Jul 18, 1995 - conditions and the concentration of hydrazine sulfate reagent. Popula- ... either ammonium sulfate+citric acid or peptone alone. Ammonium.
Biol Fertil Soils (1996) 23:292-298
9 Springer-Verlag 1996
K. Inubushi 9 H. N a g a n u m a 9 S. Kitahara
Contribution of denitrification and autotrophic and heterotrophic nitrification to nitrous oxide production in andosols
Received: 18 July 1995 To quantify the contribution of denitrification and autotrophic and heterotrophic nitrification to N20 production in Andosols with a relatively high organic matter content, we first examined the effect of C2H2 concentrations on N20 production and on changes in mineral N contents. The optimum CzH 2 concentration for inhibiting autotrophic nitrification was 10 Pa. Secondly, and Andosol taken from an arable field was incubated for 32 days at 30~ at 60, 80, and 100% water-holding capacity with or without the addition of NH~ or NO3 (200 mg N kg-1), and subsamples collected every 4-8 days were further incubated for 24 h with or without C2H2 (10 Pa). At 60 and 80% water-holding capacity with NH] added, 87-92% of N20 produced (200-250 gg NzO-N kg q ) was derived from autotrophic nitrification. In contrast, at 100% waterholding capacity with or without added NO3, enormous amounts of N20 (29-90 mg N20-N kg -1) were produced rapidly, mostly by denitrification (96-98% of total production). Thirdly, to examine N:O production by heterotrophic nitrification, the Andosol was amended with peptone or NH~ (both 1000 mg N kg-1)+citric acid (20 g C kg q ) and with or without dicyandiamide (200 mg N kg-1). Treatment with citric acid alone or with citric acid+dicyandiamide suppressed N20 production. In contrast, ~eptone increased N20 production (5.66 mg N20N k g - ) mainly by denitrification (80% of total production). However, dicyandiamide reduced N20 production to 1.1 mg NzO-N kg -1. These results indicate that autotrophic nitrification was the main process for N20 production except at 100% water-holding capacity where denitrification became dominant and that heterotrophic nitrification had a lesser importance in the soils examine. Abstract
Dedicated to ProfessorJ. C.G. Ottow on the occasion of his 60th birthday K. Inubushi ( ~ ) 9H. NaganumaI - S. Kitahara Faculty of Horticulture, Chiba University,Matsudo, Chiba 271, Japan 1 C u r r e n t address:
Kawasaki Research Center, T. HasegawaCo.Ltd., Kariyado335, Nakahara-ku, Kawasaki,Kanagawa211, Japan
N20 9 Moisture 9 Acetylene inhibition 9 Organic substances 9 Andosols Key words
Introduction N20 has a strong potential for infrared absorption and thus for inducing global warming, and it destroys the stratospheric ozone layer through NO production (Cicerone 1989). Twenty-two percent of the total N20 emission comes from agricultural lands, especially fertilized soils (Bouwman 1994). Nitrification and denitrification are the most important biological processes in the production of N20 in soil (Firestone and Davidson 1989). To distinguish between the contributions of these two processes, the C~H2 inhibition method (Hynes and Knowles 1982) has been used widely to inhibit autotrophic nitrification. However this method has not been applied in Andosols. These volcanic soils are originally acidic, containing large amounts of soil organic matter, and cover 46.5% of upland fields in Japan (Ministry of Agriculture, Fishery and Forestry 1991). Besides the autotrophic nitrifiers, heterotrophic microorganisms are also important in producing N20 (Yoshida and Alexander 1970; Hynes and Knowles 1982). N20 production by heterotrophic nitrification is particularly important in forest and grassland soils which are mostly acidic and high in organic matter (Schimel et al. 1984; Robertson and Tiedje 1987; Pennington and Ellis 1993), because heterotrophic microorganisms are less susceptible than autotrophic nitrifiers to environmental impacts such as acid precipitation. So far, however, there have been no studies on heterotrophic nitrifiers in Andosols. The objects of the present study were: (1) to evaluate the use of the C2Ha inhibition method for distinguishing between the contributions of nitrification and denitrification to N20 production in Andosots, (2) to investigate the effects of moisture and the addition of NH~ or NO~ on N20 production and the contribution of nitrification/deni-
293 trification, and (3) to e x a m i n e the possibility o f N 2 0 production by heterotrophic nitrification in the A n d o s o l s .
Materials and methods Soils Two soils from upland fields supporting arable crops were used. Ureshino soil, from the cattle-feces plot of Mie Agricultur Research Station (Light-colored Andosols, non-allophanic), had an organic C content of 44.5 g kg 1, with total N 2.2 g kg-1, NH~ With soils at 100% water-holding capacity, N20 for- od of pre-incubation were estimated from Fig. 3 as cumumation in both NH~-amended and NO;-amended soils be- lative values and are shown in Table 1. Among the three came much larger (almost three times in magnitude) and treatments, the total N20 produced was highest in NH~varied more widely than at lower moisture contents. Expo- amended soil, of which about 92% was derived from nitrinential regression, however, showed that the higher the fication. In unamended or NO~-amended soils, total N20 C2H2 concentration, the more N20 formation was sup- formation was smaller, and the contribution of nitrification was about 60 or 69%, respectively. pressed (Fig. 1).
Fig. 3 a-e N20 formation in Chiba andosol at 60, 80, and 100% water-holdingcapacity with no additional N (a), additional NH~ (b), and additional NO3-N (e), experiment2
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100% WHC
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295 Fig. 4a-e Changes in mineral N contents in Chiba Andosol at 60, 80, and 100% water-holding capacity with amendments as for Fig. 3
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Table 1 Effectsof moisture content and mineral N addition on total N20 formation, contribu: dons to nitrification and denitrification over 32 days of incubation, and numbers of nitrifiers (ammonium oxidizers) and denitriflers after incubation. Initial populations (log MPN g-l) of nitriflers and denitriflers before incubation were 3.62 and 3.6, respectively. WHC water-holding capacity, MPN most probable number
4
8
12 18 24 Days of incubation
Moisture MineralN (% WHC)
32
0
4
8
J3 12 18 24 Days of incubation
El
0 32
4
8
Total N20 formation (lag NaO-N kg-1)
Contribution Population (% in total N20 formation) (logMPN g-l)
Mean
Nitrification DenitrificationNitrifiers
Denitrifiers
SD
60
No addition Ammonium Nitrate
29.4 202 95.7
1.52 1.15 2.53
59.4 91.9 68.9
40.6 8.l 31.1
4.84 4.60 3.98
4.56 5.30 4.80
80
No addition Ammonium Nitrate
56.1 247 118
2.28 6.92 4.23
57.2 87.1 63.0
42.8 12.9 37.0
5.59 4.72 5.08
4.54 5.43 5.72
1.5 24.6 3.9
98.5 75.4 96.1
4.84 5.53 4.93
4.12 4.46 4.94
100
32
Days of incubation
No addition 29300 Ammonium 79600 Nitrate 90000
At 80% water-holding capacity, N20 formation was almost in the same ranges as that at 60% water-holding capacity but had two peaks in NH~-amended soil (Fig. 3). The contributions of nitrification and denitrification were also very similar as 60% water-holding capacity. Therefore, the total N20 produced was little more than at 60% water-holding capacity, being largest in NH2-amended soil, of which 87% was derived from nitrification. In unamended or NO3-amended soils, total N20 was smaller and the contribution of nitrification was almost the same as those at 60% water-holding capacity (Table 1). The increase in NO~ and decrease in NH~ were very similar, as at 60% water-holding capacity (Fig. 4). In contrast to the results at 60 and 80% water-holding capacity, total N20 formation was much higher at 100% waterholding capacity, especially on day 0 of incubation, immediately after adjusting the moisture content. This initial N20 formation leached 14-15 mg N kg -1 in the NH~-amended soil and unamended soil, approximately three times the magnitude of that at 60-80% water-holding capacity and almost all of this was produced through denitrification. In Ntt~amended soil, however, N20 due to nitrification became ob-
670 800 3200
vious in the later stages of incubation. In unamended soil, N20 formation with C2H2 became larger than without C2H2 after 18 days of incubation, and these data were again omitted from Fig. 3, as for the same soil at 60% water-holding capacity. In NO3--amended soil, the initial N20 formation continued for 8 days. With these changes in N20 formation, the total N20 produced was enormous (29.3-90 mg N20N kg-l), and occurred mostly by denitrification (96-98% of total production in unamended and NO3-amended soil; Table 1). NO3 disappeared rapidly during 8-12 days of incubation. NH~ did not disappear, but remained at stable levels after 8 days (Fig. 4). Because total N20 production was estimated from periodical measurements of the N20 formation rate, the values given above will be overestimates if N20 formation ceased within the first 4 days of incubation. More frequent measurements are needed for more accurate estimates. Nevertheless, most of the NO3 disappeared quickly and 68-70% of the ambient NO~ was denitrified as N20 on day 0 in unamended or NH~-amended soil, and 17% in NO3-amended soil. In NH~-amended soil, NO20 formed by nitrification was also high in the later period of incubation compared with 60 and 80% water-holding capacity.
296 Citric acid + DCD
Citric acid
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20
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Nitrification
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[
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Daysof incubation
Fig. 5a, b N20 formation (a) and changes in mineral N (b) in Chiba alone and peptone+dicyandiamide, presumably by decomAndosol amended with citric acid, citric acid+dicyandiarmde, peptone position of the peptone. Subsequently, nitrification in the and peptone+dicyandiamide at 60% water-holding capacity (experi- soil with peptone alone decreased NH~ and increased NO~ ment 3) concomitantly. In contrast, in the dicyandiamide treatment, these changes in mineral N stopped almost completely At the end of the incubation, the populations of auto- after 7 days except for a slow increase in NH~. trophic nitrifiers and denitrifiers were counted (Table 1). Autotrophic nitrifiers increased only in citric acidAlthough both increased by one or two orders of magni- amended soil after the incubation, and not in dicyanditude compared with the values before incubation, there amide-amended soil (Table 2). The addition of peptone increased the growth of denitrifiers by more than two orders was no consistent tendency among the treatments. of magnitude both with and without dicyandiamide.
Experiment 3: Effect of amendment with organic substances on N20 formation Treatment with citric acid alone or with dicyandiamide each suppressed N20 production to less than 0.5 lag N20N kg -1 throughout 28 days of incubation except on day 0 when about 3 lag N20-N kg -1 was produced, mostly by denitrification (Fig.x5). NH~ decreased in the first 7 days, probably due to immobilization, then remained almost constant until the end of the incubation. NO3 remained at a low level throughout the incubation in both treatments. There was no significant difference between the treatments with citric acid alone and with dicyandiamide. Amendment with peptone increasee N20 production considerably at the beginning of the incubation, mainly by denitrification (Fig. 5). It then remained relatively high at 33-83 lag N20-N kg -a, mostly by autotrophic nitrification. As a result, total N20 production was estimated as 5.66 mg N20-N kg -1, and 80% of the total production was derived from denitrification (Table 2). In contrast, peptone+dicyandiamide reduced the initial N20 production to one-fourth of that with peptone alone and inhibited N20 production completely after 7 days. Thus the total N20 production was 1.1 mg N20-N kg -a (Table 2). NH~ increased in the first 7 days in the treatments with peptone
Discussion Influence of C2H2 concentration on nitrification inhibition The present data confirmed that C2H2 inhibits N20 formation by nitrification in Andosols as reported for non-andosols (Sahrawat et al. 1987; Klemedtsson et al. 1988). However, to inhibit autotrophic nitrification completely, the Andosols examined needed 10 Pa C2Ha as the optimum, which is a higher concentration than that reported for non-andosols (Sahrawat et al. 1987; Klemedtsson et al. 1988). The NaO formation did not cease completely, which might be due to heterotrophic nitrification. Although this NaO formation was very low, the possibility of heterotrophic nitrification is discussed below. N20 formation by denitrification was obvious at 100% water-holding capacity, but not at lower capacities. N20 formation under wetter flooded conditions was less than at 100% water-holding capacity (data not shown), probably because N20 is further denitrified to N2 under such conditions. To inhibit this process, Andosols may require a higher C2H2 concentration than 5 kPa or a longer incubation period. Simarmata et al. (1993) reported that C2H2 (1 kPa) blockage of N20 reductase activity ceased in soils with a wider C :N ratio than 130 when lime
297 Table 2 Effect of organic matter admendments on total N20 formation, contributions to nitrification and denitrification over 28 days of incubation, and numbers of nitrifiers (ammonium oxidizers) and denitrifiers after incubation. MPN most probable number, DCD dicyandiamide
(pg N20-N kg-1)
Contribution (% of total N20 formation)
Me~
Nitrification Denitrification Nitrifiers
Denitrifiers
15.6 24.2
84.4 75.8
3.60 3.58
3.78 4.08
19.7
