The effect of nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on nitrifying organism populations under <i>in vitro</i> conditions

doi:10.4236/as.2011.23027

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Vol.2, No.3, 198-200 (2011) Agricultural Sciences

doi:10.4236/as.2011.23027

The effect of nitrification inhibitor 3,4-dimethylpyrazole

phosphate (DMPP) on nitrifying organism populations

under in vitro conditions

David Beltran-Rendon1, Kenne Rico-Fragozo1, Lina Farfan-Caceres2,

Hermann Restrepo-Diaz2*, Lilliana Hoyos-Carvajal2

1Faculty of Science, Pontificia Universidad Javeriana, Bogotá, Colombia;

2Faculty of Agronomy, National University of Colombia, Bogotá, Colombia; *Corresponding Autor: [email protected]

Received 6 April 2011; revised 23 May 2011; accepted 1 June 2011.

ABSTRACT

The application of nitrification inhibitors is a

technique to reduce the ni trate conc entration on

leachates that delay ammonium oxidation by

reducing the activity of ammonium oxidizing

bacteria in soils. Two experiments were carried

out in order to estimate the influence of DMPP

on the population of ammonium oxidization

bacteria under in vitro conditions. In both ex-

periments, three treatments were established.

The treatments were the following: a) ammo-

nium oxidization bacteria established in a grow-

ing media without fertilizers, b) ammonium oxi-

dization bacteria established in a g rowing media

with Urea, and c) ammonium oxidization bacte-

ria established in a growing media with DMPP.

Results obtained showed that the population of

the ammonia oxidizing bacteria diminished in

the DMPP treatment as compared with the urea

and control treatments. In conclusion, DMMP

influences on ammonium oxidization bacteria

activity being a useful tool in fertilizers strate-

gies to reduce the contamination by nitrates in

groundwater.

Keywords: Ammonium Oxidizing Bacteria;

Fertilizers; Nitrogen; Nitrification Inhibitors;

Winogradsky

1. INTRODUCTION

Nitrogen (N) plays an important role on the growth

and yield, since it is required in highest amounts by

plants and, hence; constitutes the basis of fertilization

strategies for agronomic and horticultural crops [1]. In

actual agricultural practices, nitrogen is usually used in

greater quantities than those needed in order to guarantee

a high yield [2]. As a consequence, nitrogen over fertili-

zation may cause environmental degradation due to ni-

trogen losses [3]. Nitrogen losses are caused by Nitrate



) and amonnium (4

H) leaching, erosion, vola-

tilization, denitrification and fixation in soil organic

matter [4]. 3



leaching from agricultural soils is one

of the important global environmental concerns [5].

These losses contribute to 3

O-N contamination of

groundwater [6]. A high 3

NO -N content in groundwater

and drinking water does harm people and livestock [5].

A technique to–



diminish NO3-N leaching into ground-

water and to conserve 4

H fertilizers applied to soils

is the retardation of biological oxidation of 4



-N to



-N [5,6]. Actually, there are compoundseffec-

inhibit nitrification when applied to soils in con-

junction with NH4

+ fertilizers or 4

that

tively

H-producing com-

pounds, such as urea or ammoniumhate [6,7]. These

compounds are called nitrification inhibitors (NIs). NIS

delay ammonium oxidation by reducing the activity of

Nitrosomonas bacteria (ammonium oxidizing bacteria)

in the soil. Ammonium oxidization bacteria transform

NH4

+ into 2

sulp



, which in turn is oxidized to 3



Nitrobacter teria [8]. Recently, DMPP haeen

introduced in Colombia to be used in nitrogen nutrition

of different crops [9]. Likewise, contamination of sur-

face water and/or groundwater by nitrates leaching has

obtained importance in managing of crops, mainly; in

rose crops in Colombia [10]. In particular, little is known

about the effect of fertilizers, especially, NIs on micro-

organisms present in tropical soils. For that reason, the

aim of this study was to estimate the influence of DMPP

on the population of ammonium oxidization bacteria

collected from tropical soil under in vitro conditions.

bacve b

2. MATERIAL AND METHODS

D. Beltran-Rendon et al. / Agricultural Science 2 (2011) 198-200

199199

ts were carried out in May

acteria were obtained by

2.1. Isolation Bacteria

In our study, two experimen

2010. Ammonium oxidization b

e preparation of Winogradsky’s columns [11]. Soil for

columns was collected on 10 December 2009 from upper

10 cm of a rose crop established in Mosquera, Colombia

(4˚42′28″ N and 74˚13′58″ W). For bacteria extraction,

10 ml from middle of Winogradsky’s column was di-

luted in 250-ml Erlenmeyer flask containing 90 ml of a

NH4

+ salt solution for ammonium oxidization bacteria

which had the following composition: NaHPO4 (13.5 g),

KH2PO4 (0.7 g), MgSO4·7H2O (0.1 g), NaHCO3 (0.5 g),

FeCl3·6H2O (0.014 g), CaCl2·H2O (0.18 g) and (NH4)2SO4

(0.5 g) per liter of water [12]. Three growing media were

established in a shaker incubator (Labline 3527, Lab-

Line instrument, Inc. USA) during 15 days at 28˚C and

150 rpm, to achieve fully aerobic conditions. After the

period of incubation, 10 ml of solution were taken from

the growth media to determine the existence of ammo-

nium oxidization bacteria by the presence of 2



-N

and 3

O-N using the Griess’s reagent, respectively [5].

Then, NH3 oxidizers were obtained by the teue

descr by Skinner and Walker [13]. Consequently,

isolated colonies of ammonium oxidization bacteria

from agar were taken by a handle. Next, those colonies

were diluted in a salt solution of NaCl at 20% w/v. To

estimate the initial concentration of inoculum, twofold

dilutions series from 102 to 106 were done realized, and

after that, inoculum was set in agar during 48 h. Subse-

quently, inoculum concentration was calculated by

counting colonies as described by Madigan et al. [14].

The initial concentrations were 6.2 × 10 5 cfu/ml and 3.7

× 104 cfu/ml for each experiment, respectively.

2.2. Treatments

chniq

ibed

f inoculum solution at 20% of NaCl

ml Erlenmeyer flask containing 90

cal Analysis

arried out on the data to

treatments. Both experi-

teria popu-

r the begin-

n the double interaction fertilizer

After that, 10 ml o

were diluted in 250-

l of a salt solution for ammonium oxidization bacteria

(concentration is mentioned above) for each experiment,

respectively. In both experiments, three treatments were

established. The treatments were the following: i) a 250-

ml Erlenmeyer flask containing 90 ml of an ammonium

salt solution and 10 ml of NaCl at 20% with Urea plus

DMMP at 1%. 37 mg of fertilizer were added by grow-

ing media. This amount is equivalent to 170 ppm that it

is the commercial dose used at fertirrigation programs in

rose crops. ii) a 250-ml Erlenmeyer flask containing 90

ml of a salt solution and 10 ml of NaCl at 20% with

Urea (37 mg of fertilizer), and iii) a 250-ml Erlenmeyer

flask containing 90 ml of a salt solution and 10 ml of

NaCl at 20% without fertilizer (control). Each treatment

was placed in a shaker incubator (Labline 3527, Lab-

Line instrument, Inc, USA) during 14 days at 28˚C and

150 rpm. Additionally, fertilizer was added to treatments

with urea or Urea + DMPP at a dose mentioned above

every 2 days during the incubation. To determine the

concentration of inoculum at each sample point, 2 ml of

each Erlenmeyer were taken to perform twofold dilution

series up to 107. Afterwards, inoculum was placed in

plates with agar. Consequently, inoculum concentration

was estimated by counting the colonies as described by

Madigan et al. [14]. Samples were done every 2 days

during 14 days. The same methodology was used in both

experiments.

2.3. Statisti

Analyses of variance were c

evaluate the effect of different

ents were analyzed together as a series of experiments.

Values were transformed using the Log10 transformation

before analysis. Data were evaluated using Statistix Ver-

sion 8.0 (Analytical Software, Tallahassee, FL, USA).

Four replicates for each treatment were used.

3. RESULTS AND DIS CUSSION

An increasing ammonium oxidization bac

lation was observed during first 4 days afte

ng of treatments. Significant differences were found on

ammonium oxidization bacteria population in both ex-

periments at 6 days after the treatments started. Ammo-

nium oxidization bacteria cultivated in a growing media

with DMPP had a less population than bacteria estab-

lished in urea or control treatments. After this period,

ammonium oxidization bacteria population started di-

minishing in all treatments. At 14 days after the begin-

ning of experiments, bacteria established in a media with

urea had a higher population than DMPP and control

treatments in both experiment 1and experiment 2 (Fig-

ures 1( a) and (b)).

Differences were found on ammonium oxidization

bacteria population i

eatments and the different experiments (Figure 2).

Treatments with DMPP showed a lower amount of am-

monium oxidization bacteria population than control and

urea treatments at 6 days after beginning both experi-

ments. DMPP inhibited the mean ammonium oxidization

bacteria population by 5% and 12% compared to urea

treatments in experiments 1 and 2, respectively. A simi-

lar trend was observed at 14 days after the treatments

started, but the DMPP had a greater percentage of inhi-

bition than at 6 days after the beginning of treatments.

DMPP reduced the mean ammonium oxidization bacte-

ria populations by 31% and 33% regarding urea in ex-

periments 1 and 2, respectively. Also, DMPP diminished

D. Beltran-Rendon et al. / Agricultural Science 2 (2011) 198-200

200

Figure 1. Evolution of ammonium oxidizing bacteria popula-

tion from control treatment (), from bacteria that received

urea (), and bacteria that received DMPP () during 14

days. Each point represents the ean four values. Vertical bars

represent ± S.E.

Figure 2. Evolution of ammonium oxidizing bacteria popula-

tion from control bacteria (), from bacteria that received urea

(), and bacteria that received DMPP () at 6 and 14 days.

Each bar chart represents the mean four values. Vertical bars

represent ± S.E.

ammonium oxidization bacteria populations by 27% and

22% compared to control treatments in experiments 1

fertilized with DMMP compared to soils fertilized with

urea in rice crops. Likewise, our results showed that

DMPP depressed the activities of ammonium oxidization

bacteria as was also stated by Zerulla et al. [15] and

Irigoyen et al. [16]. Li et al. [5] and Fernandez-Escobar

et al. [17] also concluded that the NIs inhibited ammo-

nium oxidization bacteria activity, causing NO3

–-N re-

duction in leachates. Finally, the lack of growth in bacte-

ria cultivated with DMPP during the experiment is

mainly due to the effect bacteriostatic (not bactericide)

of this molecule, since DMPP diminishes the growth of

ammonium oxidizing bacteria, causing a reduction in the

concentration of nitrate in the growing media [15].

In conclusion, the activity of the ammonium oxidiza-

tion bacteria came from a tropical soil was inhibited by

DMPP treatment as compared to the urea and control

treatments. DMPP fertilizers could be considered an

useful tool in fertilization programs of rose plants in

order to reduce the contamination in surfacewater and/or

groundwater by nitrates leaching, since studies con-

ducted by Henao and Florez [14] estimated that that

and 2, respectively. Similar observations were found by

Li et al. [5], who reported that ammonium oxidization

bacteria populations were significantly reduced in soils



-N concentrations in leachates came from rose

plants cultivated were above the drinking water quality

rds (maximum contamination limit of 10 ppm standa



-N) [18].

4. ACKNOWLEDGEMENTS

This research was supported by the research division of the National

University of Colombia Project code No 9403. Also, authors want to

thank to Dr. Marcela Franco for her cooperation during this research.

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