Eect of liquid manure on the mole fraction of nitrous oxideevolved from soil containing nitrate

R. James Stevens a,b,*, Ronald J. Laughlin a,b

a Department of Agriculture and Rural Development for Northern Ireland, Agricultural and Environmental Science Division,

Newforge Lane, Belfast BT9 5PX, UKb Department of Agricultural and Environmental Science, The Queens University of Belfast, Newforge Lane, Belfast BT9 5PX, UK

Abstract

The same emission factor is applied to fertiliser N and manure N when calculating national N2O inventories.

Manures and fertilisers are often applied together to meet the N needs of the crop, but little is known about potential

interactions leading to an increase in denitrication rate or a change in the composition of the end-products of

denitrication. We used the 15N gas-ux method in a laboratory experiment to quantify the eect of liquid manure

(LM) application on the uxes of N2 and N2O when the soil contained fertiliser15NO3 . LM increased the mole fraction

of N2O from 0.5 to 0.85 in the rst 12 h after application. More than 94% of the N2O was from the reduction of NO3 ,

probably due to aerobic nitrate respiration as well as respiratory denitrication. 2000 Elsevier Science Ltd. All rightsreserved.

Keywords: Nitrous oxide; Dinitrogen; Denitrication; Nitrogen-15; Mole fraction

1. Introduction

Major anthropogenic emissions of N2O may well

arise from animal manure (Berges and Crutzen, 1996).

An application of liquid manure (LM) to soil provides

NH4 for nitrication and organic C for metabolism.During nitrication some N2O can be produced

(Bremner and Blackmer, 1978). Metabolism of the or-

ganic C by heterotrophic micro-organisms can lower the

O2 status of the soil and create the anoxic conditions

conducive to denitrication of NO3 formed by nitri-cation or added as fertiliser (Firestone, 1982). The en-

vironmental impact of denitrication depends on

whether the end-product of NO3 reduction is N2O orN2. In the IPCC methodology for calculating national

N2O inventories, an emission factor of 0.0125 kg N2O

N kg1 N input is applied whether the inputs are syn-thetic fertilisers or animal manures (Mosier et al., 1998).

No account is taken for N2O produced by the interac-

tion between manures and fertilisers.

The mole fraction of N2O, dened as

N2O=N2ON2, produced by denitrication is vari-able and depends on environmental conditions and on

the microbial populations present (Firestone et al.,

1980). A high NO3 concentration, low pH and a readilyavailable organic C source favour the formation of N2O

relative to N2 (Firestone and Davidson, 1989). A LM

application alone should tend to favour N2 production

because it supplies organic C, has a high pH and a low

NO3 concentration. Whenever fertiliser NO3 and slurry

are applied together, the potential exists for enhanced

denitrication and an increase in the mole fraction of

N2O. Glucose addition increased the mole fraction of

N2O (Stevens et al., 1998), but the eect of LM on

the mole fraction of N2O emitted from soil containing

fertiliser NO3 has not been studied.

Chemosphere 42 (2001) 105111

* Corresponding author. Fax: +44-2890-662007.

E-mail address: jim.stevens@dardni.gov.uk (R. James

Stevens).

0045-6535/01/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved.PII: S 0 0 4 5 - 6 5 3 5 ( 0 0 ) 0 0 1 1 5 - 6

The 15N gas-ux method measures N2 and N2O ux

separately and allows nitrication to proceed (Malone

et al., 1998). We used this method in a laboratory in-

cubation experiment to investigate the eect of a LM

application on the mole fraction of N2O produced from

fertilised soil. Either LM or a solution containing an

equivalent rate of NH4 N was applied to soil containing15N-labelled NO3 . At intervals during the incubation wemeasured N2 and N2O uxes and the

15N enrichment of

the N2O. At the end of the incubation we measured the

size and enrichment of the mineral N pools. By inter-

rogation of pool sizes and enrichments we determined

the source of the N2O.

2. Materials and methods

2.1. Soil

An acid brown earth (Typic Dystrochrept) with a pH

of 6.0 and containing 48% sand, 31% silt, 20% clay and

12% organic matter was collected (010 cm deep) from a

long-term pasture site at the Agricultural Research

Institute, Hillsborough, Northern Ireland in November

1997. The soil was bulked, partially air-dried (0.22 g

H2O g1 oven-dry soil), sieved (

indophenol blue method. Nitrite was determined by a

manual colorimetric method based on the sulphanila-

midenaphthylethylenediamide procedure (Keeney and

Nelson, 1982). The 15N contents of the NO3 , NO2 and

NH4 in the extracts were determined by methods basedon their conversion to N2O (Stevens and Laughlin, 1994;

Laughlin et al., 1997).

2.7. Statistical analysis

Analysis of variance was carried out using Genstat 5

Release 3.1 to determine the signicance of treatments

on the uxes of N2O, N2 and CO2, the sizes and en-

richments of the NH4 , NO2 and NO

3 pools, and the

rate of nitrication. Analytical procedures applicable to

a repeated measure design were used for the data on gas

uxes. Values of least signicant dierence for P < 0:05(LSD) were calculated for comparing any two mean

results.

3. Results and discussion

3.1. Eect of liquid manure on respiration

On average, LM signicantly (P < 0:001) increasedthe rate of CO2 emission. The average ux rate increased

from 139 to 363 nmol C g1 h1 at LM1, and from 162to 449 nmol C g1 h1 at LM2. The eects during theobservation period are shown in Fig. 1. Fluxes were

greatest after 3 h and then generally declined with time.

These ux proles are consistent with the metabolism of

readily degradable components such as volatile fatty

acids (Beauchamp et al., 1989).

3.2. Eect of liquid manure on nitrous oxide emission

LM signicantly (P < 0:001) increased the ux ofN2O. The average of the measured rates increased from

0.090 to 0.635 nmol N g1 h1 at LM1 and from 0.060 to1.400 nmol N g1 h1 at LM2. The eects over time areshown in Fig. 2. Fluxes from control treatments, C1 and

C2, were not signicantly dierent (P > 0:05) betweenobservation times. The eects of LM were immediate

and lasted only for 48 h after LM application. Measured

uxes were signicantly higher (P < 0:001) than controlson each occasion between 3 and 24 h after manure

application. The uxes with LM2 were always higher

than with LM1 during the rst 24 h. Over the 72 h

observation period, the total cumulative loss of N2O

increased from 2.2 to 16.1 nmol N g1 at LM1 and from1.5 to 39.1 nmol N g1 at LM2.

3.3. Eect of liquid manure on dinitrogen emission

LM signicantly (P < 0:001) increased the ux of N2.The average of the measured rates increased from 0.067

to 0.249 nmol N g1 h1 at LM1 and from 0.067 to 0.879nmol N g1 h1 at LM2. The eects over time are shownin Fig. 3. Fluxes from control, treatments, C1 and C2,

were not signicantly dierent (P > 0:05) betweenobservation times. Measured uxes were signicantly

higher (P < 0:001) than controls after 12 and 24 h forLM1, and after 6, 12 and 24 h for LM2. The eects of

LM were greatest at 24 h after manure application. Over

the 72 h observation period, the total cumulative loss of

N2 increased from 2.0 to 8.0 nmol g1 at LM1 and from

2.0 to 33.2 nmol g1 at LM2.

3.4. Sources of nitrous oxide

Nitrous oxide can be formed during oxidation of

NH4 and during reduction of NO3 . Since the NO

3

pool was labelled, the relative contributions of nitri-

cation and denitrication can be quantied from the

measurements of 45R and 46R in N2O (Arah, 1997).

Values for the fraction of the N2O originating by

denitrication from the labelled pool and the enrich-

ment of the labelled pool are shown in Table 1. In the

LM1 treatment, the calculated values for the fraction

from the labelled pool were not signicantly dierent

from 1.0 so the N2O always originated from one pool

whose enrichment averaged 56.6 at.% excess. This

average value was between the values found for the

Fig. 1. The eects of LM at two rates (LM1 and LM2) on the ux of carbon dioxide in comparison with control treatments of

ammonium bicarbonate (C1 and C2).

R. James Stevens, R.J. Laughlin / Chemosphere 42 (2001) 105111 107

NO3 pool at time zero and after 72 h of incubation(Table 2), so all of the N2O was produced by the re-

duction of the NO3 pool. In the LM2 treatment, mostof the N2O was again produced by the reduction from

the NO3 pool (Table 1). Only after 6 h when the valueof the fraction from the enriched pool was 0.94, there

was any evidence that a fraction (0.06) of the N2O

originated from an unlabelled pool. Nitrication was

occurring during the incubation. By calculation from

the size and dilution of the NO3 pool at time zero andafter 72 h of incubation (Kirkham and Bartholomew,

1954), the rates of nitrication were the same at C1

and LM1 (0.28 lmol g1 d1) and at C2 and LM2(0.35 lmol g1 d1). Nitrication responded to NH4concentration rather than organic C and could have

therefore produced some N2O (

3.5. Eect of liquid manure on mole fraction of nitrous

oxide

LM increased the mole fraction of N2O evolved be-

tween 3 and 12 h after manure addition, the eects being

similar at both LM rates (Fig. 4). Average values during

the rst 12 h were 0.85, otherwise the mole fraction of

N2O was ca. 0.5. In studies, where the15N gas-ux

method was used to measure N2 and N2O uxes, the

mole fraction of N2O ranged from 1 to

N2O. These organisms have two NO3 reductases, a

membrane-bound reductase responsible for respiratory

denitrication under anaerobic conditions and a peri-

plasmic reductase involved in aerobic NO3 respiration(Bell et al., 1990). The relative rates of expression of

these reductases depend on the nature of the C substrate

and the O2 concentration. Conditions favouring the

periplasmic reductase and aerobic NO3 respiration oc-cur during the oxidative metabolism of reduced C sub-

strates such as butyrate (Sears et al., 1997). The liquid

manure application supplied reduced C substrates as

volatile fatty acids, one of which was butyrate. Hence

aerobic NO3 respiration may have been partly respon-sible for the pulse of N2O in the rst day after LM

application.

3.7. Recovery of 15N label

The recovery of 15N added as NO3 averaged 91% inall measured pools and was not signicantly dierent

between treatments (Table 3). Assimilatory nitrate re-

duction by bacteria would have been inhibited since

NH4 concentrations were >1 mM (Rice and Tiedje,

1989), but nitrate assimilation by fungi may have

occurred. This process has been suggested as being im-

portant in N cycling in forest soils (Stark and Hart,

1997) and in aquatic ecosystems (Caraco et al., 1998).

Alternatively, there may have been luxury uptake of

NO3 by denitrifying micro-organisms. Soil micro-or-ganisms under anaerobic conditions were found to take

up NO3 in excess for their immediate requirements forenergy generation (Ellis et al., 1996), probably storing it

in liquid vacuoles (Jorgensen and Gallardo, 1999).

Mineralisation of assimilated NO3 may have been re-sponsible for the

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