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The importance of autotrophic versus heterotrophic oxidation of atmospheric ammonium in forest ecosystems with acid soil
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.
REFERENCES [1] van Breemen, N., Burrough, P.A., Velthorsto E.J., van Dobben, H.F., de Wit. T.. Ridder. T.B. and Reijnders, H.F.R. (19821 Soil acidification by atmospheric ammonium sulphate in forest canopy throughfalL Nature 299. 548-550. [21 van Breemen, N., Mulder, J. and van Grinsven. J.J.M. (19871 Impacts of acid atmospheric deposition on woodland soils in the Netherlands: II. Nitrogen transformations. Soil Sci. SOc. Am. J. 51, 1634-1640. [31 van Breemen. N., de Visser. P.H.B. and van Grinsven J.J.M. (19861 Nutrient and proton budgets in four soilvegetation systems underlain by pleistocene alluvial deposits. J. Geol. Soc. London 143, 659-666, [41 Stams. A.J.M., Booltink, H.W.G.. Lutke-Schipholt. I.J., Beemsterboer, n.. Woittiez, J. and van Breemen°N. (19901 Field study on the fate of atmospheric ammonium in an acid forest soil. Biogeochemistry (submitted). [51 Belser, L.W. and Schmidt, E.L. (19781 Diversity of the amalor,ia-oxidizing population of a soil. Appl. Epviron. Microbiol. 36, 584-588. 161 Belser. L.W. (19791 Population ecology of autotrophic nitrifying bacteria. Ann. Rev. Microbiol. 33, 309-333. [71 Hankinson. T.R. and Schmidt. E.L. (19841 Examination of an acid forest soil for ammonia- and nitrite-oxidizing autotrophic bacteria. Can. J. Microbiol. 30, !125-1132. 181 Josserand, A. and Bardin, R. (19811 Nitrification en sol acide. I.-Mise en evidence de germes autotrophes nitrifiants (genre Nitrobacter) dans un sol torestier sous resineux. Rev. Ecol. Biol, Sol. 18, 435-445. [91 Martikainen. P.J. and Nurmiaho-Lassila. E.L. (19851 Ni;rosospira. an important ammonium-oxidizing bacteti-
344 um in fertilized coniferous forest soil. Can. J. Microbiol. 31,190-197. [10] Adams, J.A. (1986) Identification of heterotrophic nitrification in strongly acid larch humus. Soil Biol. Biochem. 18, 339-341. [11] Killham, K. (1986) Heterotrophic nitrification, in Nitrification (Prosser, J.l., ed.), pp. 117-126. IRL Press, Oxford. [12] Lang, E. and Jagnow, G. (1986) Fungi of a forest soil nitrifying at low pH values. FEMS Mierobiol. Ecol. 38, 257-265. LI3] Remacle, J. (1977) Microbial trans~'ormation of nitrogen in forests. Oecol. Plant. 1, 33-43. [14] Schimel, J.P., Firestone, M.K. and Killham, K. (1984) Identification of heterotrophic nitrification in a Sierran forest soil. Appl. Environ. Microbiol. 48, 802-806. [15] Stroo, H.F., Klein, T.M. and Alexander, M. (1986) Heterotrophic nitrification in an acid forest soil and by an acid-tolerant fungus. Appl. Environ. Microbiol. 52,11071111. [16] Focht, D.D. and Verstraete, W. (1977) Biochemical ecology of nitrification and denitrification. Adv. Mierobiol. Ecol. 1,135-214.[17] van Breemen, N. and Jordens, E.R. (1983) Effects of atmospheric ammonium sulfate on calcareous and noncalcareous soils of woodlands in the Netherlands, in Effects of Accumulation of Air Pollutants in Forest Ecosystems (Ulrich. B. and Pankrath, J., eds.), pp. 171-182, D. Reidel Publishing Company. [18] Schmidt, E.L. and Belser, L.W. (1982) Nitrifying bacteria, in Methods in Soil Analysis (Page, A.L., Miller, R.H. and Keeney, D.R,, ¢ds.), pp. 1027-1042. Agronomy 9, part 2, 2nd edn., Madison, WI. [19] Alexander, M. (1982) Most Probable Number method for microbial populations, in Methods in Soil Analysis (Page,
A.L., Miller, R.H. and Keeney, D.R., eds.), pp. 815-820. Agronomy 9, part 2, 2nd edn., Madison, WI. [20] Eylar, O.R., and Schmidt, E.L. (1959) A survey of heterotrophic microorganisms from soil for ability to form nitrite and nitrate. J. Gen. Microbiol. 20, 473-481. [21] Gode, P. and Overbeck, J. (1972) Untersuchungen zur heterotrophen Nitrifikation lm See. Z. AIIg. Mikrobiol. 12, 567-574. [22] Verstraete, W. (1972) Mechanism of nitrification by Arthrobacter sp. J. Bacteriol. 110, 962-967. [23] Martin, J.P. (1950) Use of acid, rose bengal, and streptomycin in the plate method for estimating soil fungi. Soil Sci. 69, 215-232. [24] Doxtader, K.G. and Alexander, M. (1966) Nitrification by growing and replacement cultures of Aspergillus. Can. J. Microbiol 12, 807-815. [25] Keeney, D.R. and Nelson D.W. (1982) Nitrogen inorganic forms, in Methods in Soil Analysis (Page, A.I.., Miller, R.H. and Keeney, D.R., ~ds.), pp. 643-698. Agronomy 9, part 2, 2rid edn., Madison, WI. [26] Hauek, R.D. (1982) Nitrogen-isotope-analysis, in Methods in Soil Analysis (Page, A.L., Miller, R.H, and Keeney, D.R. eels.), pp. 735-780. Agronomy 9, part 2, 2nd edn., Madison, WI. [27] de Boer, W., Duyts, H. and Laanbroek, H.J. (1988) Autotrophic nitrification in a fertilized, acid heath soil. Soil Biol. Bi~chem. 20, 845-850. [28] Keeney, D.R. (1986) Inhibition of nitrification in soils, in Nitrification (Prosser, J.l., ed.), pp. 123-124, IRL Press, Oxford. [29] Hynes, R.K. and Knowles, R. (1982) Effect of acetylene on autotrophic and heterotrophic nitrification. Can. J. Microbiol. 28, 334-340.
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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