Almeida, Vanessa1; Fraga, Irene2; Bajouco, Rui1; Alves, Ricardo3, Coutinho, João1*
1CQVR, Dep Biologia e Ambiente, ECVA, UTAD, 5000-801 Vila Real, Portugal, *j_coutin@utad.pt 2CITAB, Dep Biologia e Ambiente, ECVA, UTAD, 5000-801 Vila Real, Portugal
3Laboratório de Solos e Plantas,UTAD, 5000-801 Vila Real, Portugal
Abstract
The present work studied the effect of the incubation time forthe evaluation of the actual nitrification rate, alterna- tively to the soil potential nitrification rate described by the international standard procedure ISO 15685. In this work, both actual and potential soil nitrification rates were determined by monitoring the N-NO2- production kinet-
ics over a 72-h period.
It was observed, that the N-NO2- (nitrite) production has a linear increase with the incubation time (R2 = 0.998)
during the first 10-h of incubation for both alternatives. Notwithstanding, a non-proportional relation of N-NO2-
production with incubation time was verified, complying a logistic function (R2 = 0.983), which cannot be attributed
to substrate limitation. On opposition to the method described in literature (24-h incubation), the results indicate that the evaluation of the actual nitrification rate of the soil should be performed based on a 5-h incubation period, similar to that used when assessing the potential nitrification rate.
Keywords: nitrogen, soil, nitrification rate. Resumo
Foi estudado o efeito do tempo de incubação para a avaliação da taxa efetiva de nitrificação do solo, em alterna- tiva à determinação do potencial de nitrificação, método descrito na norma ISO 15685. Para tal, foram determi- nadas a cinética de nitrificação e o potencial de nitrificação pela monitorização da cinética de produção de de N- NO2- por um período de 72 h.
Em ambas as alternativas, a produção de N-NO2-durante as primeiras 10 h de incubação apresentou um aumen-
to linear em função do tempo (R2 = 0,998). Após este período, a relação da produção de N-NO2- com o tempo
passa a ser menos proporcional, obedecendo a uma função logística (R2 = 0,983), não se podendo atribuir esta
quebra de proporcionalidade à limitação de substrato. Ao contrário do indicado na literatura (24 h), os resultados obtidos indicam que a avaliação da taxa efetiva de nitrificação de um solo deverá ser realizada com base num período de incubação de 5 h, semelhante ao utilizado para o potencial de nitrificação.
128 Introduction
Nitrogen (N) is a limiting nutrient for plant growth and production in most of the natu- ral and agricultural terrestrial ecosystems. Microbiology plays a decisive role in the soil ecosystem functions, such as trans- formations ofthe different N forms.Among those transformations, the nitrificationpro- cesshas great agro-environmental im- portance [1].Understandsoil nitrification fluxes and anticipate ratescanhelp on promotingthe efficient uptake of the N by- crops.
Nitrification is an aerobic process that is carried out by separate groups of bacteria: the ammonia and the nitrite oxidizers.The biochemistry of ammonium oxidation is a two-step process: in the first stage, ammo- nia is oxidized to hydroxylamine, which is mediated by the membrane-bound enzyme ammonia monooxygenase. Hydroxylamine is further oxidized to nitrite by the hydroxyl- amine oxidoreductase.Afterwards, nitrite- oxidizing bacteria produces nitrate in a quickly process mediated by the nitrite oxi- doreductase [1, 2].As nitrite oxidation is blocked by sodium chlorate, soil nitrification rate in the laboratory may be assessed as the nitrite production during an incubation period of the soil.
For a given soil, the main factors that regulate nitrification rate are temperature, moisture, soil reaction, O2 and CO2 availa-
bility andthe substrate (ammonium) sup- ply[3]. When the decomposition/N mineral- ization is low, or when NH4+uptaken and
N-immobilization by plants or by hetero- trophs is high, nitrification rate decreases. Equally, any ecosystem disturbance or changes in soil management practices that alters soil N mineralization will usually modify nitrification rates [4]. Therefore, in laboratorial non limiting NH4+ conditions,
the nitrification rate at near-optimal tem- perature (25 ºC), called potential nitrifica-
tion (PN) and representing Vmax, is consid-
ered to be a measured of the ammonium oxidizer population [4]. Nevertheless, the
actual nitrification rate (ANR) may be as-
sessed, despite the size of the nitrifyer population present in the soil, if natural NH4+ abundance and temperature condi-
tions are maintained.
The aim of this preliminary work was to study the nitrification kinetics at near-optimal and suboptimal conditions in order to main- tain or alter the procedures described in the literature, which indicate a 5-h incubation period for PN, and a 24-h period for ANR determinations [3].
Material and methods
Soil sampling and pre-treatment
A pool of a dystric Anthrosol subsamples (0- 20 cm) was collected during late Spring on a vineyard located at the University campus, Vila Real.The sample was immediately transported to the laboratory facilities,where it was treated without being driedor fro- zen.Fresh soil sample was sieved by a 2 mm mash, homogenized and stored at 4 ºC. The soil is derived from schist and presents a pH(H2O) of 5.7 and an organic carbon
content of 7.8 g kg-1. Its actual moisture con-
tent was 255 g kg-1.
Actual nitrification rate and potential nitri- fication
Both assays were performed by the meth- ods described by [3], using eight periods of incubation (0; 2.5; 5; 7.5; 10; 24; 48 and 72hours) with an orbital shaking set at 180 rpm. ANR was conducted at 15 °C (Vila Real average annual temperature), without NH4+ addition. PN was conducted at 25
°C, with substrate addition (equivalent to 125 mg N-NH4+ kg-1 dry soil). Both deter-
minations were destructive assays, with 4 replications for each incubation period. Incubations were performed in 100 mL Er- lenmeyer flasks with 6 g of fresh soil with 20 mL of water (for ANR) or 1 mM(NH4)2SO4
(for NP) and 0.1 mL of 1.5 MNaClO3. After
each incubation period, 5 mL of 2 MKCl was added and the suspension was centrifuged at 2500 rpm for 5 min. The N-NO2- content in
the supernatantwas quantified by the Griess-Ilosvay reaction, usinga segmented flow analyzer (SanPlus, Skalar) set with a configuration presenting a limit of detection of 4 µg L-1.
Non-linear regressions were adjusted to the obtained data for both PN and ANR kinetic alternatives, using a discontinuous model.
Results and Discussion
As expected, values for PN are much higher than ANR, as observed in the Figure 1, as a result of the temperature and the initial NH4+
concentration effects on ammonium oxida- tion. Besides this effect, the difference be- tween the two rates tends to increase: after 2.5-h, ANR represents about 58% of PN, but after the 72-h period, ANR only represents about 31% of PN, which can be due to the crescent substrate limitation over time in ANR.
Nevertheless, the trends of both kinetics curves are similar. During the first 10-h of incubation, we can observe that N-NO2- pro-
duction has a linear increase with time (fig- ure), with highly significant correlation (r) values for both parameters (0.999 and 0.991, respectively). The linearity of nitrite accumulationduring this first period may be interpreted as the result of two factors: (i) the maintenance of the lag period of ammonium oxidizer population, in accordance to the minimum regeneration period of 10-h indi- cated by [4], and; (ii) the non-limitation of the flux by oxygen diffusion rates.
Figure 1 - Actual Nitrification rate (square points) and Potential Nitrification (round points). (mean ± SD, n=4)
After 10-h, a nonlinear relation of N-NO2-
production with the incubation period was verified for PN and ANR, as also shown in the figure. To the observed values for both rates, a logistic model,frequently fitted to nitrite production kinetics [5], is highly adapted (r=0.991 and 0.992 for ANR and PN, respectively). As the same trend was observed for PN, where an amount of 125 mg N kg-1 was supplemented, the non-
linearity in ANR should not be attributed only
to substrate limitation. Consequently, it can be pointed out that oxygen depletion may occur in the liquid phase after a 10-h incuba- tion, limiting the ammonium oxidation flux, despite the fact that suspensions were vig- orously and continuously shaken.
Based on the results, the 24-h incubation- period recommended by [3] should not be used to evaluate the ANR in the laboratory, since it seems to underestimate it. For both measurements, the incubation should not exceed a period of 10-h. Moreover, a period of 5-h, as indicated for the PN by [2,3] andby the ISO 15685[6] may be adopted for both determinations, since this time period allows the incubation and N-NO2- determination
within one working day. Nevertheless, this option requires methods with low detection limits for N-NO2- quantification, considering
the possibility of very low concentrations of nitrite in solution, such as the case of soils similar to that used in the present study.
Conclusion
Taking into consideration the available re- sults for the soil under study, it may be con- cluded that:
• kinetics of both ANR and PN fit an iden- tical non-continuous model, with a linear step for time periods till 10-h, followed by a curvilinear step during the subsequent 62-h period;
• unlike the indicated in the literature, it is recommended to evaluate the actual nitri- fication rate in soils within a period of 10- h of incubation;
• if analytical feasible, a general 5-h incuba- tion period, the midpoint of the linear phase of nitrite production, is recommended to estimate the soil ANR, since this time period is already prescribed by several authors and by the international standard procedure to assess the PN of the soils.
Acknowledgments
Vanessa Almeidais granted by a research fellowship BI/UTAD/INTERACT/VW/214/ 2016. The study was supported by the re- search projectsVitalityWINE/INTERACT/ /NORTE-01-0145-FEDER-000017, and in- novine & wine/ /NORTE-01-0145-FEDER- 000038.
130 References
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