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Developing a Technique to Quantify Heterotrophic andAutotrophic Nitrification in Acidic Pasture SoilsA. Islam a; D. Chen a; R. E. White aa Faculty of Land and Food Resources, School of Resource Management, Universityof Melbourne, Victoria, Australia

Online Publication Date: 01 October 2007To cite this Article: Islam, A., Chen, D. and White, R. E. (2007) 'Developing aTechnique to Quantify Heterotrophic and Autotrophic Nitrification in Acidic PastureSoils', Communications in Soil Science and Plant Analysis, 38:17, 2309 - 2321To link to this article: DOI: 10.1080/00103620701588437

URL: http://dx.doi.org/10.1080/00103620701588437

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Developing a Technique to QuantifyHeterotrophic and Autotrophic Nitrification

in Acidic Pasture Soils

A. Islam, D. Chen, and R. E. White

Faculty of Land and Food Resources, School of Resource Management,

University of Melbourne, Victoria, Australia

Abstract: A series of laboratory incubation experiments were conducted on soils from

Maindample and Ruffy in northeast Victoria and from Whittlesea in the Plenty Valley,

north of Melbourne, Victoria, Australia, to develop a technique for quantifying both

autotrophic and heterotrophic nitrification in acidic pasture soils. The use of a

specific inhibitor of the autotrophic ammonium oxidizers (N-serve) did not completely

inhibit autotrophic nitrification in its commonly recommended concentrations (10 and

20 mg g21 soil) in these soils. The N-serve concentration, which completely inhibited

autotrophic nitrification, was found to be 60–80 mg g21. Varying soil types, pHs, and

organic-matter contents affected the optimum dose of N-serve required for complete

inhibition of autotrophic nitrification. Mixing the inhibitor with the soil after appli-

cation was also important for immediate inhibition of autotrophic nitrification. Using

N-serve in combination with 15N-labeled glycine in the Maindample soil showed

that heterotrophic organisms were using the organic route for nitrification, and

N-serve did not affect heterotrophic nitrification. A lag of 12 to 24 h in complete

inhibition of autotrophic nitrification by N-serve may have occurred suggesting

nitrification studies using N-serve should include pre-incubation of the soils with

N-serve for at least 1 day.

Keywords: Acid soils, N-serve, glycine, nitrification

Received 30 March 2006, Accepted 2 December 2006

Address correspondence to D. Chen, Faculty of Land and Food Resources, School

of Resource Management, University of Melbourne, Victoria 3010, Australia. E-mail:

delichen@unimelb.edu.au

Communications in Soil Science and Plant Analysis, 38: 2309–2321, 2007

Copyright # Taylor & Francis Group, LLC

ISSN 0010-3624 print/1532-2416 online

DOI: 10.1080/00103620701588437

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INTRODUCTION

Soil organisms may be classified according to their mode of nutrition.

Broadly, the heterotrophs, which include many species of bacteria and all

the fungi, require carbon (C) in the form of organic molecules for growth.

However, the autotrophs, which include the remaining bacteria and most

algae, can synthesize their cell substrate from the C of atmospheric carbon

dioxide (CO2), harnessing the energy of sunlight (in the case of photosynthetic

bacteria and algae) or chemical energy from the oxidation of inorganic

compounds (the chemoautotrophs) (Paul and Clark 1996).

Nitrification in soils has been one of the most intensively studied processes

in the nitrogen (N) cycle. The mainly autotrophic nature of nitrifying bacteria in

most soils is generally accepted, and reviews of the process and microbiology of

nitrification have centered largely on autotrophic bacteria (Haynes 1986; De

Boer, KleinGunnewiek, and Laanbroek, 1995; Hart, Binkley, and Perry,

1997). The process is mainly carried out by soil bacteria (the chemoautotrophs)

that prefer a pH close to neutral (Killham 1990). Previous studies in acidic

pasture soils at Maindample and Ruffy in northeast Victoria gave unusually

high rates of nitrification (Islam, White, and Chen, 2000), because their pHs

of 4.8 to 5.3 (1:5 in H2O) were less than the assumed optimum pH range of

5.8–8.5 for autotrophic ammonium oxidizers (Haynes 1986; Henriksen and

Kemp 1988; Watson et al. 1989). Nitrification combined with nitrate leaching

is one of the main causes of acidification in these pasture soils.

High nitrification rates at low pHs in the acidic pasture soils of northeast

Victoria suggested that heterotrophic nitrification might play a role in these

soils, because it is known to be less affected by low pH than autotrophic nitri-

fication (Focht and Verstraete 1977; Haynes 1986; Prosser 1989; Pennington

and Ellis 1993; Paul and Clark 1996), although its contribution is still incon-

clusive (De Boer and Kowalchuk 2001). A number of techniques have been

developed to study heterotrophic nitrification in soils (Duggin, Voigit, and

Borman 1991; Pennington and Ellis 1993; Barraclough and Puri 1995;

Pedersen, Dunkin, and Firestone 1999). However, an effective technique for

quantifying both autotrophic and heterotrophic nitrification has been lacking.

The use of selective biochemical agents for inhibition of autotrophic nitri-

fiers has been widely used for separating autotrophic and heterotrophic nitri-

fication pathways. A number of inhibitors have been identified and used as

tools for biochemical blocks for ammonium (NH4þ) and nitrite (NO2

2)

oxidizers (Prosser 1989; Barraclough and Puri 1995; Pedersen, Dunkin, and

Firestone 1999). The majority of inhibitors are specific to ammonia

oxidation, although they often inhibit nitrite oxidation at high concentrations

(Prosser 1989). Among the inhibitors of autotrophic nitrification, nitrapyrin

(N-serve) and acetylene are effective inhibitors of the oxidation of ammonia

to hydroxylamine, the first step in autotrophic nitrification (McCarty and

Bremner 1986; Henriksen and Kemp 1988). N-serve is widely believed to

be innocuous with respect to heterotrophic microorganisms even at

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concentrations higher than those commonly used for inhibiting autotrophic

nitrification (Kutuzova and Tribis 1989; Duggin, Voigit, and Borman 1991;

Barraclough and Puri 1995).

The most commonly used concentrations of N-serve for the effective inhi-

bition of the ammonium oxidizers are 10 and 20 mg g21 soil (Crawford and

Chalk 1992; Barraclough and Puri 1995). However, application rates greater

than the recommended concentrations have been reported (Bremner,

Blackmer, and Bundy 1978; Duggin, Voigit, and Borman 1991). Nitrapyrin

was less effective in organic-rich soils because of sorption on soil colloids

and loss due to volatilization (Lewis and Stefanson 1975; Hendrickson and

Keeney 1979; Sahrawat, Keeney, and Adams 1987). Using higher concen-

trations of N-serve than the recommended doses would diminish the

negative effect of sorption in soils of high organic-matter content.

The objective of this investigation was to develop a technique based on

the use of nitrapyrin to block the autotrophic ammonia oxidizers for quantify-

ing both autotrophic and heterotrophic nitrification in acidic pasture soils in

Victoria. Laboratory incubations were carried out on three soils to

determine the optimum dose of N-serve, the best method of application,

and the effect of the optimum dose on the heterotrophic nitrification of a15N-labeled amino acid.

MATERIALS AND METHODS

Soils

The soils used in the study were a Brown Sodosol (Isbell 1996) on Palaeozoic

sedimentary rocks from Maindample (pH 5.1 in 1:5 soil–H2O), a bleached-

mottled magnesic Yellow Kurosol on Palaeozoic granite at Ruffy (pH 4.6),

and a Black Vertosol on Tertiary basalt at Whittlesea with a field pH of 6.2.

The first two soils were from northeast Victoria, and the Vertosol was from

the Plenty Valley, north of Melbourne, Victoria. Bulk surface samples (0–

5 cm) were collected from these sites in April 2000, brought immediately to

the laboratory, sieved to less than 2 mm, thoroughly mixed, and immediately

stored in polyethylene bags at 48C in a cool room. The cold storage of the soil

was intended to reduce the effect of soil mixing on N transformations and

changes in N-availability assays (Hart et al. 1994).

Experimental

Nitrification Inhibition

N-serve 24E (240 g of active ingredient of N-serve L21 in petroleum ether)

was applied together with NH4þ to all three soils in concentrations ranging

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from 0 to 100 mg g21 soil to find the N-serve concentration required for

complete inhibition of NH4þ oxidation. Nitrapyrin was applied to the Black

Vertosol at 0, 10, 20, 40, and 80 mg g21 soil and to Maindample and Ruffy

soils at 0, 20, 40, 60, 80, and 100 mg g21 soil. Ammonium sulfate was

applied to all treatments at 10 mg N g21 soil, although there was NH4þ

initially present in small quantities (15–20 mg N g21 soil) in these soils. All

the solutions were applied as uniformly as possible to the soil surface in a

fine spray with a 23-gauge syringe. In another experiment, N-serve in its

maximum concentrations required for nitrification inhibition and the NH4þ

were applied as previously described and then thoroughly mixed to find the

effect of mixing NH4þ and inhibitor with the soil on nitrification. The soil

was first spread over a plastic sheet to form a thin layer, and NH4þ together

with the N-serve in solution was uniformly applied to the soil surface,

followed by thorough mixing of the soil in a beaker using a spatula. The appli-

cation of substrate and inhibitor to Maindample and Ruffy soils and their sub-

sequent mixing took approximately 5 h, which was followed by the zero time

sampling.

Using 15N-Glycine (15N-Labeled Organic Substrate)

Ammonium sulfate at 100 mg N g21 soil and 15N-labeled glycine (9.50 atom%)

at 30 mg N g21 soil were applied to the Maindample soil, combined with

inhibitor in the concentration required for complete inhibition of the autotrophic

nitrifiers. Control treatments (without inhibitor) were also included. Both the15N-label and inhibitor were applied to the soil in a fine spray with a

23-gauge syringe followed by thorough mixing as described earlier, followed

by zero time sampling.

The experiment was conducted in 250-mL incubation vials (68 mm

diameter � 75 mm height) containing the fresh soil equivalent of 20 g of

oven dry/soil. The soil depth within the vials was not more than 1 cm. The

soil moisture content was maintained at 70% of field capacity and was replen-

ished every day. All the vials were covered with parafilm with four pinholes

and incubated at 228C in an automated incubator for 0 and 7 days for nitrifica-

tion inhibition experiments and for 0, 0.5, 1, 2, 4, and 5 days for the exper-

iment using 15N-labeled glycine. In each experiment, three replicated

subsamples of each treatment at each sampling time were collected and

extracted with 100 mL of M potassium chloride (KCl).

Chemical Analysis

Nitriteþ nitrate (NO32) in the KCl extracts were measured colorimetrically

using a modified Griess–Ilosvay method, following reduction of NO32 to

NO22 on a copperized cadmium column (Chen, Chalk, and Freney 1990), on

an Alpkem 501 analytical auto-analyser for nitrification inhibition exper-

iments. NH4þ and NO2

2þNO3

2 in the 15N-labeled extracts were measured

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by steam distillation (Keeney and Nelson 1982). The 15N–14N isotope ratios

in the NH4þ and NO2

2þNO3

2 fractions were determined on N2 generated by

hypobromite oxidation (distillates) (Chen, Chalk, and Freney 1990). Isotope

ratios were measured on a VG Isogas (Sira 10) mass spectrometer equipped

with dual inlets and triple collectors. All the data were presented as

mg N g21 soil, on an oven-dry basis.

Nitrification rates (n) were estimated by the rate of change in total

NO22þNO3 (NT), that is, n ¼ dNT/dt (Crawford and Chalk 1992). Nitrifica-

tion inhibition (NI) percentages were calculated as NI (%) ¼ (NTcontrol2

NTþinhibitor � 100)/NTcontrol (Bundy and Bremner 1973).

RESULTS AND DISCUSSION

Optimizing the N-serve Concentration for Maximum Nitrification

Inhibition

Considerable nitrification was observed with the application of the rec-

ommended common dose of N-serve (Goring 1962) in the Black Vertosol

(Figure 1). The nitrification in this soil was expected to be predominantly

autotrophic because of its relatively high pH (6.2). Even at the recommended

doses of N-serve at 10 and 20 mg g21 soil, the nitrification inhibition in this

soil was only 47 and 63%, respectively (Figure 1). The effect of increasing

concentrations of N-serve on nitrification showed a successive increase in

inhibition with each increment increase. No nitrification in the soil was

noted when the N-serve concentration was increased to 80 mg g21

(Figure 1). On the other hand, nitrification in the two more acidic soils

attained a constant rate at 80 to 100 mg g21 of N-serve (Figure 1), which

Figure 1. Net nitrification rates in Maindample (ML) and Ruffy (RL) soils and a

Black Vertosol with different N-serve concentrations. A three-replicate sample set is

shown for all soils.

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suggested that all the autotrophic nitrification was blocked at that range of

N-serve concentration. From this series of experiments, it was observed

that all autotrophic nitrification was suppressed when the N-serve concen-

tration was 80 mg g21 soil (Figure 1). N-serve application rates greater

than the recommended doses (Goring 1962; Bremner, Blackmer, and

Bundy 1978) have been reported in a number of studies. Duggin, Voigit,

and Borman (1991) observed complete inhibition of autotrophic nitrification

when the N-serve was used at 50 mg g21 soil in studies on hardwood forest

soils, and they observed that autotrophic and heterotrophic nitrifiers were

equally important in NO32-N production in fresh soil from the forest floor.

Kutuzova and Tribis (1989) also used N-serve at 50 mg g21 for complete

inhibition of autotrophic nitrification in pure culture studies. N-serve has

been the inhibitor most favored by investigators aiming to quantify hetero-

trophic and autotrophic nitrification because of its known specificity for

the autotrophic nitrifiers (Oremland and Capone 1988; Bedard and

Knowles 1989; Kutuzova and Tribis 1989; Duggin, Voigit, and Borman

1991; Barraclough and Puri 1995). Using higher concentrations of N-serve

than the recommended doses would diminish the negative effect of

sorption in soils of high organic-matter content.

Effect on Nitrification Rate of Mixing Substrate and Inhibitor

with Soil

Inhibition of the net rates of nitrification after thorough mixing of the

substrate and inhibitor were 91% and 92% in the Maindample and Ruffy

soils, respectively (Table 1). Inhibition in the nitrification rates was

increased by about 25% and 7% more than the inhibition attained when

80 mg g21 soil of N-serve was not mixed into the Maindample and Ruffy

soils, respectively (Figure 1). These results suggested that mixing the

Table 1. Net nitrification rates (n) in Maindample and Ruffy soils (mg NO32-N g21

h21) after mixing of NH4þ and N-serve inhibitor at 80 mg g21 soil

Maindample soil Ruffy soil

Treatment

Incubation

time

(days)

NO22þNO3

2

(mg N g21

soil) na

NO22þNO3

2

(mg N g21

soil) na

Control 0 29.5+ 0.4 27.7+ 0.2

7 59.7+ 0.4 0.180+ 0.001 49.3+ 0.5 0.128+ 0.004

N-serve 7 32.2+ 0.4 0.016+ 0.000 29.5+ 0.4 0.010+ 0.001

an, net rate of nitrification (mg NO32-N g21 soil h21). Values are means of three

replicates+ standard errors.

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inhibitor and NH4þ substrate with the soil, so that they were uniformly dis-

tributed, was the preferred method of application. It is also possible that

by mixing, the optimum dose of N-serve might have been found to be

lower than 80 mg g21 soil and close to the recommended dose reported in

the literature. Some previous laboratory incubation studies on nitrification

in soils have also preferred the mixing of the substrate and nitrification

inhibitor with the soil for even distribution and instant inhibition of nitrifica-

tion (Myrold and Tiedje 1986; Hart, Binkley, and Perry 1997; Pedersen,

Dunkin, and Firestone 1999).

Use of 15N-Glycine to Study the Pathway of Heterotrophic Nitrifiers

in the Maindample Soil

The Maindample soil was incubated with 15N-labeled glycine, with and

without N-serve at 80 mg g21. There was a negligible effect of N-serve at

this relatively high concentration on the mineralization of 15N-labeled

glycine (Figure 2). The mineralization trend of the 15N-labeled glycine

showed that almost all the mineralization of applied glycine was complete

in 2 days, and no 15N-labeled amino acid was left for further oxidation by

the heterotrophic microorganisms (Figure 2). The 15N-labeled glycine was

the only source of 15NH4þ ions in this soil. Only 64–67 % of the added

15N-labeled glycine was mineralized and appeared as 15NH4þ ions. The

glycine that did not appear in the mineral phase could have been assimilated

by the soil microorganisms (Figure 3). Barak et al. (1990), Barraclough

(1997), Gibbs and Barraclough (1998), and O’Dowd, Barraclough, and

Hopkins (1999) have all provided evidence that mineralization of the N

from amino acids occurs via a direct route in which organic N compounds

Figure 2. Mineralization of 15N-labeled glycine in the Maindample soil. A three-

replicate sample set is shown for the soils with and without N-serve.

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are assimilated by soil microorganisms, and then only excess N is released as

NH4þ. The results from this study showed consistency with the previous

studies in the sense that not all of the applied glycine was mineralized

and/or nitrified.Although it has been claimed that N-serve inhibits the growth of auto-

trophic nitrifiers immediately after addition without affecting the hetero-

trophic nitrifiers (Powell and Prosser 1985), the appearance of 15NO32 in

the early stage could be because of a lag in the inhibitory effect of

N-serve. During the first 12 h, the 15NO32 produced in both the control and

the N-serve treatment was very similar (Table 2 and Figure 4), which

could be because autotrophic nitrification was not completely inhibited,

and autotrophic nitrifiers were producing 15NO32 by direct coupling with

N-mineralizing microorganisms, as suggested by De Boer and Kowalchuk

(2001). Between 12 and 24 h, the rate of nitrification in the N-serve-

treated soil fell markedly relative to the control and subsequently dropped

to a constant low level (Figure 4), well less than the level maintained in

the control (Figure 4). This suggested that N-serve inhibition of autotrophic

nitrifiers was complete from 24 h onward and that the continued low rate of15NO3

2 production in this treatment was due to heterotrophic nitrifiers. There

was little labeled glycine left after 2 days (Figure 2), so the appearance of15NO3

2 after 2 days of incubation in the N-serve-treated soil could have

been due to the oxidation of microbially assimilated organic 15N by the het-

erotrophic nitrifiers, because the heterotrophs use only organic N as a source

of energy. The apparent decrease in the rate of 15NO32 appearance after 1 day

(Figure 4) probably reflects the cessation of autotrophic nitrification (as

N-serve becomes fully effective) and continued slow heterotrophic

nitrification.

Figure 3. Cumulative mineral 15N (15NH4þ and 15 NO3

2) production from the applied15N-labeled glycine over the incubation period in Maindample soil. A three-replicate

sample set is shown for the soils with and without N-serve.

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There was no increase in the 15NH4þ pool after 1 day of incubation

because of cessation of mineralization of 15N-labeled glycine (Figure 2).

The supply of total NH4þwas maintained because of mineralization of the indi-

genous soil organic N and remained higher in the N-serve-treated soil because

nitrification was inhibited in that treatment (Table 2).

Table 2. Total and labeled ammonium and nitrate in the Maindample soil labeled

with 15N-enriched glycine

Incubation

time(days) ATa (mg g21)

15NH4þ-N

(mg g21) NTb (mg g21)

15NO32-N

(mg g21)

Control

0 106+ 1.1 0.8+ 0.1 2.4+ 0.1 0.09+ 0.02

0.5 107+ 2.7 10.1+ 0.8 5.7+ 0.9 0.28+ 0.00

1 120+ 4.9 19.0+ 0.8 6.4+ 0.7 0.68+ 0.04

2 116+ 3.6 18.6+ 0.6 17.0+ 0.6 1.43+ 0.05

4 115+ 5.1 17.3+ 0.4 18.3+ 3.3 2.78+ 0.03

5 109+ 0.5 16.4+ 0.1 19.0+ 1.2 3.45+ 0.10

þN-serve 24E

0 106+ 1.2 0.5+ 0.0 2.4+ 0.4 0.07+ 0.01

0.5 121+ 0.0 12.1+ 0.0 4.2+ 0.1 0.24+ 0.02

1 116+ 1.8 18.6+ 0.4 4.8+ 0.3 0.44+ 0.02

2 110+ 2.0 18.0+ 0.5 5.4+ 0.5 0.56+ 0.01

4 117+ 4.9 17.8+ 1.0 5.7+ 0.4 0.76+ 0.01

5 120+ 9.0 17.3+ 1.3 6.0+ 0.4 0.80+ 0.05

aTotal NH4þ-N (labeledþ unlabeled).

bTotal NO32-N (labeledþ unlabeled). Values are means of three replicates+

standard errors.

Figure 4. Nitrification of 15N-labeled glycine and 15NH4þ produced in the mineraliz-

ation of 15N-labeled glycine during incubation in the Maindample soil. A three-

replicate sample set is shown for the soils with and without N-serve.

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CONCLUSIONS

Because the effect of N-serve varies with the soil type and soil organic-matter

content, the concentration for complete inhibition of the nitrifiers was deter-

mined in the given soils. When N-serve was tested on the Maindample and

Ruffy soils and a Black Vertosol, it was concluded that its normal rec-

ommended doses of 10 to 20 mg g21 soil did not completely inhibit auto-

trophic nitrification in the two acidic soils with high organic C contents.

N-serve completely inhibited autotrophic ammonium oxidation at a concen-

tration of 60–80 mg g21 soil. Although the need for this unusually high con-

centration may have been related to the non mixing of N-serve with the soil,

soil properties were also a factor, especially soil pH, because a concentration

of 40 mg g21 soil of N-serve achieved 78% reduction in nitrification in the

Black Vertosol compared with only 37 and 63% reduction in the Maindample

and Ruffy soils, respectively.

These studies revealed that the inhibitor should be thoroughly mixed with

the soil after application to ensure that it reached all the sites where nitrifiers

reside to inhibit autotrophic nitrification immediately. The mixing technique

used here ensured the uniform distribution of the inhibitor and NH4þ

(labeled and/or unlabeled) and rapid inhibition of the autotrophic

ammonium oxidizers. Even then, there was evidence in the 15N-labeled

glycine experiment that some autotrophic nitrification occurred in the first

24 h after the inhibitor was applied.

There was a slight increase in the labeled NO32 pool in the Maindample

soil treated with N-serve and 15N-labeled glycine in the first 2 days, after

which it was constant (Table 2 and Figure 4). This early oxidation of15NH4

þ derived from the labeled glycine in the presence of N-serve could be

the result of nitrification of the newly immobilized 15N-organic N by the het-

erotrophic nitrifiers or a lag in the inhibition of autotrophic nitrification. Also,

oxidation of inorganic 15NH4þ by heterotrophic nitrifiers (Stroo, Klein, and

Alexander 1986) could have been responsible for the increase in 15NO32 in

the early incubation period of the N-serve-treated Maindample soil.

These results were encouraging because it indicated that N-serve had no

deleterious effect on glycine mineralization and the glycine decomposing

microorganisms. The rate of 15NO32 production in the Maindample soil was

slow in the first 12 h of incubation, and the 15N-labeled NO32 produced in

both the control and N-serve treatments was similar. Thus, we cannot rule

out the possibility of a contribution to nitrification by the autotrophic nitrifiers

in the early incubation period due to coupling of nitrification with the direct

mineralization of glycine. The production of 15NO32 in the N-serve-treated

Maindample soil after 2 days of incubation, when N-serve inhibition was

fully functional and the mineralization of glycine had been completed, was

attributed to heterotrophic nitrification of the N from glycine that did not

appear in the mineral form and was thought to be assimilated by the microor-

ganisms. The inclusion of 15N-labeled glycine helped determine that organic

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N could have been the only substrate for the heterotrophic nitrifiers. Although

previous studies have reported no lag phase in inhibition of nitrification with

the use of nitrapyrin (Powell and Prosser 1985), a lag of 12 to 24 h may have

occurred here. Results from the given studies would have been more conclus-

ive if the soils were pre-incubated with the N-serve to avoid any possible lag

time. From the changes in the rate of 15NO32 production with and without

N-serve after 2 days of incubation, it is also possible to estimate the ratio of

potential heterotrophic nitrification to total nitrification in this soil.

ACKNOWLEDGMENTS

This work was funded by a Melbourne International Research Scholarship

(MIRS) and assisted by staff at the Rutherglen Research Institute, Department

of Primary Industries, Victoria. The authors appreciate the very helpful discus-

sions with Peter Janssen of the University of Melbourne.

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Barak, P., Molina, J.A.E., Hadas, A., and Clapp, C.E. (1990) Mineralization of aminoacids and evidence of direct assimilation of organic nitrogen. Soil Science Society ofAmerica Journal, 54: 769–774.

Bedard, C. and Knowles, R. (1989) Physiology, biochemistry, and specific inhibitors ofCH4, NH4

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