FEMS MicrobiologyEcology 74 (1990) 337-344
Published by Elsevier
337
FEMSEC 00304
The importance of autotrophic versus heterotrophic oxidation
of atmospheric ammonium in forest ecosystems with acid soil
A l f o n s J.M. Stams, E. M a r i n k a F l a m e l i n g and Emile C.L. M a r n e t t e
Department of Microbiology, Wageningen Agricultural University. kVageningen, The Netherlands
Received 10 May 1990
Revisionreceived26 August 1990
Accepted 31 August 1990
Key words: Acid rain; Ammonium deposition; Autotrophic nitrification; Heterotrophic nitrification;
Penicillium; Nitrogen cycle
1. S U M M A R Y
The role of autotrophic and heterotrophic
nitrifying microorganisms in the oxidation of
atmospheric ammonium in two acid and one
calcareous location of a Dutch woodland area was
investigated. In soil slurries nitrate formation was
completely inhibited by acetylene, a specific inhibitor of autotrophic ammonium-oxidizing bacteria. A survey of nitrifiers in the forest soils showed
that both autotrophic ammonium- and nitriteoxidizing bacteria were present in high numbers
and evidence was obtained that autotrophic
bacteria are able to nitrify below pH 4. These
results show that autotrophic nitrifying bacteria
may account for most of the nitrification in the
examined soils. To assess the contribution of heterotrophic nitrifiers, about 200 strains of heterotrophic bacteria and 21 morphologically distinct
fungal strains were isolated from the acid soil
locations and tested for their ability to nitrify.
Only one Penicillium strain produced nitrate in
Correspondence to: A.J.M. Stams, Department of Microbiology, Wageningen Agricultural University, Hesselink van
Suchtelenweg4, 6703 CT Wageningen, The Netherlands.
test media, but its nitrate formation when added
to acid soils was poor. These findings indicate that
in the investigated soil heterotrophs are of minor
importance in the oxidation of atmospheric ammonium.
2. I N T R O D U C T I O N
The increased intensity of agricultural activities
in the Netherlands over the past ten years has led
to escalating levels of atmospheric ammonium and
ammonia. The deposition of this atmospheric
nitrogen causes direct and indirect harmful effects
on ecosystems. In forest ecosystems the deposition
is particularly high, because the deposition of ammonium sulfate, which is formed by a reaction of
ammonia with sulfur oxides, mainly takes place at
the surface of vegetation [1]. The average atmospheric input of ammonium in Dutch forest soils is
about 3 kmol per hectare per year, whereas in the
vicinity of the emitting source the deposition can
even be much higher (4-6 kmol h a - t yr-t).
Atmospheric ammonium will cause strong soil
acidification if it is oxidized to nitrate. Flux measurements of ammonium and nitrate in soil profiles of some Dutch forest soils near the Hackfort
0168-6496/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
338
estate have shown that strong nitrification occurs
both in calcareous soils and in acid soils with a
pH as low as 3.5 [1-3], and from a field study
with [~SN]-ammonium at one of the acid locations
it became clear that nitrate is formed directly from
ammonium and not from organic nitrogen compounds [4].
Autotrophic bacteria are the most important
nitrifying microorganisms in neutral and calcareous soils [5,6] and they can be isolated from acid
soils [7-9]. Autotrophic ammonium-oxidizing
bacteria, however, were shown not to be active
below pH 4. Nitrification in acid soils might be
explained in different ways: (i) nitrification is
performed by normal autotrophs present in less
acidic micro-niches, (ii) autotrophs with unusual
physiological properties are involved, or (iii) nitrification is carried out by heterotrophic microorganisms (heterotrophic nitrification). The third
possibility is proposed for soils in which either
very low numbers of antotrophs are counted or in
which specific inhibitors of autotrophs have no or
little effect [10-16]. Heterotrophic nitrification is
carried out by both heterotrophic bacteria and
fungi. These organisms oxidize either ammonium
to nitrite or nitrate via an organic pathway, or
oxidize the nitrogen of special organic nitrogencontaining compounds like, e,g., hydroxamates or
oximes [161.
In this study the role of autotrophic and heterotrophic nitrifiers in the oxidation of atmospheric
ammonium in acid forest soils was investigated.
Results of soil slurry incubations in the presence
and absence of specific inhibitors and a survey of
the types of nitrifiers show that nitrification of
atmospheric ammonium, a process which takes
place in the leaf litter [4], is mainly autotrophic.
3. MATERIALS A N D M E T H O D S
3.1. Preparation of the soils
The soil samples which were taken consisted of
a mixture of the H layer (humus layer just above
the mineral soil) and the A~ layer (first 5 cm of the
mineral soil). Samples were taken from two acid
and one calcareous forest soil location near the
Hackfort estate. From the acid locations the leaf
litter was also sampled. The locations have been
referred to as plot A, B and D [17]. The characteristics of the soils and the vegetation of the area
were described previously [3,17]. Pooled samples
were sieved (2 mm) and homogenized. Part of
each soil sample was used directly for counting the
microorganisms and the remainder was stored at
4°C in perforated plastic bags until use. Unless
stated otherwise the incubations described below
were done at 20°C in the dark.
3.2. Enumeration of autotrophic and heterotrophic
nitrifiers
For the enumeration of microorganisms, 10 g
soil was diluted to 100 ml with 1 mM K 2 H / K H 2
PO 4, pH 6 (an appropriate mixture of the acid and
the basic phosphate) and blended twice for 30 s in
a Waring blender. Then, the soil slurry was sonifled twice for 10 s in an ultrasonic waterbath at
150 W and immediately diluted in decimal steps in
the same buffer. Autotrophic ammonium- and
nitrite-oxidizing bacteria were enumerated with
the most probable number (n = 5) method [18].
The medium for ammonium oxidizers contained
(in raM): (NH4)2SO 4, 0.75; CaCI 2, 0.1; MgSO 4,
0.15; K 2 H / N a H 2 P O 4, 5" 1 ml trace elements
solution per liter. The phosphate buffer was sterilized separately and added afterwards to the sterilized medium. The trace elements solution had the
following composition (raM): HCI, 50; FeSO4,
7.5; MnCI 2, 0.5; MgCI 2, 0.5; CuSO 4, 0.1;
Na2MoO 4, 0.1; ZnSO4, 0.5; H3BO3, 1.0; NiCI 2,
0.1; Na2WO4, 0.1 and Na2SeO 3, 0.1. The pH of
the medium was 7.5 due to the appropriate ratio
of K2HPO 4 and NaH2PO 4. The medium for nitrite
oxidizers was the same except that it contained 0.1
mM of NaNO,, instead of ammonium, the MgSO4
concentration was 0.75 mM, and the pH was 6.0.
In initial experiments with an acid forest soil from
the Hackfort B location it was found that pH 7.5
and 6 gave the highest numbers for ammoniumand nitrite-oxidizing bacteria after 8 weeks of
incubation. Diluted soil samples (1 ml) were added to tubes containing 5 mi sterile medium, incubated for 10 weeks and analyzed for nitrate plus
nitrite (ammonium oxidizers) or for nitrite (nitrite
oxidizers) as described previously [18]. From the
339
pattern of positive and negative tubes the numbers
of bacteria were calculated [191.
The plate count method was used for the enumeration of heterotrophs. A medium with the
following composition was used for bacterial
counts (in mM): glucose, 10; NH4Ci, 5; MgSO 4.
1; CaSO 4. 1; K2SO 4, 1; N a 2 H / N a H 2 P O 4. 10, 1
mi trace elements solution; 0.02% yeast extract
(Difco); and 1.2% agar. The pH of the medium
was 7. Pure cultures were obtained by repeated
plating of single colonies. Isolated strains were
tested for their ability to nitrify. For this purpose
several media were prepared, including the
medium mentioned above with either ammonium
or 0.1% yeast extract + 0.2% peptone as nitrogen
source and the media described by Eylar and
Schmidt [20]. For some strains additional media
[21,22] were used. Incubations were done in 100-ml
flasks with 40 ml medium or in 300-ml flasks with
100 ml medium. After 14 days of incubation at 50
rpm, the presence of nitrite and nitrate was measured.
Fungi were counted and isolated as described
by Martin [23], and their ability to nitrify was
examined in described media [20,24l.
3.3. Soil slurry incubations
Ten g of soil was added to 300-ml flasks containing 100 ml medium with the following composition (in mM): (NH4)2SO4. 5: N a H 2 P O 4, 10;
KCi, 5; CaSO4, 1: MgSO 4. and 1 ml trace elements solution, pH was adjusted to 4.5 or 7.5 with
1 H NaOH. Flasks were shaken at 100 rpm and
after various periods of time samples of 5 ml were
taken and the pH measured. Before sampling demineralized water was added to correct for
evaporation. After centrifugation and filtration
samples were analyzed for a m m o n i u m and nitrate.
To discriminate between autotrophic and heterotrophic nitrification, incubations were performed in the presence of 20 ppm nitrapyrin (2chloro-6-trichloromethyl pyridine) or 57o acetylene
in the gas phase. Nitrapyrin was added as a 20%
solution in ethanol, and experiments with acetylene
were performed in closed 600-ml bottles instead of
in 300-ml flasks. Somc incubations with the acid
soils were performed with 9% 1SN-enriched
(NH4)2SO 4 at an initial pH of 4.5. In these ex-
periments, 40 g soil was added to 600-ml flasks
containing 200 m| medium. At the beginning and
after 160 days of incubation the percentage 15N
enrichment in ammonium and nitrate was determined. For this purpose, KCI was added to a
final concentration of 1 M and the flasks were
vigorously shaken for 3 h. After centrifugation
and filtration the supernatant was first analyzed
for ammonium and nitrate and then separated by
distillation methods [25]. To 100 ml of the supernatant 10 ml of 8 M N a O H was added, the
ammonia was distilled off and trapped into 10 ml
0.1 N H2SO 4. To assure complete removal of the
[tSN]-.ammonium, (NH4)2SO 4 was added to the
residue and the ammonium was distilled off again;
this procedure was repeated three times. Then, 0.5
g Devarda's alloy (Merck) was added to reduce
the nitrate and the ammonia was distilled as described. Appropriate methods were used for cleaning the still [26]. The percentage 15N was determined in a Finnigan M A T 271/251 gas-mass
spectrometer as described elsewhere [4].
3.4. Enrichment cultures
Autotrophic nitrifying bacteria from the acid
Hackfort B location were enriched at pH 6.5 in
the same medium as described for the soil slurry
incubations. To investigate the pH dependency of
autotrophic nitrifiers, enrichment cultures were
centrifuged and added to media with different
initial pH values, in these experiments the concentration of phosphate was only 5 mM. At various periods of time, samples were taken and
analyzed for pH, a m m o n i u m and nitrate. To investigate whether enriched nitrifiers have a stimulatory effect on the nitrification rate in soils, cell
suspensions were, also added to soil slurries.
3.5. Analyses of nitrogen compounds and pH
Qualitative analysis of n i t r a t e + nitrite and
nitrite were done by spot tests [18], whereas
quantitative analysis was performed colorimetrically with a Technicon autoanalyzer. A m m o n i u m
was determined with the indophenol-blue method
[25]. Nitrate was first reduced with cadmium to
nitrite, which was then determined by a modified
Griess-method [25]. The pH of the soils was determined in slurries of soil samples in demineral-
340
ized water (ratio 1 to 1 for mineral soils and 1 to 2
for leaf litter).
2.0
8
4. RESULTS
4.1. Survey of autotrophic and heterotrophic nitri-
tiers
In both the calcareous and acid soil locations,
autotrophic ammonium- and nitrite-oxidizing
bacteria were present (Table 1). Especially in the
leaf litter layer the numbers were high and only a
factor 10 lower than were found in the calcareous
soil. Heterotrophic bacteria were present in the
acid soils in numbers of 10 ~ ~o 3 x 107 per g dry
soil. About 200 heterotrophic bacterial strains were
isolated from the acid soils and tested for their
ability to form nitrite or nitrate in the test media
with either ammonium or yeast extract-peptone as
the nitrogen source. Although sterile media upon
incubation in time tested slightly positive for
nitrite, none of the strains appeared to form significant amounts of nitrite or nitrate after two
weeks of incubation.
Based on phenotypic appearance, 23 different
fungal strains were isolated and tested on their
ability to form nitrite or nitrate. One fungus isolated from one of the acid soil locations was able
to form nitrate in a glucose-ammonium medium
Table 1
Numbers of autotrophic nitrifyingbacteria counted in two acid
and one calcareous forest soil with the most probable number
technique
Location
Soil
Date
Hackfort D
soil
Hackfort A
leaf litter
Hackfort A
soil
Hackfort B
Ir.af litter
Hackfort B
soil
N H 4-
N O 2-
oxidizers oxidizers
(numbersg- 1dry soil)
pH
7.9
sep. '85
167000
940000
3.3
sep. '86
13500
21400
3.4
mar. "86
3.3
sep. "86
3.5
3.4
sep. '85
mar. '86
1000
>15000
30
30
19000
180000
18000
2000
1.0
oH/
pH
Fig. 1. Nitrate formationby a Penicilliumnigricansstrain in a
medium with glucoseas carbon source and ammonium as sole
nitrogen source.The pH refers to the initial pH of the medium.
The incubation time was 14 days.
[24]. The strair~ which was classified by the
Centraai Bureau voor Schimmelcultures (Baarn;
The Netherlands) as Penicillium nigricans, formed
nitrate in :media with initial p H values of as low as
3.3 (Fig. 1), a property which could also be demonstrated with Aspergillus flavus (ATCC 10124)
(data not shown). Nitrate formation by the fungus
was not inhibited by nitrapyrin nor by acetylene.
Addition of glucose-grown fungal biomass to
heat-sterilized soil led to some nitrate formation;
however, in non-sterile soils nitrate formation was
much higher. Addition of the fungus to non-sterile
soils gave no increase in the nitrate formation
above the natural occurring nitrate formation, and
the fungal biomass was rapidly degraded.
4.2. Soil slurry incubations
To elucidate the role of autotrophic and heterotrophic nitrifying microorganisms in the nitrification in forest soils, soil slurry incubations were
performed in the presence and absence of specific
inhibitors of autotrophic nitrifiers. Fig. 2A shows
the nitrification of the calcareous Hackfort (D)
soil. A fast degradation of ammonium with a
concomitant formation of nitrate was observed.
The addition of 20 ppm nitrapyrin at the start of
the incubation inhibited nitrate formation completely.
Incubation of the acid Hackfort B soil in media
8
7
6
5
~
A
A~A"~,~A~A~A
B
C
*--l~ A
\. ....
[
A--A'A
~
._...._A
~
a
12s-o - - ' 0 / 0 ~ *
10
E
~
s
8.
6
3
/
2
/\/
\
•.
.
.
.
.
/\f
,
....../
~
, _- ,
,
,-.-r .•I'
20 /.0 60 80 100 120 0 20 ~,0 60 80 100 120 0 20 g0 ~
lime {doys}
80 100 120
Fig. 2. Course of ammonium (ms).nitrate (O) and pH (A) in a slurry of a calcareous soil from Hackfort D incubated at a high ptl (A)
and of an acid soil from Hackfort B incubated at a high pH (B) and a low pH (C).
with an initial p H of 7.5 led, after an a d a p t a t i o n
time of a b o u t 30 days, to a fast a m m o n i u m oxidation a n d nitrate f o r m a t i o n (Fig. 2B). The addition
of nitrapyrin was also inhibitory here. A m u c h
slower nitrate f o r m a t i o n was observed when this
acid soil was incubated at an initial p H of 4.5
(Fig. 2C). In c o n t r a s t to the incubation at high
p H , this incubation did not lead to a m e a s u r a b l e
decrease in the a m m o n i u m concentration. H o w ever, also at this low p H nitrate was formed from
a m m o n i u m . This b e c a m e clear w h e n in a separate
experiment the s a m e acid soil from Hackfort was
i n c u b a t e d with [tSN]-ammonium. Table 2 s h o w s
the percentage tSN e n r i c h m e n t in a m m o n i u m and
nitrate before and after incubation. As the a t o m
percentage tSN in a m m o n i u m and nitrate are in
the same range, it is evident that at least the m a j o r
part of the nitrate is formed f r o m a m m o n i u m and
not from other sources. In c o n t r a s t with the in-
Table 3
Nitrate formation (in mM) and final pH of two incubation
experiments with acid soil from the Hackfort B location at an
initial pH of 4.5 with and without 10 mM ammonium in the
medium, and in the presence and absence of 20 ppm nitrapyrin
or 5% acetylene in the gas phase
In the first experiment soil (sampled September 1985) was
incubated for 90 days and in the second (sampled March 1986)
for 50 days.
Table 2
Concentration (raM) of ammonium and nitrate and percentage
*5N enrichment in ammonium and nitrate before and after the
incubation of soil from Hackfort A and B in a mineral medium
with 10 mM 9~ enriched [t>N]-ammonium
Time
Hackfort A
Hackfort B
(days) concert- pereen- concert- percentration tage ISN tration rage15N
Ammonium
0
Ammonium 160
Nitrate
0
Nitrate
160
9.83
9.72
0.06
1.04
8.33
6.68
0.365
7.48
9.72
9.60
0.08
0.60
8.30
7.67
0.365
6.67
Amendment
Experiment 1
none
mtrapyrin
acetylene
Experiment 2
none
mtrapyrin
acetylene
Without ammonium With ammonium
Nitrate
formed
Final
pH
Nitrate
formed
Final
pH
12.1 +0.8
0.0
0.0
3.7
4.2
4.3
41.5±3.4
1.2 ± 1.2
0.0
3.8
4,0
4,1
4.6 + 1.1
4.3 + 0.2
0.0
3.7
3.8
4.0
8.3 _+1.7
7.9 +_0.3
0.0
3,7
3.7
3.9
342
cubations at high pH, nitrapyrin turned out to be
an unreliable inhibitor in incubations of the acid
soils at low pH. Table 3 shows the results of two
incubations of soil from the same location in the
presence and absence of inhibitors of autrophic
nitrification. Several separate incubations at low
pH were done with soil from the acid location B.
In three experiments nitrapyrin caused a complete
inhibition of nitrate formation, whereas no inhibition at all was observed in four other incubations.
Acetylene, another inhibitor of autotrophic nitrification, inhibited nitrification completely in the
four experiments in which it was applied.
At the beginning and at the end of the incubations given in Fig. 2B and C, the numbers of
autotrophic nitrifying bacteria were counted with
the MPN method. In case of the incubation at
high pH, a significant increase in both autotrophic
ammonium- and nitrite-oxidizing bacteria was
found (Table 4). After incubation of the soil at
low pH the numbers had even decreased.
The effect of the addition of enriched autotrophic bacteria to soil slurries was tested. As
counted with the MPN method, approximately
6 × 10 3 ammonium- and 2 × 10 6 nitrite-oxidizing
bacteria were added per g soil. A significant increase in the nitrification rate was observed at
high pH. At low pH, however, the addition had no
effect on nitrate formation (results not shown).
In order to investigate the pH tolerance of
autotrophic nitrifying bacteria, enrichment cultures were made in a medium with an initial pH of
6.5. Washed cell suspensions were added to media
with different initial pH values, and pH (Fig. 3A)
and nitrite and nitrate formation (Fig. 3B) were
measured over time. The rate of nitrification in-
Tab!e 4
Numbers of autotrophic ammonium- and nitrite-oxidizing
bacteria before and after incubation at high and low pH given
in Figs. 2B and 2C. respectively
Before
N H 4-oxidizers.
NO2-oxidizers
(numbers g- i dry soil)
30
18000
After
high pH
low pH
5500
< 30
1200000
2000
5 o.o'.:'-:r--..-..o~o ....
3
B
-- 210.5
0
~'7-----'--~ pHPH
7.97.2
25 50 75 100 125 150
time(days)
Fig. 3. Course of pH (A) and nitrate+nitrite formation (B) by
an enrichment culture of autotrophic nitrifying bacteria in
mineral ammonium-containingmedia with different initial pH
values,
creased with increasing initial pH. Below a p H of
5.3 no nitrification occurred, whereas at an initial
pH of 5.7 and 6.3, nitrification proceeded until a
pH of 3.8 and 3.7, respectively, was reached. In
these incubations nitrate was the only product. In
"aedia with an initial pH of 7.2 and 7.9, nitrification stopped at a pH of about 5.8. At p H 7.2
nitrite was formed intermediately, whereas at pH
7.9 nitrite was the only product.
5. DISCUSSION
'The presence of high numbers of both autotrophic ammonium- and nitrite-oxidizing bacteria
in the leaf litter together with their ability to
nitrify below p H 4 strongly suggests that these
343
bacteria are the main n i t r i ~ i n g organisms involved in the oxidation o f atmospheric a m m o n i u m
in these forest ecosystems. This is in agreement
with the findings m a d e earlier that the major part
of surface-applied [15N]-ammonium is oxidized in
the leaf litter and not in the mineral soil [4]. A
relatively high p H which is essential for the activation of the autotrophs may also occur in the
natural ecosystem where: (i) the p H o f the rainand throughfaU water is between 4 and 6 and
therefore significantly higher than the p H o f the
soil water, (ii) mineralization which is mainly associated with the leaf litter layer is a process which
leads to locally elevated p H values, and (iii) nitrate
uptake by plants leads to aikalization.
Three different types of arguments are normally used to show the involvement o f heterotrophic microorganisms in nitrification: (i) the
presence of only low n u m b e r s of autotrophic
bacteria. (ii) the stimulation of nitrification by
organic nitrogen c o m p o u a d s , and (iii) the inactivity o f specific inhihitors of autotrophic nitrifiers.
This study shows that n o n e o f these are fully
reliable. High n u m b e r s of a u t o t r o p h s may be present in the fresh leaf litter but not in the organic
layer or the mineral soil. In most studies in which
autotrophie nitrifying bacteria are counted, it is
not clearly stated which p a r t of the soil is analyzed
[7-12]. It is likely, however, that material which is
difficult to homogenize, like leaf material, is discarded. Stimulation of nitrification by the addition o f organic nitrogen c o m p o u n d s and inhibition o f nitrification by a m m o n i u m may also not
be taken as evidence for heterotrophic nitrification. Degradation o f organic nitrogen c o m p o u n d s
leads to ~n increase of the pH, which may stimulate autotrophic bacteria, whereas the addition of
a m m o n i u m may lead to a decrease o f p H due to
an ion exchange with p r o t o n s from soil particles.
In addition, it w~s recently found that a u t o t r o p h s
are directly stimulated by the mineralization process [27]. T h e specificity o f 'specific' inhibitors
may be questioned too. This and other studies (for
a review see ref. 28) show that nitrapyrin is not an
effective inhibitor in soils with a high organic
matter content. Acetylene is also thought to be a
specific inhibitor o f autotrophic nitrification.
However, so far only one Arthrobacter strain [29],
o n : Aspergillus flavus strain [14] and our Peniciliium nigricans strain were tested and shown not to
be affected by acetylene. Acetylehe is also an
inhibitor of N 2 0 reduction in the denitrification
process, and it is not k n o w n w h e t h e r heterotrophic
nitrification by denitrifying bacteria is also inhibited by acetylene.
ACKNOWLEDGEMENTS
This research was financed by the D u t c h
Ministry of Housing, Physical Planning a n d Environmental Control and was p a r t o f the D u t c h
Priority P r o g r a m m e on Acid Rain. Drawings were
m a d e by N. Slotboom. 15N analysis was performed by the N e t h e r l a n d s Energy Research
F o u n d a t i o n , Petten.
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