Short communication
Microbial control of nitrate concentrations in an agricultural soil
treated with dairy waste compost or ammonium fertilizer
Wei Shi
1, Jeanette M. Norton*
Department of Plants, Soils, and Biometeorology, Utah State University, Logan, UT 84322-4820, USA
Received 4 June 1999; received in revised form 9 November 1999; accepted 14 February 2000
Abstract
We conducted a 112-day laboratory incubation of an agricultural soil treated with dairy-waste compost or ammonium sulfate ((NH4)2SO4) to examine the role of microbial production and consumption of NO
ÿ
3 in controlling soil NO
ÿ
3 concentrations.
Inorganic N, net N process rates, nitri®cation potentials and gross N process rates were measured at various time periods in the treated soils. Microbial consumption ofNOÿ
3 was not an important process in controlling soilNO
ÿ
3 concentrations in these soil
systems. Transient growth in the nitri®er population was observed with ammonium sulfate but not compost addition. Nitri®cation rates were signi®cantly correlated with and comprised about 50% of the gross N mineralization rates, suggesting that nitrifying bacteria were not weaker competitors for soil NH
4 than heterotrophs in these systems. 7 2000 Elsevier Science
Ltd. All rights reserved.
Keywords:N mineralization; Nitri®cation;15Npool dilution; Microbial nitrate consumption; Gross N process rates
Control of soil NOÿ
3 concentrations has both agri-cultural and environmental importance. Appropriate agricultural N management should control NOÿ
3 con-centrations at levels meeting crop N requirements without excessive NOÿ
3 accumulation in soil, because excess NOÿ
3 is susceptible to loss by leaching or deni-tri®cation, andNOÿ
3 loss decreases N-fertilizer use e-ciency. Microbial production and consumption of
NOÿ
3 occur simultaneously and their relationship con-trols soilNOÿ
3 concentrations. Microbial assimilation of NOÿ
3 has not been con-sidered an important process in controlling soil NOÿ 3 concentrations in most agricultural soils, because microorganisms generally prefer NH
4 for their growth (Jansson et al., 1955; Jansson, 1958; Jones and Richards, 1977), and NH
4 even at relatively low
con-centrations (i.e., <1 mg N gÿ1
soil) may decrease mi-crobial utilization of NOÿ
3 (Rice and Tiedje, 1989). Nitrate accumulation is often observed in many agri-cultural soils. One explanation is that microbial assimi-lation of NOÿ
3 is negligible in those soil systems, and that nitri®cation is the dominant process controlling soil NOÿ
3 concentrations. In contrast, it may be that signi®cant microbial assimilation of NOÿ
3 does occur, but that production of NOÿ
3 greatly exceeds NO ÿ 3 con-sumption by both microorganisms and plants. The de-termination of the gross rates of simultaneous production and consumption requires the use of isoto-pic methods.
The functional groups of soil microorganisms act variously as producers, consumers, and competitors of the dierent forms of soil N. For example, hetero-trophs not only decompose soil organic matter, supply-ing NH
4 to soil nitri®ers, but may also compete with nitri®ers for soil NH
4 (Verhagen and Laanbroek, 1991). Though soil organic amendments such as com-post signi®cantly increase the activities of soil hetero-trophs (SchnuÈrer et al., 1985; Fauci and Dick, 1994),
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1
Current address: Dept. of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA.
* Corresponding author. Tel.: 2166; fax: +435-797-2117.
the eects of this enhanced microbial activity on ammoni®cation, nitri®cation, microbial N assimilation, and the interactions of these processes have not been well characterized. Our objectives were to examine if microbial consumption ofNOÿ
3 was an important pro-cess controlling soil NOÿ
3 concentrations, and whether soil nitri®ers can strongly compete for NH
4 with soil heterotrophs in N amended soils.
Timpanogos silt loam soil (®ne-loamy, mixed, super-active, mesic Calcic Argixeroll) was collected from the 0±15 cm surface layer in bulk from the Blue Creek Farm of Utah State University. Ammonium sulfate was used as the inorganic N source, while mature dairy-waste compost (M.G. Pace, unpub. MS thesis, Utah State University, 1996) was used as the organic N source. Soil and dairy-waste compost were sieved (2 mm) and stored at 48C until use. Selected properties of the soil and the compost are given in Table 1. Three soil treatments in this laboratory incubation exper-iment were (1) control, soil without additions; (2)
NH
4, soil with the addition of (NH4)2SO4at 50 mg N
kgÿ1
soil (equivalent to 100 kg N haÿ1
); and (3) com-post, soil with the addition of dairy-waste compost at 2.0 g (dry wt.) per 100 g soil (equivalent to 40 Mg dry wt. haÿ1
). Treated soil samples (20 g dry wt. equival-ent) were weighed into 120-ml specimen containers and placed in an incubator at 208C. The gravimetric water content of soil samples was adjusted to 19% (60% of ®eld capacity) every 4±6 days. Three samples of each treatment were randomly withdrawn at 0, 7, 25, 40, 70, and 112 days for measuring inorganic N and nitri-®cation potential (Hart et al., 1994). The nitrinitri-®cation potential soil slurries were continuously shaken for 24 h at 200 rpm (Stark, 1996), and the pH of the soil slur-ries was monitored and adjusted four times to main-tain the pH near 7.5.
Gross N process rates were measured by 15N pool
dilution techniques (Hart et al., 1994) for the three soil treatments, at the four labeling dates (incubation-day 7, 40, 70, and 112). For measuring gross nitri®cation and microbial assimilation of NOÿ
3 at each labeling date, three pairs of soil samples per treatment (as three replications) were withdrawn, and each soil sample received 1 mg N kgÿ1
soil as K 15NOÿ
3 solution (99% enrichment, 20 mgNOÿ
3±N L ÿ1
). For measuring gross N mineralization and microbial assimilation of NH
4, we used the same procedure as described above, except that soil samples were labeled with 15NH4Cl:
Depend-ing on the labelDepend-ing date, the amount of 15NH4Cl
injected varied from 1 to 5 N mg kgÿ1
soil, along with the enrichment decreasing from 99% to 50%. For each pair of labeled soil samples, one was extracted 15 min after and the other 24.25 h after the injection. A diusion procedure (Stark and Hart, 1996) was used to prepare samples for 15N analysis. The 15N
enrich-ments in theNH 4 orNO
ÿ
3 pools were analyzed by con-tinuous-¯ow direct combustion and mass spectrometry with ANCA 2020 system (Europa Scienti®c, Cincin-nati, OH).
The 15N excesses and recoveries measured shortly and one day after 15N injection were compared by two-way ANOVA with the labeling dates and treat-ments as factors. If the 15Nexcesses measured one day
after 15N injection were signi®cantly lower than those
measured initially, the gross N process rates were cal-culated by the equations of Kirkham and Bartholo-mew (1954). Eects of treatments and incubation days on soil process rates of N mineralization, nitri®cation, microbial N assimilation, and the ratios of these rates were analyzed using two-way ANOVA with the treat-ments and sampling dates as factors. All statistical analyses were performed using SuperANOVA software (Abacus Concepts, 1995, Berkeley, CA).
Throughout the 112-day incubation, NH
4±N con-centrations in the control soil and soil added with compost were very low (<1 mg kgÿ1
soil), whereas
NOÿ
3±N concentrations were ten to hundred times
NH
4±N concentrations and increased almost linearly with the incubation (Fig. 1). In the soil with added (NH4)2SO4, NH4±N concentrations rapidly decreased
and reached levels similar to the control soil or the soil with compost at 40 days, while the NOÿ
3 ±N concen-trations increased rapidly and non-linearly (Fig. 1).
We measured the recoveries of 15NOÿ
3 one day after the 15N injections and found that they did not dier
from those measured shortly after the 15Ninjections (p
= 0.26). The 100% recovery of 15NOÿ
3 combined with the accumulation of soil NOÿ
3 suggests that microbial assimilation of NOÿ
3 and denitri®cation were both very low, and that they can be ignored as important pro-cesses controllingNOÿ
3 concentrations under the exper-imental conditions. Additionally, the 15N excess measured one day after the 15N injection did not sig-ni®cantly dier from those measured shortly after injection (p = 0.10), which indicates that gross nitri®-cation rates could not be measured by the15Npool
di-Table 1
The selected properties of Timpanogos soil and dairy-waste compost Organic C (g kgÿ1
) Organic (g kgÿ1
) N C:N ratio EC (ds mÿ1
) pH
Soil 14 1.6 9.0 0.2 6.8
lution method and that the observed net rates rep-resent nitrate production. Rice and Tiedje (1989) docu-mented that NH
4 could decrease microbial assimilation of NOÿ
3 even at relatively low concen-trations (<1 mg N gÿ1
soil). They suggested that mi-crobial assimilation of NOÿ
3 would not be an important process in most agricultural soils. Wichra-masinghe et al. (1985) showed that there was no mi-crobial assimilation of NOÿ
3 in agricultural soils with 4% organic C and C:N ratios of 13. Recous and Mary (1990) also reported that microbial assimilation of
NOÿ
3 in cultivated soil was negligible when KNO3was
added at 50mg N gÿ1
soil without the addition of glu-cose C. In contrast, when gluglu-cose at 500 mg C gÿ1
soil was added along with the same amount of KNO3,
mi-crobial assimilation of NOÿ
3 occurred. The absence of microbial assimilation of NOÿ
3 in our experiment suggests a C limitation of the heterotrophic microor-ganisms even in the compost treated soil. While several studies have documented the importance of microbial
NOÿ
3 consumption in forest and grassland soils (Stark and Hart, 1997; Jackson et al., 1989; Schimel et al., 1989; Davidson et al., 1990), our data supports the paradigm of a C-limited but N sucient microbial bio-mass in agricultural soils. Additional in situ N cycle experiments in similarly treated ®eld soils also support this interpretation (data not shown).
Soil nitri®ers get their energy solely from the oxi-dation of NH
4 to NO ÿ
3: Increased nitri®cation rates
with increasing additions of mineral NH
4 have been reported in studies of nitri®cation kinetics (Darrah et al., 1985; Nishio and Fujimoto, 1990). Nishio and Fujimoto (1990) observed that an increase in nitri®cation rate was attributed to the growth of nitri®ers when NH
4 was added at levels >50 mg N kgÿ1
soil. The increased nitri®cation rates and poten-tials along with the addition of (NH4)2SO4 in this soil
system (Fig. 2) support the observation that available
NH
4 limited both the nitri®cation rate and nitri®er population size. Initially, the existing nitri®er
popu-Fig. 1. Inorganic N accumulation patterns in the control soil, soil with the addition of the compost, and soil with the addition of (NH4)2SO4.
Values are means with standard errors (n= 3). When error bars are not visible they are smaller than the symbol. Nitrate N in the control and compost treatments was ®t to a linear model. Nitrate N in theNH
lation responded to NH
4 addition with an increase in nitri®cation rate (Fig. 2) resulting in an increased ratio of nitri®cation rate to nitri®cation potential (Table 2). The metabolic energy produced exceeded the mainten-ance requirement of the population and allowed for population growth (Fig. 2). An apparent speci®c growth rate for nitri®ers in the (NH4)2SO4treated soil
was calculated based on the assumption that nitri®er population growth was represented by an exponential increase in the nitri®cation potential between days 7
and 25 (Fig. 2). The apparent speci®c growth rate cal-culated was 0.01 dayÿ1
, equivalent to a doubling time of 69 days. The observed speci®c growth rate is far below maximum speci®c growth rates reported for soil nitri®er populations under non-limiting NH
4 supply (Belser, 1979; Darrah et al., 1985) suggesting that growth remained NH
4 limited under these experimen-tal conditions. As the addedNH
4 was depleted, nitri®-cation rates and the ratio of nitri®nitri®-cation rate to nitri®cation potential began to decrease (Fig. 2, Table 2). The supply of mineralized NH
4 could not maintain the larger nitri®er population. Nitri®cation potentials began decreasing at day 40 until they were equivalent to those of the control soil by the end of the incubation (Fig. 2).
In the control soil or the soil receiving compost, N mineralization was the primary source of NH 4 available to soil nitri®ers and was the rate limiting process for subsequent nitri®cation. N mineralization supplied substrate approximately equivalent to or less than the maintenance requirement of the exist-ing nitri®er population.
Gross N mineralization rates were signi®cantly dierent at the dierent incubation times (p< 0.01) and among the three soil treatments (p < 0.01) (Table 3). Generally, gross N mineralization rates decreased with incubation, and the soil receiving com-post had higher gross N mineralization rates than the control soil. Because of low NH
4 concentrations in the control soil or the soil added with compost, the ad-dition of NH
4 from 15N injections even at 1 mg N kgÿ1
soil could enhance the rates of those N processes that utilizeNH
4:Our data (Table 3) showed that NH 4 consumption rates were much higher than the gross N mineralization rates, implying that the NH
4 addition enhanced the combined consumption by nitri®ers and immobilizing heterotrophs. Since the enhanced nitri®-cation rates were almost equal to the enhanced NH 4 consumption rates (data not shown), we conclude that nitri®ers were the main consumers of the added NH 4 in these systems.
The ratios of nitri®cation rates to gross N mineraliz-ation rates were not signi®cantly dierent among the
Fig. 2. The patterns of nitri®cation rates and potentials with incu-bation days in the control soil, the soil receiving the compost, and the soil receiving (NH4)2SO4. Values are means with standard errors
(n= 3). When error bars are not visible they are smaller than the symbol.
Table 2
Ratios of gross N process rates in the control soil, the soil receiving the compost, and the soil receiving (NH4)2SO4
Incubation days Nitri®cation rate/gross N mineralization rate Nitri®cation rate/nitri®cation potential Control Compost NH
4 Control Compost NH 4
7 0.52 (0.03)a 0.51 (0.03) 0.57 (0.11) 0.03 (0.00) 0.08 (0.01) 0.24 (0.01) 40 0.45 (0.11) 0.26 (0.02) 0.50 (0.16) 0.02 (0.00) 0.03 (0.00) 0.09 (0.02) 70 0.68 (0.03) 0.63 (0.15) 0.50 (0.48) 0.04 (0.00) 0.05 (0.00) 0.03 (0.01) 112 3.53 (1.50) 0.50 (0.09) 3.06 (0.17) 0.05 (0.01) 0.02 (0.00) 0.06 (0.00)
a
three soil treatments (p = 0.10) (Table 2). Nitri®ca-tion rates were about 50% of the gross N mineraliz-ation rates or higher. High ratios of nitri®cmineraliz-ation rates to gross N mineralization rates in all the three soil treatments throughout the 112-day incubation (Table 2) further indicate that nitri®ers are strong competitors forNH
4 in these systems.
Acknowledgements
This research was ®nancially supported by the Utah Agricultural Experiment Station (Project 323) and a grant from the USDA NRI-CGP (#9600839). We thank the Isotope Analysis Laboratory of Utah State University for 15N analysis, and Dr. J. M. Stark for
helpful discussion.
References
Belser, L.W., 1979. Population ecology of nitrifying bacteria. Annual Review of Microbiology 33, 309±333.
Darrah, P.R., Nye, P.H., White, R.E., 1985. Modeling growth re-sponses of soil nitri®ers to additions of ammonium sulphate and ammonium chloride. Plant and Soil 86, 425±439.
Davidson, E.A., Stark, J.M., Firestone, M.K., 1990. Microbial pro-duction and consumption of nitrate in an annual grassland. Ecology 71, 1968±1975.
Fauci, M.F., Dick, R.P., 1994. Soil microbial dynamics: short- and long-term eects of inorganic and organic nitrogen. Soil Science Society of America Journal 58, 801±806.
Hart, S.C., Stark, J.M., Davidson, E.A., Firestone, M.K., 1994. Nitrogen mineralization, immobilization and nitri®cation. In: Weaver, R.W., Angle, S., Bottomley, P., Bezdicek, D., Smith, S., Tabatabai, A., Wollum, A. (Eds.), Methods of Soil Analysis. Part III: Microbiological and Biochemical Properties. Soil Science Society of America, Madison, pp. 985±1018.
Jackson, L.E., Schimel, J.P., Firestone, M.K., 1989. Short-term par-titioning of ammonium and nitrate between plants and microbes in an annual grassland. Soil Biology and Biochemistry 21, 409± 415.
Jansson, S.L., 1958. Tracer studies on nitrogen transformations in soil with special attention of mineralization-immobilization re-lationships. Annals of the Agricultural College of Sweden 24, 101±361.
Jansson, S.L., Hallam, M.J., Bartholomew, W.V., 1955. Preferential utilization of ammonium over nitrate by micro-organisms in the decomposition of oat straw. Plant and Soil 6, 382±390.
Jones, J.M., Richards, B.N., 1977. Eect of reforestation on turnover of 15N-labeled nitrate and ammonium in relation to changes in
soil micro¯ora. Soil Biology and Biochemistry 9, 383±392. Kirkham, D., Bartholomew, W.V., 1954. Equations for following
nutrient transformation in soil utilizing tracer data. Soil Science Society of America Proceedings 19, 189±192.
Nishio, T., Fujimoto, T., 1990. Kinetics of nitri®cation of various amounts of ammonium added to soils. Soil Biology and Biochemistry 22, 51±55.
Recous, S., Mary, B., 1990. Microbial immobilization of ammonium and nitrate in cultivated soils. Soil Biology and Biochemistry 22, 913±922.
Rice, C.W., Tiedje, J.M., 1989. Regulation of nitrate assimilation by ammonium in soils and in isolated soil microorganisms. Soil Biology and Biochemistry 21, 597±602.
Schimel, J.P., Jackson, L.E., Firestone, M.K., 1989. Spatial and tem-poral eects of plant-microbial competition for inorganic nitrogen in a California annual grassland. Soil Biology and Biochemistry 21, 1059±1066.
SchnuÈrer, J., Clarholm, M., Rosswall, T., 1985. Microbial biomass and activity in an agricultural soil with dierent organic matter contents. Soil Biology and Biochemistry 17, 611±618.
Stark, J.M., 1996. Shaker speeds of aerobic soil slurry incubations. Communications in Soil Science and Plant Analysis 27, 2625± 2631.
Stark, J.M., Hart, S.C., 1996. Diusion technique for preparing salt solutions, kjeldahl digests, and persulfate digests for nitrogen-15 analysis. Soil Science Society of America Journal 60, 1846±1855. Stark, J.M., Hart, S.C., 1997. High rates of nitri®cation and nitrate
turnover in undisturbed coniferous forests. Nature 385, 61±64. Verhagen, F.J.M., Laanbroek, H.J., 1991. Competition for
am-monium between nitrifying and heterotrophic bacteria in dual energy-limited chemostats. Applied and Environmental Microbiology 57, 3255±3263.
Wichramasinghe, K.N., Rodgers, G.A., Jenkinson, D.S., 1985. Transformations of nitrogen fertilizers in soil. Soil Biology and Biochemistry 17, 625±630.
Table 3
Rates of gross N mineralization andNH
4 consumption in the control soil, the soil with the addition of the compost, and the soil with the
ad-dition of (NH4)2SO4
Incubation days Gross N mineralization rate (mg N kgÿ1
soil dayÿ1
) NH
4 consumption rate (mg N kg
ÿ1
soil dayÿ1
Control Compost NH
4 Control Compost NH 4
7 0.44 (0.03)a 1.23 (0.06) 3.30 (0.53) 1.10 (0.07) 2.00 (0.06) 8.56 (0.62) 40 0.38 (0.10) 0.85 (0.14) 2.15 (0.82) 1.66 (0.19) 2.45 (0.13) 3.52 (0.57) 70 0.32 (0.03) 0.52 (0.11) 0.60 (0.18) 2.27 (0.10) 2.25 (0.06) 2.24 (0.10) 112 0.09 (0.03) 0.30 (0.06) 0.11 (0.01) 1.26 (0.02) 2.07 (0.11) 1.11 (0.04)
