with an initial pH of
7, but the lowest value was
obtained for cultures at pH 7.5, reflecting
the low rate of ammonium
reduction referred to above. At pH values
greater than or equal to 7, the nitrite oxidation rate reflects
both rates of urea hydrolysis and
oxidation of extracellular ammonium.
At all initial pH values studied,
final nitrite concentrations
were greater than those in cultures grown
on ammonium. Growth
in the presence of
Nitrobacter sp.
strain NHB1 did not significantly
affect product formation (Table
1),
suggesting a lack of nitrite
inhibition.
Growth on urea and ammonium at constant pH.
Growth on
Nitrosospira sp. strain NPAV in coculture with
Nitrobacter sp. strain NHB1 was studied in a pH-controlled
fermentor at pH values of 5, 7, and 8, with urea as the substrate, and
at 5, 6, 7, and 7.5, with ammonium as the substrate. At pH 5, changes in urea, ammonium, and nitrite concentrations were similar to those in
unbuffered medium (Fig. 3). Urea
hydrolysis was completed within 150 h and was accompained by
oxidation of ammonium, which ceased when urea was fully converted. A
lag in the growth of Nitrobacter led to an accumulation of
nitrite, which was subsequently oxidized completely to nitrate. At pH
7, growth on urea was followed by growth on ammonium (Fig. 3b). No
growth occurred on urea at pH 8 during incubation for 450 h, but
urea hydrolysis and ammonia oxidation commenced immediately when the pH
control was switched off and the pH was slightly reduced. With ammonium
as a substrate, nitrite was produced exponentially at pH 7 and 7.5, but
no growth occurred at pH 6 or lower.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 3.
Changes in urea, ammonium, nitrite, and nitrate
concentrations during growth of Nitrosospira sp. strain NPAV
and Nitrobacter sp. strain NHB1 in liquid batch culture on
urea in a pH-controlled fermentor at pH values of 5 (a) and 7 (b).
|
|
Growth was assessed in buffered medium at pH 6.2 on urea (Fig.
4a) and medium containing urea and
ammonium (Fig.
4b). In both
cases, growth continued on ammonium after
the urea had been utilized
but was eventually inhibited, presumably by
the low pH. Growth
in this medium on ammonium alone was not possible.
Thus, cells
actively growing on urea could oxidize free ammonium after
complete
urea hydrolysis at a pH that was inhibitory for growth on
ammonium.
While final nitrite concentrations were significantly
different,
specific nitrite production rates were not significantly
different
in the presence of urea and ammonium during exponential
product
formation (Table
1).
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 4.
Changes in urea, ammonium, and nitrite concentrations
during growth of Nitrosospira sp. strain NPAV in liquid
batch culture on buffered medium at pH 6.2 containing urea (a) or urea
and ammonium (b).
|
|
| |
DISCUSSION |
This study provides a further demonstration of the inability of
pure cultures of ammonia-oxidizing bacteria to grow on ammonium at low
pH in liquid batch culture. Growth of Nitrosospira sp. strain NPAV was possible at pH 7 but not at pH 6.2 or lower. The most
likely mechanism for inhibition of growth is ionization of NH3 to NH4+ at low pH and either
the lack of an active ammonium transport system or insufficient energy
for active transport. An alternative explanation is inhibition by
nitrite, but the pH minimum for growth was not altered in this study by
coculture with the acidophilic nitrite oxidizer, Nitrobacter
sp. strain NHB1, which removed nitrite at all pH values tested.
Although ammonia oxidation was eventually inhibited by acidification of
the medium, the final pH was lower than the pH minimum for batch
growth, suggesting that actively growing cells may be capable of
ammonia oxidation at lower pH values than stationary-phase cells.
Further evidence for this came from growth in buffered medium at pH
6.2, in which nitrite production continued for some time after complete
urea hydrolysis. In this case, nitrite formation was not exponential
without urea, and growth was dependent on the presence of urea, as
nitrite was not produced with ammonium as the sole, nitrogen source. In
unbuffered medium there was no ammonium-dependent growth below pH 7. The ability of actively growing cells to grow at pH values lower than stationary-phase cells has also been observed for
Nitrobacter in nitrite-limited continuous culture
(18) and may be of significance in natural environments in
which the activity of ammonia oxidizers is maintained by low but
continuous rates of substrate supply. The ability of this strain to
hydrolyze urea did, however, enable significant growth at pH values as
low as 4 both in the presence and absence of Nitrobacter sp.
strain NHB1.
The changes in urea, ammonia, and nitrite concentrations during growth
at low pH suggest a mechanism for urea-linked ammonia oxidation. Urea
hydrolysis led to the simultaneous appearance of ammonia and nitrite in
the growth medium. Ammonia concentration increased at a greater initial
rate than nitrite concentration, but increases in both were exponential
and are therefore likely to be linked to growth. Urea hydrolysis and
nitrite formation were tightly linked. In unbuffered medium at pH
values of 6 and below, nitrite production ceased when urea hydrolysis
was completed, even though there was sufficient ammonia present in the
medium for growth. This suggests the following mechanism for
acidophilic growth on urea. The preferred form for uptake of ammonium
is NH3, which can diffuse passively across the cell
membrane. As pH decreases, NH3 ionizes to form
NH4+, which requires active transport. There is
limited evidence for active transport of NH4+
by ammonia oxidizers (12), but even if it were possible,
active transport would require energy which the cell may not be able to
supply at a low pH. Evidence of urea transport in other microorganisms suggests uptake by simple diffusion (24). Urea can
therefore be taken up by cells at a low pH, through diffusion, and is
then hydrolyzed intracellularly to ammonia. A proportion of this
ammonia diffuses into the medium, but some remains in the cell and is oxidized to nitrite, which then also diffuses into the medium. The
ammonia released into the medium ionizes and is no longer available for
uptake and oxidation by the cell and accumulates. Growth and nitrite
production therefore cease when urea hydrolysis is completed.
A further influence of urea hydrolysis is in increasing pH through
release of ammonia. In our liquid culture experiments, the ammonia
produced was not sufficient to raise the pH above 7, even when the
initial pH of the medium was 6. This was, in part, due to acidification
of the medium by nitrous acid produced during ammonia oxidation. In
contrast, cultures at initial pH values of 7 and 7.5 showed increases
in pH value, which could be attributed to urea hydrolysis. Alkanization
by release of ammonia may contribute to ammonia oxidation in acid soils
and may provide microenvironments favorable for nitrification
(29). The difficulties in growing Nitrosospira
on urea at a pH above 7.5 in buffered or pH-controlled media suggested
that urea may be inhibitory to growth under these conditions. Rates of
urea hydrolysis were similar at initial pH values of 4.5, and 6 and
were similar to those of ammonia oxidation, suggesting that urea
hydrolysis in these situations may not limit ammonia oxidation.
This study therefore demonstrates the ability of ureolytic autotrophic
ammonia oxidizers to contribute to nitrification at pH values as low as
4 when supplied with urea, without the requirement for surface growth
or microenvironments of higher pH. This may be of importance for
nitrogen cycling in acid soils in which urea is produced, although
there is strong competition from other ureolytic microorganisms for
urea. The study also demonstrates that urea-linked ammonia oxidation at
acid pH values results in diffusion of non-oxidized ammonia from the
cell which cannot be utilized further by autotrophic ammonia oxidizers
and which may provide a supply of ammonium to other soil organisms.
| |
ACKNOWLEDGMENT |
Simon Burton acknowledges receipt of a Natural Environment
Research Council Postgraduate Studentship.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular and Cell Biology, University of Aberdeen, Institute of
Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, United Kingdom.
Phone: 44 1224 273148. Fax: 44 1224 273144. E-mail:
j.prosser{at}abdn.ac.uk.
Present address: Department of Bioscience and Biotechnology,
University of Strathclyde, Royal College Building, Glasgow GI 1XW,
United Kingdom.
| |
REFERENCES |
| 1.
|
Adams, J. A.
1986.
Identification of heterotrophic nitrification in strongly acid larch humus.
Soil Biol. Biochem.
18:339-341[CrossRef].
|
| 2.
|
Allison, S. M., and J. I. Prosser.
1991.
Urease activity in neutrophilic autotrophic ammonia-oxidising bacteria isolated from acid soils.
Soil Biol. Biochem.
23:45-51.
|
| 3.
|
Allison, S. M., and J. I. Prosser.
1993.
Ammonia oxidation at low pH by attached populations of nitrifying bacteria.
Soil Biol. Biochem.
25:935-941[CrossRef].
|
| 4.
|
Bazin, M. J.,
A. Rutili,
A. Gaines, and J. Lynch.
1991.
Humic acid relieves pH-inhibition of nitrification in continuous flow columns.
FEMS Microbiol. Ecol.
85:9-14[CrossRef].
|
| 5.
|
Boswell, J. G.
1955.
The microbiology of acid soils.
New Phytol.
54:311-319[CrossRef].
|
| 6.
|
Bruns, M. A.,
J. R. Stephen,
G. A. Kowalchuk,
J. I. Prosser, and E. A. Paul.
1999.
Comparative diversity of ammonia oxidizer 16S rRNA gene sequences in never-tilled, tilled, and successional soils, Appl.
Environ. Microbiol.
65:2994-3000.
|
| 7.
|
de Boer, W.,
H. Duyts, and H. J. Laanbroek.
1988.
Autotrophic nitrification in a fertilized heath soil.
Soil Biol. Biochem.
20:845-850[CrossRef].
|
| 8.
|
de Boer, W., and H. J. Laanbroek.
1989.
Ureolytic nitrification at low pH by Nitrosospira spec.
Arch. Microbiol.
152:178-181[CrossRef].
|
| 9.
|
de Boer, W.,
H. Duyts, and H. J. Laanbroek.
1989.
Urea stimulated autotrophic nitrification in suspensions of fertilised, acid heath soil.
Soil Biol. Biochem.
21:349-354[CrossRef].
|
| 10.
|
de Boer, W.,
P. J. A. Klein Gunnewiek,
M. Veenhuis,
E. Bock, and H. J. Laanbroek.
1991.
Nitrification at low pH by aggregated chemolithotrophic bacteria.
Appl. Environ. Microbiol.
57:3600-3604[Abstract/Free Full Text].
|
| 11.
|
de Boer, W.,
A. Tietema,
P. J. A. Klein Gunnewiek, and H. J. Laanbroek.
1992.
The autotrophic ammonium-oxidizing community in a nitrogen-saturated acid forest soil in relation to pH-dependent nitrifying activity.
Soil Biol. Biochem.
24:229-234[CrossRef].
|
| 12.
|
Glover, H. E.
1985.
The relationship between inorganic nitrogen oxidation and organic carbon production in batch and chemostat cultures of marine nitrifying bacteria.
Arch. Microbiol.
142:45-50[CrossRef].
|
| 13.
|
Hankinson, T. R., and E. L. Schmidt.
1984.
Examination of an acid forest soil for ammonia-and nitrite-oxidizing autotrophic bacteria.
Can. J. Microbiol.
30:1125-1132.
|
| 14.
|
Hankison, T. R., and E. L. Schmidt.
1988.
An acidophilic and a neutrophilic Nitrobacter strain isolated from the numerically predominant nitrite-oxidising population of an acid forest soil.
Appl. Environ. Microbiol.
54:1536-1540[Abstract/Free Full Text].
|
| 15.
|
Hayatsu, M.
1993.
The lowest limit of pH for nitrification in tea soil and isolation of an acidophilic ammonia oxidising bacterium.
Soil Sci. Plant Nutro.
39:219-226.
|
| 16.
|
Jiang, Q. Q., and L. R. Bakken.
1999.
Comparison of Nitrosospira strains isolated from terrestrial environments.
FEMS Microbiol. Ecol.
30:171-186[CrossRef][Medline].
|
| 17.
|
Keen, G. A., and J. I. Prosser.
1987.
Interrelationship between pH and surface growth of Nitrobacter.
Soil Biol. Biochem.
19:665-672[CrossRef].
|
| 18.
|
Keen, G. A., and J. I. Prosser.
1987.
Steady state and transient growth of autotrophic nitrifying bacteria.
Arch. Microbiol.
147:73-79[CrossRef].
|
| 19.
|
Killham, K.
1986.
Heterotrophic nitrification, p. 117-126.
In
J. I. Prosser (ed.), Nitrification. Society for General Microbiology special publication 20. IRL Press, Oxford, United Kingdom.
|
| 20.
|
Klemedtsson, L.,
Q. Q. Jiang,
Å. Kasimir-Klemedtsson, and L. R. Bakken.
1999.
Autotrophic ammonium-oxidising bacteria in Swedish mor humus.
Soil Biol. Biochem.
31:839-847[CrossRef].
|
| 21.
|
Koops, H.-P.,
B. Böttcher,
U. C. Möller,
A. Pommerening-Röser, and G. Stehr.
1991.
Classification of eight new species of ammonia-oxidising bacteria: Nitrosomonas communis sp. nov., Nitrosomonas ureae sp. nov., Nitrosomonas aestuarii sp. nov., Nitrosomonas marina sp. nov., Nitrosomonas nitrosa sp. nov., Nitrosomonas eutropha sp. nov., Nitrosomonas oligotropha sp. nov. and Nitrosomonas halophila sp. nov.
J. Gen. Microbiol.
137:1689-1699.
|
| 22.
|
Lang, E., and G. Jagnow.
1986.
Fungi of a forest soil nitrifying at low pH values.
FEMS Microbiol. Ecol.
38:257-265[CrossRef].
|
| 23.
|
Martikainen, P. J.
1985.
Nitrification in forest soil of different pH as affected by urea, ammonium sulphate and potassium sulphate.
Soil Biol. Biochem.
17:363-367[CrossRef].
|
| 24.
|
Mobely, H. L. T., and R. P. Hausinger.
1989.
Microbial ureases: significance, regulation, and molecular characterisation.
Microbiol. Rev.
53:85-108[Abstract/Free Full Text].
|
| 25.
|
Olsen, C.
1929.
Nitrogen cycling in the soil.
Nature (London)
123:144.
|
| 26.
|
Pennington, P. I., and R. C. Ellis.
1993.
Autotrophic and heterotrophic nitrification in acidic forest and native grassland soils.
Soil Biol. Biochem.
25:1399-1408[CrossRef].
|
| 27.
|
Powell, S. J., and J. I. Prosser.
1986.
Inhibition of ammonium oxidation by nitrapyrin in soil and liquid culture.
Appl. Environ. Microbiol.
52:782-787[Abstract/Free Full Text].
|
| 28.
|
Prosser, J. I.
1989.
Autotrophic nitrification in bacteria.
Adv. Microbiol. Physiol.
30:125-181[Medline].
|
| 29.
|
Schmidt, E. L.
1982.
Nitrification in soil, p. 253-288.
In
F. L. Stevenson (ed.), Nitrogen in agricultural soils: agronomy 22. American Society for Agronomy, Madison, Wis.
|
| 30.
|
Schmidt, E. L., and L. W. Belser.
1982.
Nitrifying bacteria, p. 1027-1042.
In
A. L. Page, R. H. Miller, and D. R. Keeney (ed.), Methods of soil analysis agronomy 9. American Society for Agronomy, Madison, Wis.
|
| 31.
|
Skinner, F. A., and N. Walker.
1961.
Growth of Nitrosomonas europaea in batch and continuous culture.
Arch Microbiol.
38:339-349.
|
| 32.
|
Stephen, J. R.,
G. A. Kowalchuk,
M.-A. V. Bruns,
A. E. McCaig,
C. J. Phillips,
T. M. Embley, and J. I. Prosser.
1998.
Analysis of  -subgroup ammonia oxidizer populations in soil by DGGE analysis and hierarchical phylogenetic probing.
Appl. Environ. Microbiol.
64:2958-2965[Abstract/Free Full Text].
|
| 33.
|
Stephen, J. R.,
A. E. McCaig,
Z. Smith,
J. I. Prosser, and T. M. Embley.
1996.
Molecular diversity of soil and marine 16S rDNA sequences related to -subgroup ammonia-oxidizing bacteria.
Appl. Environ. Microbiol.
62:3922-3928[Abstract].
|
| 34.
|
Troelstra, A.,
W. De Boer,
L. Riemer, and J. M. Verstraten.
1992.
Nitrate production in nitrogen-saturated acid forest soils: vertical distribution and characteristics.
Soil Biol. Biochem.
24:235-240[CrossRef].
|
| 35.
|
Walker, N., and K. N. Wickramasinghe.
1979.
Nitrification and autotrophic nitrifying bacteria in acid tea soils.
Soil Biol. Biochem.
11:231-236[CrossRef].
|
| 36.
|
Watson, S. W.
1971.
Taxonomic considerations of the family Nitrobacteraceae Buchanan: requests for opinions.
Int. J. Syst. Bacteriol.
21:254-270[Abstract/Free Full Text].
|
| 37.
|
Weber, D. F., and P. L. Gainey.
1962.
Relative sensitivity of nitrifying organisms to hydrogen ions in soils and in solution.
Soil Science
94:138-145.
|
| 38.
|
Weier, K. L., and J. W. Gilliam.
1986.
Effect of acidity of nitrogen mineralization in Atlantic coastal plain soils.
Soil Sci. Soc. Am. J.
50:1210-1214[Abstract/Free Full Text].
|
Applied and Environmental Microbiology, July 2001, p. 2952-2957, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.2952-2957.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Norton, J. M., Klotz, M. G., Stein, L. Y., Arp, D. J., Bottomley, P. J., Chain, P. S. G., Hauser, L. J., Land, M. L., Larimer, F. W., Shin, M. W., Starkenburg, S. R.
(2008). Complete Genome Sequence of Nitrosospira multiformis, an Ammonia-Oxidizing Bacterium from the Soil Environment. Appl. Environ. Microbiol.
74: 3559-3572
[Abstract]
[Full Text]
-
Gieseke, A., Tarre, S., Green, M., de Beer, D.
(2006). Nitrification in a Biofilm at Low pH Values: Role of In Situ Microenvironments and Acid Tolerance.. Appl. Environ. Microbiol.
72: 4283-4292
[Abstract]
[Full Text]
-
Tarre, S., Green, M.
(2004). High-Rate Nitrification at Low pH in Suspended- and Attached-Biomass Reactors. Appl. Environ. Microbiol.
70: 6481-6487
[Abstract]
[Full Text]
-
Koper, T. E., El-Sheikh, A. F., Norton, J. M., Klotz, M. G.
(2004). Urease-Encoding Genes in Ammonia-Oxidizing Bacteria. Appl. Environ. Microbiol.
70: 2342-2348
[Abstract]
[Full Text]
Top
Abstract
Introduction
