soil denitrification in sealed soil-plant systems

Plant and Soil 33, 113-127 (1972) Ms. 1779

I.

SOIL D E N I T R I F I C A T I O N IN S E A L E D

S O I L - P L A N T SYSTEMS

EFFECT OF PLANTS, SOIL WATER CONTENT AND SOIL ORGANIC MATTER CONTENT

by R. C. STEFANSON

Department of Agricultural Biochemistry and Soil Science, Waite Agricultural Research Institute, University of Adelaide,

Glen Osmond, South Australia 5064

SUMMARY

Evolut ion patterns and constituent components of denitrificatioI1 have been measured in sealed soil-plant systems. In two samples of the Urrbrae red-brown earth containing 1.6 and 2.3% organic C, respectively, the growth of the plant consistently increased the amounts of N20 and N2 from the soil at all water contents below field capacity. At soil water contents above field capacity, the total losses of soil nitrogen were limited in the pasture soil (org. C 2.3Yo) by the amount of nitrate substrate and in the crop soil by the lack of easily decomposible soil organic matter. The yields of plant tops were low in these treatments.

In the presence of plants, N2 was evolved preferentially, while in their absence N20 accounts for most of the soil nitrogen loss. This trend was most pronounced in the crop soil.

The pore space relationships measured at the end of the experiments showed that potential oxygen diffusion pathways were more restrictive in the crop soil than in the pasture soil. The plant effect on soil denitrification was two-fold, firstly by increasing the demand for oxygen in the soil and secondly by supplying easily decomposible organic matter.

INTRODUCTION

Losses of soil nitrogen as volatile products have been shown to be increased by the growth of plants in pots 16 and in sealed growth chambers 14. W o l d e n d o r p 16 found that plant roots which sup- ported an active rhizosphere population stimulated the loss of soil nitrogen as denitrification products. In sealed growth chambers 14 considerable amounts of nitrogen and nitrous oxide appeared in the gaseous phase of the soil-plant atmosphere under a wide range of

1 14 R. C. STEFANSON

soil conditions. Where the soil water content was less than field

capaci ty, the growth of plants increased the quanti t ies of ni trogen

and nitrous oxide. In these systems, quant i ta t ive est imation of the volatile forms of nitrogen was possible as well as changes in the

evolution pat terns which occurred with differing soil conditions. In

bo th the open and closed systems, the denitrification was p robab ly a

biological process occurring in anaerobic microsites in a par t ia l ly

aerobic soil ~. B u r f o r d and M i l l i n g t o n 4 have shown the presence of N20 in

the surface soil of the Urrbrae red-brown ear th after rain. Because

of the diverse na ture of the source and diffusion rates, these authors

were unable to make an accurate est imate of the losses of soil nitro-

gen as nitrous oxide in the field. In later work ~, the determinat ions

of ni trogen to argon ratios did not permit the evaluat ion of N2 as a

denitrification product in field soils. In t h e present experiment, sealed growth chambers have been

used to establish the effect of soil organic ma t t e r content and soil

water content on the evolution pa t te rn and the const i tuents of

ni t rogenous gases evolved from a soi l -plant system.

MATERIALS AND METHODS Soils

The Urrbrae red-brown earth 9 was sampled from contiguous areas which had been either under an annual pasture of Tri[olium subterraneum and Lolium rigidum for the previous 10 years or had been cropped continuously with cereals for the last 10 years. Both sites had received annual top-dressings of superphosphate. These soils are referred to in the text as pasture and crop soils, respectively. The 0-10 cm samples were collected in an air-dry state and passed through a 0.6-cm rotary sieve.

Sealed growth chambers The sealed growth chambers, ancillary equipment and the operational

techniques have been described elsewhere 12

Methods o/ nitrogen analyses The total soil nitrogen including inorganic nitrogen was determined by

Kjeldahl digestion 1 on air-dried samples taken before and after the experimental period. The nitrogen contents of the plant material were estimated by micro-Kjeldahl. The water vapour from evapotranspiration was condensed and passed through a cation exchange resin. Ammonium was determined in the washings from the resin at the end of the experiment. A vial of 40% w/v KOH was included in each chamber to absorb the CO2 evolved from the

SOIL DENITRIFICATION. I 1 15

respiration of the soil after wetting. Total nitrogen content of the KOH solution was measured at the conclusion of the experiment, by neutralization of the excess KOH by bubbling with CO2 and by distillation with Devarda's alloy into 0.01 N HC1 followed by boiling, cooling and t i trat ion with 0.01 N NaOH.

Preparation o/the pots o/soil Eight kg of soil were packed to a bulk density of 1.45 g/cc. Basal dressings

of 1.3 g Ca(H2POd)2.H20 and 0.005 g NaMoOd.2H20 per pot were spread 1 cm below the surface of the soil.

In the pasture soil 10 seeds of Tricticum aestivum var. Heron were placed just below the surface of the soil and covered with 1 cm of coarse gravel. These pots were purged with 80% argon -- 20% oxygen gas mixture until the ef- fluent contained less than 100 ppm v/v of nitrogen. Approximately 100 ppm of nitrogen as Ca(NOa)~ in the appropriate quant i ty of water was forced under a slight positive pressure of argon into tile top of the purging tanks, Tiffs process caused a structural collapse of the surface crop soil and prevented the emergence of seedlings. For the crop soil the seeds were placed in water- saturated Pertite, which was placed on the surface after adding water and nitrogen. The techniques for transferring the pot containing the soil to the sealed growth chambers and exchanging the internal atmosphere for one of argon/oxygen have been described 12. Changes in the internal atmosphere were monitored daily for the first 10 days and then three times per week. The analyses were made by gas chromatography 2.

Measurement o/bulk density and porosity At the conclusion of each experiment the soils were sampled for moisture

content, dried at 36°C, ground and subsampled for the total nitrogen determinations. Before grinding, several 2-3 cm clods were selected randomly for the determination of bulk density, total porosity and pore space relationships. During the collection of the field samples, similar clods were collected for comparative studies. The clods were saturated with kerosene while under vacuum. After measurement of the total volume displacement 6, the clods were transferred to an alunclum base on a sintered glass funnel. The excess kerosene was removed at 2 cm suction and the pF curve determined to 0.2 bar.

RESULTS

(i) The effect o/soi l water content on valotile losses o/soil nitrogen

I n r e c o n s o l i d a t e d samples of b o t h soils, t he soil w a t e r c o n t e n t s

a t 0, 0.2 a n d 15 b a r were 45, 24 a n d 6 per cen t , r espec t ive ly . Af t e r

38 days , e q u i l i b r i u m levels of N 2 0 a n d N2 were e s t ab l i shed in the

sealed g r o w t h c h a m b e r s for all t he i m p o s e d e x p e r i m e n t a l c o n d i t i o n s

(Figs. 1A a n d 1B). U n d e r local f ield cond i t ions , d e n i t r i f i c a t i o n t a ke s

] 16 R. C. STEFANSON

• 12C

11t

100

801 o

z 70;

A ; ~°i

z

• P l an t s J

0 No P tan t s

• P l an t s

0 No Plants

m

B

so

, - 20

10

SOIL WATER CONTENT( ° / o )

Fig. 1. Effect of soil water content and plant growth on the evolut ion of soil n i t rogen as N 20 a n d N2 f rom A t he U r r b r a e p a s t u r e soil grid B ti le U r r b r a e

crop soil..

place under a water regime fluctuating around field capacity (240//0) To simulate these conditions in the sealed growth chambers, the volume of the syphon was adjusted to hold an equivalent of 8% soil water content. The water in the syphon was returned to the surface of the soil. In the drier treatments the 640 ml of water soaked into the soil within a few minutes; the comparative time in the wettest treatment was about an hour. Spatial determinations of tile soil water content in each pot at the end of the experiment showed that tile water content was within 3% of the calculated content throughout the pot of soil.

SOIL D E N I T R I F I C A T I O N . I 1 17

In the absence of plants, a change in average soil water content from 20-32% caused a large rise in the total amounts of nitrogenous gases evolved from both soils (Figs. 1A and 1B). All the added nitrate- N and 25 ppm of soil nitrate-N was reduced to N~O and N2 from the pasture soil at the highest soil water contents (Fig. 1A).

Figure 1B shows that, although the production of N20 and N2 increased from the crop soil with changes in the soil water content, soil denitrification was limited by other factors. The unfavourable pore space relationships (Table 5) and high soil water contents did not increase the levels of denitrification products beyond 20 ppm of soil nitrogen (Fig. 1B).

(ii) The e]/ect o] soil organic matter on de~itri/ication

It is well known that denitrifying bacteria have a high require- ment for energy which may be supplied by decomposible soil organic matter. Figure 1A, compared with Figure 1B, shows a strong inter- action between soil organic matter content and soil water content as determinants of soil denitrification. At soil water contents of 8-16, 12-20 and 16-24 per cent, the final level of denitrification products was similar for both the crop and pasture soils which were not sup- porting plant growth (Figs. 1A and 1B). At these soil water contents, the energy or substrate sources were not rate limiting for denitrification. Figures 1A and 1B show that organic substrate restricted the reduction of nitrate-N in th e crop soil at high soil water contents, whereas in the pasture soil denitrification increased rapidly at soil water contents above field capacity.

The patterns of evolution of N20 and N2 from the crop and pasture soils at 20-28 per cent soil water content are shown in Figures 2 and 3. After a short lag period of 1-2 days, N20 was evolved rapidly for 20 days from the pasture soil (Figs. 2A and 2B). By contrast, N20 was evolved slowly over a longer period from the crop soil (Fig. 3A).

Nitrogen gas was evolved from both soils at a steady rate during the first 20 days of the experimental period (Figs. 2A and 3A). The rate of gas evolution was related to the soil water content and organic matter content. At low soil water contents both N20 and N2 were evolved slowly for a long period. As soil water content was increased, N2 was released more rapidly but at a constant rate for approximately 20 days, while NgO was evolved more rapidly after the initial lag period. Figure 3B shows that the concentration of

l 18 R. C. STEFANSON

A

B

¢3

o

= A

• • N=O

• N 2

• • • Pasture Soil wi thout Plants

Pasture Soil wi th Plants

• N20 • N:z

A ~ I - ' e

T I M E IN D A Y S

Fig. 2. Evolution pa t te rn of Ne and N20 from the pasture soil at a fluctu- at ing soil water content of 20-28% in A the absence of plants and B the presence of plants. The broken lines show the measured values of NeO and N~

and the solid line indicates the corrected values of N20 and N2.

n i t r o g e n was n o t a t e q u i l i b r i u m a t 38 d a y s . Th i s s i t u a t i o n was ex-

c e p t i o n a l ; h o w e v e r , t i le r e su l t s s t r e n g t h e n t h e i m p o r t a n c e of p l a n t s

in p r o d u c i n g c o n d i t i o n s f a v o u r a b l e to d e n i t r i f i c a t i o n .

SOIL DENITRIFICATION. I 1 19

A

B

10 =

0

==

Crop Soil without plants • N 2 0

• N 2

Crop Soil with Plants

iND iCATES USE OF H 2 BURNER P

I

t t

• N 20 • NZ I

, j I . 1 ( S

t F S

. .,:. _

. _ _ . . . . . r . . c - q - . . , , T . . . . t"T-: 2 '~ 6 8 10 12 1/* 16 tE 20 22 24 26 21~ 30 32 3/~ 36 38

T i M E I N D A Y S

Fig. 3. Evolution pat tern of N~ and N20 from the crop soil at a fluctuating soil water content of 20-28% in A the absence of plants and B the presence of plants. Tile broken lines show the measured values o5 N20 and N2 and the

solid line indicates the corrected values of N20 and N~.

(iii) The e//ect o/ the plant on soil denitri/ication

The effect of the plant on the evolution pattern of nitrogen and nitrous oxide was two-fold; firstly, at all soil water contents at or below field capacity the plant increased the amount of denitrification products appearing in the internal atmosphere of the sealed growth chambers (Tables I-4); secondly, in the presence of plants relatively more of the nitrate-N was reduced to N2 as compared to

120 R.C. STEFANSON

the N20 evolved in the absence of plants. The experimental design does not permit separation of the N20 and N2 evolved from the soil or the plant. However, the evolution patterns of N2 and N20 with changing soil water content and soil organic matter content suggest that the majority of these gases are evolved from the soil-plant root system rather than from the plant alone.

At all soil water contents below field capacity in the pasture soil, the presence of plants consistently increased the losses of soil nitrogen compared to those occurring in their absence (Tables 1 and 2). These denitrification losses were maximal in the chambers with the highest yields of plant tops (Table 2). The yield of tops in the driest t reatment was small because of low soil water contents (Table 2). The wheat variety Heron is particularly susceptible to excess soil water, and thus poor plant growth resulted in the wet treatments (Table 2). In the pasture soil, the plant induced the largest increase in denitrification in 16-24 per cent soil water t reatment (Fig. 1 A) where the plant yield was high (Table 2) and soil aeration was restricted by soil water in the larger pores. Except in the two wettest treatments on the crop soil, the actively growing plant caused a very large increase in the volatilization of soil nitrogen (Fig. 1B). In the wettest treatments the total evolution of N20 and N2 was similar in the presence and absence of plants; however, plant yields were low in these treatments (Table 4). The highly significant increase in denitrification occurring in the crop soil and the pasture soil may be the result of changes in the oxygen status of the soil atmosphere induced by the visually abundant root growth and the addition of organic substrate in the form of root exudates.

Nitrate may be reduced directly to nitrogen under conditions of very low partial pressure of oxygen. Also under these conditions nitrous oxide may be absorbed and reduced to nitrogen 5. No evi- dence was found for absorption and reduction of N20 to N2 in the sealed growth chambers. Removal of the excess oxygen evolved from photosynthesis was carried out with a hydrogen burner. This operation decomposed the N20 to Ns in chambers filled with pure gases. This decomposition was not completely quantitative under experimental conditions because of the restriction on diffusion of N20 from the wet soil mass. Except for Figures 2 and 3, the results have been corrected for this decomposition.

Nitrogen was evolved preferentially to N20 from the crop soil in

SOIL DENITRIFICATION. I 121

T A B L E 1

N i t r o g e n b a l a n c e shee t fo r pasture soil without p l a n t s (nag N pe r k g o v e n - d r y soil)

T r e a t m e n t 1 2 3 4 5 5 W a t e r c o n t e n t 2 8 - 3 6 % 2 4 - 3 2 % 2 0 - 2 8 % 16 2 4 % 1 2 - 2 0 % 8 - 1 6 %

Be/ore experiment

T o t a l soil * 1480

Ca(NO~)~ 94

After 38 days

N~O 70.5 88.0 46.0 0.7 0.5 0.5 N~ 43.0 30.0 5.2 3.1 1.7 1.5

K O H ( N O 2 ) 0.1 0 0 0 0 0 N H ~ 0.1 0.2 0.1 0 .2 0.2 0.2

W a t e r f ron l s y p h o n 0.2 0.1 0 0.1 0 0.1

T o t a l soi l** 1498 1488 1538 1595 1488 1488

A p p a r e n t t o t a l n i t r o g e n b a l a n c e + 3 8 + 2 2 + 15 + 2 5 + 16 + 16

* M e a n of 12 d e t e r m i n a t i o n s .

** M e a n of 4 d e t e r m i n a t i o n s .

T A B L E 2

N i t r o g e n b a l a n c e shee t fo r pasture soil with p l a n t s (rag N pe r k g o v e n - d r y soil)

T r e a t m e n t 1 2 3 4 5 6 W a t e r c o n t e n t 2 8 - 3 6 % 2 4 - 3 2 % 2 0 - 2 8 % 1 6 - 2 4 % 1 2 - 2 0 % 8 - 1 6 %

Be/ore experiment

Soil * 1527

Ca(NOB) ~ 98 Seeds 1.4

Alter 38 days

N 2 0 80.0 62.9 21.0 16.9 0.7 1.0

N2 41.0 52.0 ~21.0 7.2 2.1 1.5 K O H ( N 0 2 ) 0.1 0. I 0.1 0 0.I 0 N H a 0.2 0.2 0.1 0.1 0.1 0 W a t e r f r o m s y p h o n 0.1 0 0 0. i 0 0.1 P l a n t t o p (gin) 0.65 0.70 4 .02 6.68 8.59 0.67

P l a n t % N ** 3 .22 3.37 4.57 4 .62 4 .62 4.50 P l a n t u p t a k e 2.6 3.0 23,0 88.5 49.6 3.8

Soil * ** 1475 1593 1596 1596 1550 1604

A p p a r e n t t o t a l

n i t r o g e n b a l a n c e - - 27 - - 29 + 35 + 32 - - 4 - - 22

* Mean of 12 rep l i ca te s . ** M e a n of 3 r ep l i ca te s . *** M e a n of 4 r ep l i ca t e s .

122 R.c. STEFANSON

the presence of plants (Tables 3 and 4). Soil water content did not affect this trend. The comparable situation in the pasture soil shows that relatively more N2 was evolved, but not a predominance of N2

in the planted treatment (Tables 1 and 2). Figures 2 and 3 show the evolution patterns for N~O and N2 for both the crop and pasture soils, with and without plants grown at 20-28 per cent soil water content. In both soils the presence of plants has increased the relative evolution of N2 gas, while the majority of volatilization of soil nitrogen occurs as N20 in the unplanted treatments.

(iv) Nitrogen balance sheets Tables 1 to 4 show the nitrogen balance sheets in the four experi-

ments. Within the limitations of total soil nitrogen determinations, the only major volatile losses were N~O and N2. Trace amounts of total nitrogen were found in the KOH solution in the water re- maining in the syphon and some NH4 + was detected on the cation exchange column. In this case no discernible trend existed between the amount of nitrogen and the experimental treatment. Although the amount of nitrogen ill the KOH was small, this suggests that some NO or NO~ was evolved from the soil system. The NH4 + collected from the condensed water of evapotranspiration was not related to treatment, and may have evolved from either the plant or the soil.

(v) Physical properties o! the soils Table 5 shows the bulk densities, total porosities and the pores

drained at 200 cm H20 suction of 2-3 cm clods taken at the conclusion of the experiments and from the field at the time of sampling. No differences could be detected in the physical measurements of the clods taken from several planted and unplanted chambers of the same soil. Bulk density and total porosity of the clods were similar for both the field and pot soils. The pots were packed to an average bulk density of 1.45 g per cc, but solne consolidation during the flushing process of alternating vacuum and slight positive pressure increased the density of the wet soil.

Clods taken from the crop soil contained a smaller proportion of pores which drained at 0.2 bar than those taken from the pasture soil (Table 5). For both soils, disturbance and repacking of the soil increased the volume of fine pores (Table 5). The decrease in large

S O I L D E N I T R I F I C A T I O N . I

T A B L E 3

N i t r o g e n b a l a n c e shee t fo r crop soil without p l a n t s (mg N p e r k g o v e n - d r y soil)

123

T r e a t m e n t 1 2 3 4 5 6 W a t e r c o n t e n t 2 8 - 3 6 % 2 4 - 3 2 % 2 0 - 2 8 % 1 6 - 2 4 % 1 2 - 2 0 % 8 - 1 6 %

Be/ore experiment

Soil * 1000

Ca(NO3) 2 99

A/ter 38 days

N 2 0 18.9 18.7 16.1 2.9 0.7 0.3

N2 4.6 3.4 3.4 2.2 2.0 1.4 K O H ( N O ~ ) O. 1 0 0 0.1 O. 1 0 N H a 0.1 0.1 0.1 0.1 0.1 0.1 W a t e r f r o m s y p h o n 0.1 0 0 0 0 0

Soil ** 1050 1050 1100 1130 1090 1090

A p p a r e n t t o t a l

n i t r o g e n b a l a n c e - - 2 5 - - 2 5 + 2 0 + 3 5 - - 1 0 - - 1 0

* M e a n of 12 d e t e r m i n a t i o n s .

** M e a n of 4 d e t e r m i n a t i o n s .

T A B L E 4

N i t r o g e n b a l a n c e shee t fo r crop soil with p l a n t ( rag N per k g o v e n - d r y soil)

T r e a t m e n t 1 2 3 4 5 6

W a t e r c o n t e n t 2 8 - 3 6 % 2 4 - 3 2 % 2 0 - 2 8 % 1 6 - 2 4 % 1 2 - 2 0 % 8 - 1 6 %

Be/ore experiment

Soil * 990

Ca(NOa)2 98 Seeds 2

Alter 38 days

N 2 0 2.7 4.6 7.1 7.7

N2 14.8 14.5 30.1 31.8 K O H ( N O ~ ) 0 0 0 0

N H 3 0.2 0.8 0.1 0.1 W a t e r f r o m s y p h o n 0 0 0 0.1 P l a n t t ops (g) 0.2 2.0 3.58 1.96

P l a n t N c o n t e n t % ** 1.44 1.17 1.29 1.22 P l a n t u p t a k e

( m g / k g soil) 0.4 3.0 5.8 3.0

Soil *** 1040 1040 1010 1030

A p p a r e n t t o t a l n i t r o g e n b a l a n c e - - 30 - - 25 - - 35 - - 10

0.9 1.0 8.1 8.4

0 0 0.1 0.1 0 0 2.13 0.76 1.64 2.17

4.4 1.6

1040 1050

- - 3 5 - - 3 2

* Mean of 12 rep l i ca te s . ** M e a n of 3 r ep l i ca te s . *** M e a n of 4 r ep l i ca te s .

124 R . C . STEFANSON

TABLE 5

Bulk densities and porosities of clods taken from the field and the experimental pots

Bulk densi ty Total porosity Volume of pores Soil (g]ec) (per cent) drained between

0-0.2 bar

Mean * Range Mean * Range Mean * Rarlge

Crop field 1.59 1.53-1.68 40.3 36.6-43.8 10.2 7.9-12.3 Pot 1.66 1.58-1.71 36.2 33.1-40.9 3.3 2.4- 4.5

Pasture field 1.65 1.59-1.74 38.6 36.6-41.2 10. I 6.9-14.9 Pot 1.63 1.56-1.74 39.4 37.1-40.7 7.0 4.8- 7.9

* Means are of ten replicates.

pores or the increase in tortuosity is more pronounced in the crop soil. Thus, the potential for anearobic microsites is greater in the potted soils, particularly in the crop soil.

DISCUSSION

The dissimilation of nitrate to N20 and N~ in the soil-plant system may occur in the soil or internally in the plant ~5. Measure- ment of the accumulation of N20 and N2 in the atmosphere of the sealed growth chamber estimates the sum of these processes, less any assimilation of these gases. The concentration of N2 within the chambers at the end of the experiment was a net balance of N2 evolved and of any N2 fixation that may have occurred during the experimental period. Twenty hours before the end of the experimental period some acetylene was added to all chambers. Ethylene concentrations were determined at times 0 and 20 hours to assay for N2 fixation s. Small differences in ethylene concentration suggested that some potential fixation of N2 could have occurred in the chambers. These results are not conclusive because the soil had been in an atmosphere low in N for at least 3 months and N2 may be an acti- vator in N2 fixation. Residual fertilizer and N20 are known to inhibit N fixation.

More denitrification products were evolved with increasing soil water content, with higher levels of native soil organic matter and in the chambers containing plants, suggesting that the majority of N20 and N2 appearing in the sealed growth chambers was of biological


and not of chemical origin. Incubating samples of the same soils with nitrate-N, B u r f o r d 3 showed that only traces of N20 and N2 were evolved in the sterile flasks and that large amounts of N2 and N20 appeared in the non-sterile flasks. The denitrification mechanisms in these systems were largely biological.

The close proximity of nitrous acid and amino acids within the plant may lead to some evolution of N2 within the plant 15. The importance of this reaction as a source of N2 in the sealed chambers was probably very small. Losses of nitrogen from plants grown in open sterile sand culture 10 were negligible while in the same experiments M i c h o u s t i n e et al.lO showed considerable losses of nitrogen when the same system had been innoculated with soil denitrifying bacteria. Earlier, W o l d e n d o r p 16 had shown that losses of nitrogen from the soil-plant system occurred from the soil, not the plant. The results in this paper suggest that nitrate-N was reduced biologically in the soil or rhizosphere. Partial confirmation of this would come from a comparison between sterile and non-sterile soil and culture solution systems, and this is probably not feasible in the sealed growth chambers.

Denitrifying bacteria can utilize nitrate ions as a terminal electron acceptor provided the partial pressure of oxygen is very low and a suitable source of hydrogen donors exists in the soil v. The first requisite is met if demand for oxygen exceeds the diffusion of oxygen through the soil to the sites of respiration. Supply of oxygen is controlled by the pore space relationships and soil water content. Hydrogen donors may come from the soil organic matter or root exudates. Thus, plants may affect both of these factors which control denitrification in the soil-plant system. The smaller proportion of large pores in the crop soil (Table 5) would restrict the rapid diffusion of oxygen in the soil gases. At a similar soil suction th e crop soil would have a potentially larger anaerobic volume, provided soil respiration rates were similar. However, in the absence of plants in the crop soil, the denitrification losses were limited by the lack of an energy source for the denitrifying bacteria. Plants increased the total demand for oxygen and were able to supply an energy source in the form of root exudates, resulting in large relative increases in N20 and N2 (Fig. 1B) evolved, particularly at the lower average soil water contents. Although soil respiration was probably higher in the pasture soil, the more favourable porosity (Table 5) maintained the

126 R.C. STEFANSON

flux throughout the soil. The introduction of the plant in the sealed growth chambers containing this soil at low and high soil water contents only slightly increased the evolution of N20 and N2, except at the critical soil water content of field capacity (Fig. IA). At the end of the experiment the soil water was evenly distributed through the soil mass. At the higher soil water contents, the water from the syphon laid on the surface of the soil for periods of up to one hour. In later experiments 13 the water from the syphon was evenly distributed through the pot of soil via a ceramic candle. The total denitrification was similar in both cases.

In the soil-plant system there are at least two pathways for the reduction of nitrate-N to N20 and N2, because plant growth favoured the direct reduction of nitrate-N to N2 (Table 4) and to N20 in the absence of plants. Even at 3.6 per cent N20 no reabsorption and further reduction occurred in the pasture soil at high but unsaturated soil water contents 11. In both, the predominant gas was evolved rapidly after a short lag period. The reduction of nitrate-N to N2 appears to be directly influenced by the rhizosphere, while the second shows that N20 is not an obligatory precursor of N2 evolution. However, conditions in the unplanted chamber are very different from those occurring in incubation flasks 11 _ namely, in geometry, gas movement and evaporation, which reached 400 ml per day in the wetter treatments containing plants.

ACKNOWLEDGEMENTS

I wish to t h a n k t he S o u t h A u s t r a l i a n W h e a t I n d u s t r y R e s e a r c h C o m m i t t e e which f inanced th i s project . M a n y helpful suggest ions were m a d e d u r i n g t he e x p e r i m e n t a l per iod b y Professor D. J. G r e e n l a n d a n d Dr. J. R. B u r f o r d , while du r ing t he p r e p a r a t i o n of t he m a n u s c r i p t the help of Professor D. J. D. N i c h o l a s a n d Dr. D. G. L e w i s is acknowledged .

Received July 20, 1971

REFERENCES

1 13remner, J. M., Total nitrogen. In: Black, C. A. (ed.), Methods of Soil Analysis. No. 9 ill Agron. Series, Am. Soc. Agron., Madison, Wisc. 2, 1149-1176 (1965).

2 Bur fo rd , J. R., Single sample analysis of N2-N20-CO2-A-O2 mixtures by gas chromatography. J. Chromat. Sei. 7, 760-762 (1969).

3 B u r f o r d , J. R., Gaseous losses of nitrogen from soils by denitrifieation. Ph.D. Thesis, Adelaide (1969).


4 B u r f o r d , J. R. and M i l l i n g t o n , R. J., Nitrous oxide in the atmosphere of a red- brown earth. 9tll Intern. Congr. Soil Set. Trans., Adelaide, 505-511 (1968).

5 Cady, F. B. and B a r t h o l o m e w , W. W., Sequential products of anaerobic denitrifieation in Norfolk soil material. Soil Sci. Soc. Am. Proc. 24, 477-482 (1960).

6 Cur r i e , J. A., The volume and porosity of soil-crumbs. J. Soil Sci. 17, 24-35 (1966). 7 G r e e n w o o d , D. J., Nitrification and nitrate dissimilation in soil. II. Effect of

oxygen concentration. Plant and Soil 17, 378-391 (1962). 8 H a r d y , R. F. W., H o l s t e n , E. K., J a c k s o n , E. K. and B u r n s , R. C., The acety-

lene-ethylene assay for N2 formation : Laboratory and field evaluation. Plant Physiol. 43, 1185-1207 (1968).

9 L i t c l l f i e ld , W. H., Soil survey of the Waite Agricultural Research Institute. C.S.I.R.O. Australia Div. Soils, Divl. Rep. 2[Sl, 1-37 (1951).

10 M i c h o u s t i n e , E. N., H a k i m , A., A b a z l i e v i t e l l , S. D. and Legg, J. O., Le processus de la denitrification et les pertes del ~zote par le sol. Ann. Inst. Pasteur 109 supp. no. 3, 235-246 (1965).

11 No m mik, H., Investigations on denitrification in soil. Acta Agr. Seand. 6, 195 228 (1956).

12 S t e f a n s o n , R. C., Sealed growth chambers for studies of the effects of plants on the soil atmosphere. J. Agr. Eng. Research 15, 295-301 (1970).

13 S t e f a n s o n , R. C., Soil denitrification in sealed soil-plant systems. II . The effect of soil water content and form of applied nitrogen. Plant and Soil 37, 129-140 (1972).

14 S t e f a n s o n , R. C. and G r e e n l a n d , D. J., Measurement of nitrogen and nitrous oxide evolution from soil-plant systems using sealed growth chambers. Soil Sci. 109, 203-206 (1970).

15 S t e w a r d , F. C. and D u r z a n , D. B., Metabolism of nitrogenous compounds. In: S t e w a r d , F. C. (ed.), Plant Physiology. Academic Press, New York, 4A, 379-680 (1965).

16 W o l d e n d o r p , J. W., The influence of living plants on denitrifieation. Meded. LandbHoogesch. Wageningen 63 (13), 1-100 (1963).

soil denitrification in sealed soil-plant systems

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