Determination of the Number of Ammonification Bacteria and Activity of the Ammonification Process in Soils and their Relevance for the Development of the of Soil Health Parameter

The results of daily counting of the colony forming units (CFU) number of bacteria capable of the ammonification (AMM) and activity of the AMM process using ammonium concentrations after induction of the AMM by peptone in soil samples from fallow and intensively cropped plots of gray forest soil are presented. Statistical analysis of visually observed wave-like dynamics of CFU revealed statistically significant harmonics in CFU of these soils. The harmonics of the dynamic of CFU of different soils were fairly close by their quantitative characteristics. This indicates not only the dynamic similarity, but also a comparable orderliness in the functioning of the ammonifying component in the differently used soils. The daily dynamics of the ammonium concentration in the soil samples was manifested in the form of curve accumulation of NH4 in time. The presentation of the results of the dynamics of NH4 in the form of subtraction differences between the subsequent concentration of NH4 and the previous one showed that the dynamics of daily production of NH4 just like the dynamics of CFU, has an oscillating nature. The observed differences in the dynamics of NH4 in the compared soil ecosystems (SE) the fallow and intensively cropped plots are very minor. Thus, even detailed monitoring the dynamic of AMM in SE do not allow to reveal significant differences in AMM of genetically identical but differently used soils. Therefore, the process of ammonification cannot be used to identify differences in the “nitrogen” parameters of the soil health.


Introduction
The main natural processes of nitrogen inputs into the soil ecosystem (SE) are nitrogen fixation and ammonification. The latter process is often called mineralization [1]. The nitrogen fixation by bacteria is a unique biological process that has been thoroughly studied and still continues to be investigated. According to the calculation, the total annual potential nitrogen inputs into the soil ecosystem by free-living nitrogen fixers can range from tens to hundreds of kilograms per hectare. Associative nitrogen fixation is estimated to be about 10-25 kg/ha a year for soils of central Europe and up to 50-100 kg/ha in the subtropical and tropical zones of 780 identified and as expected was wave-like. Such discrepancy shows that the nitrogen fixation is not "everyday" or "ubiquitous" process, but really an "exclusive" biological process. Therefore, the process of nitrogen fixation must be taken into account with partiality to many conditions [4].
Another important natural process of SE nitrogen supplyammonification (AMM) -is being paid less and less attention, although it takes place both in natural and agrarian ecosystems and is a "universal and perpetual engine" for maintaining nitrogen circulation in various components of terrestrial ecosystems. In soil ecosystems -the ammonification expresses the decomposition and metabolism of polymeric and monomeric nitrogen-containing substances with the release of a key substance -ammonium [7].
Small attention to the process of AMM can be explained by several reasons. High evidence of the prevalence of the process as well as a greater or smaller ability of all microorganisms to "participate" in this process, at least as a necromass. The complexity and multicomponent nature of the AMM itself, its flow both in the cell of the microorganism, and beyond. High reactivity and short "life time" of the intermediate products of the ammonification process in a free state. Significant influence on this process by both physicochemical factors and conditions, and a broad microbial diversity [8][9][10].
Even the concentration of such a stable final product of AMM, as ammonium, can quickly and dramatically change in the soil, and it is difficult to be quickly tracked by chemical methods. As a result, the estimated concentration in the soil is far from full, i.e. only in the form of residual quantities [11].
The literature suggests and describes with varying degree of detail mainly qualitative methods for detecting and monitoring ammonification [1,[12][13][14]. Ammonification can be observed and detected by composting organic (plant) residues, which requires a long incubation time [15]. For the observation of AMM, incubation of moist soil with a nitrogen-containing substrate or even without it is used more often. Incubation lasts from several days to several tens of days [16,17]. Ammonification registration is carried out mainly at the level of quantitative registration of CFU on meatpeptone agar (MPA) and/or on starch-ammonia agar (SAA), after seeding the corresponding tenfold dilutions of the selected sample of the ammonifying substrate. Observation of the ammonification process is performed visually via changing the color of the litmus paper placed under the plug of the tube with the test sample. Of course -this is an indirect method and, in essence, it controls the dynamics of pH in the sample medium changes [14]. As a consequence, the results of ammonification in the overwhelming number of modern (especially Russian-language) publications are represented by the determination of only the number of CFU of bacteria attributed by the authors to ammonifiers. In this case, as a rule, the determination of CFU in the test sample is performed once, and if several times, then with a large time interval between the measurement's definitions [18,19].
It should be noted that there are publications containing the dynamics of daily long-term recording of bacteria attributed by the authors to ammonifiers. In these publications, the dynamics have a pronounced oscillatory character, although the authors do not discuss these features, let alone the reasons and significance of such dynamics for a deeper understanding of the process [20,21]. In literature, quantitative methods for determining the ammonification process are described. The methods consist of determining the concentration of ammonification products by the amount of NH 3 and NH 4 + in the substrate that is analyzed. For example, one quantitative method for determining NH 3 is titrating the acid that has absorbed NH 3 , released from the substrate, known as Conway's method [14]. Another method for determining NH 4+ is based on a photocolorimetric evaluation of NH 4 + concentration, for example, with a Nessler reagent, usually in salt extract from the soil [13,14,22,23]. However, information containing a detailed description of the temporal, and especially the spatial dynamics, of the process of ammonification, and not only the number of CFU ammonifiers, is still short, shallow, and, therefore, little informative. The conclusions about the ammonification process are mainly constructed as supposedly generalizing, which does not allow using them for prognostic purposes and mathematical models, especially to apply them in the development of methods for determining soil health in relation to nitrogen [24]. Moreover, in the publications with the registration of this process in the form of a quantitative determination of one of the ammonification products -ammonium, the registration of ammonium concentration in the soil is often represented, as it is manifested, i.e. in the form of dynamics of ammonium accumulation in time. On the merits, it is a demonstration of the lack of connection between the dynamics of the ammonification process and the dynamics of the number of bacteria, which introduces delusions [6,17,21,25].
The purpose of this study was to investigate the long-term daily dynamics of the number of CFUs in fallow and intensively cultivated soils and to track the daily ammonification process in these soils in terms of ammonium concentration, after initiation of the microbial community (MC) of soils by peptone application.
The tasks were set to experimentally verify: a) The existence of wave-like dynamics in CFU ammonifiers, as is the case for any determination of the total number of CFU in the soil by seeding on solid media; b) The dynamics of the process, designated as ammonification, which is fixed by detection of ammonium in the soil is also oscillatory, and ammonium accumulation dynamics revealed in the soil is a consequence of the "summation" of the current (initiated) ammonification process and previous ammonifying processes in the soil, leading to dynamic accumulation; c) To determine the capabilities of the methods, traditionally

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used to determine ammonification in soils, to identify differences in the dynamics of ammonification in the soils which are genesis and landscape similar but differently used.

Soil Selection and Preparation
To determine the daily dynamics of the number of CFUs and the activity of ammonification, the same type of soil (gray forest) was used, but with a different prehistory. At the same time, it was conditionally assumed that the soil for the experiment, as an intensively used soil of a field, was designated as convention soil, and the control soil -the follow soil was meant as organic soil.
Samples of the soil were selected in the village of Zaoksky, Tula region (1 st Rudnevsky passage, 13), from the potato field of the Mittlider training center in September 2013. The soil of the field was treated according to an intensive system of agriculture with the maximum load of mineral fertilizers: N180P180K180 [26].
The duration of the use of soil in agricultural production was more than 20 years. The control soil -a fallow, was selected at a distance of about 100 m from the field soil. The description of the fallow area is given in [4]. The sod layer of the fallow soil (about 5 cm), represented by intensive grassy vegetation was cut before the sampling. Soil samples were collected from a depth of 5-20 cm for fallow and 0-20 cm of the field, respectively. Samples were transported to the laboratory in new plastic bags, dried in a dry ventilated room at room temperature. Dried to an air-dry state, the samples were freed from the roots, sifted through a sieve with a hole's diameter of 2 mm and stored in an air-dry state. The initial soil pH is 5.5-6.0 for fallow, fields -7.2-7.6.

Quantitative Determination of Ammonification Activity with Nessler's Reagent
The soil was moistened to 40% and packaged in vials of 5g.
Simultaneously with moistening, peptone was introduced in an amount of 40μg / g of air-dry soil. The vials were incubated at +28 °C under a SaranWrap film in a desiccator to avoid drying out of the soil. Daily for 11 days, 3 vials from each sample were randomly used and the concentration of N-NH 4 + in the salt extract from the soil was determined by the colorimetric method using Nessler's reagent.
Used photo colorimeter FEC-56 PM, the optical step of the cuvette was 20 mm, the pH of the fallow soil is 6.0, the field is 7.2, at 22º.
As the initial (zero) point, the N-NH 4 + content in the soil was used before the samples were incubated. The concentration of nitrogen of ammonium in the solution was determined by the calibration curve, producing a recalculation for 100 g of soil according to the formula: where: a -the amount of NH 4+ found on the calibration scale, mg/ml; V -the total volume of the extract, ml; 100 -the value for conversion to 100 g of soil; k -soil hygroscopicity coefficient; V 1the volume of the hood, taken to determine; g -soil sample.

Processing of the Results
The obtained results were processed using Excel. The results of daily counts of CFU and ammonium concentrations were analyzed mathematically using the method of harmonic analysis [6,27].     Table   1). Both in fallow and in the field soils, by harmonic analysis of the dynamics of light CFUs, one fifth order harmonic was identified. In the dynamics of dark-colored CFUs (forming H2S), also one harmonic was detected, but for the fallow soil it was third-order harmonic, and for the field -only the first. The amplitude of oscillations of the CFU number of light colonies in the field soil was only 3 times higher than the amplitude of oscillations in the fallow soil, i.e. they are close, and the period, phase and frequency of oscillations are simply equal ( Table 1). The amplitude of oscillations of darkcolonies in the field soil also exceeds (2.6 times) the amplitude of oscillations in the fallow soil, and the phase, period, and frequency of the oscillations of dark colonies for the samples of the fallow and field soils differ more ( Figure 3).

Daily Dynamics of Ammonification Activity
Daily determination of the amount of N-NH 4+ by the method     The process of ammonification, especially in the conditions of our experiments, is purely microbiological. The dynamics of CFUs of ammonificators was oscillatory, wave-like. The dynamics of known microbiological exometabolites, primarily gaseous, such as CO 2 , N 2 O and even CH 4 , as well as the growth dynamics of microbial biomass, are wave-like, which has been shown many times [21,28,29,5,6].
Even the process of nitrogen fixation was truly wave-like [4].
Therefore, it was logical to expect that the production of N-NH 4 + into the soil system would also be uneven, oscillatory. However, the , the graphical results obtained by means of the subtraction of the previous day data N-NH 4+ from the following day data, i.e. the rate of ammonium production per day, are presented (differential results!) ( Figure 5). Indeed, the uneven activity of the N-NH 4 + formation per day was revealed, and the daily dynamics of N-NH 4 + was oscillatory ( Figure 5). Thus, the wave-like dynamics of N-NH 4 + production by ammonifiers in soil with three peaks was detected. One peak generated 3-4 days ( Figure 5).

Discussion
The daily dynamics of CFU of bacteria on such media, which researchers always use to account for ammonifiers, is wave-like.
Such dynamics were expected. It is already known that daily, longterm counting of both CFU and direct microscopic counting of microbial cells, especially in natural ecosystems, always reveals wave-like dynamics microorganisms, which corresponds to one of the laws of growth and existence of biological populations [6,30]. The four-day "lag-period" in the dynamics of light colonies of ammonificators from the fallow soils, apparently, is due to the fact that in the MC of the natural ecosystem by the environment were "selected" such populations, which correspond to the K-strategy concept. Physiologically this is due to the consequence of the adaptation of bacteria to the new substrate. The same reason explains the presence of "lag-period" in the growth of dark colonies from all soils. In the MC of the field soil, r-strategists apparently prevail. In the environmental aspect, the differences in the dynamics of AMM populations in the compared soils can be interpreted as a "quieter" response to the disturbing of the MC fallow soil than MC of field soil. This has already been observed for soil relevant to "organic" and "convention system" systems [31].
Harmonic analysis allowed more clearly and convincingly identify the existing similarities and differences between the dynamics of CFU of ammonification of the fallow and field soil.
Similarities in the harmonics order indicate that in the fallow and field soils bacterial communities mainly inhabit with similar growth values (periods and frequencies), and also, it can be assumed, with the same functional characteristics (Table 1) The results of the dynamics of ammonium production per day were also subjected to harmonic analysis ( Figure 5). However, the presence of significant harmonics even at a statistical level of 0.1 was not detected. This may be due to several reasons. The number of analyzed values is too short, however, for the harmonic analysis, the values of deviations from the "mean" are primarily important.
A more objective reason for this is that not the dynamics of the to the same disturbing is almost the same and wave-like ( Figure   5). This indicates that the MCs of these soils are always equally "ready" to the ammonification process. Based on these results, the conclusion suggests itself that even detailed dynamic results in the form of the number of CFU and the ammonification process activity do not allow to reveal significant differences between natural SE (follow soil) and intensively exploited soil.
Thus, the traditional method of determining the ammonification of SE is not sensitive enough to use it for the rapid, routine detection of differences between two genesically and landscapeclose, but radically different in exploitation soils. As a result, the ammonification as well as nitrogen fixation [4], as the processes to the influx of nitrogen into SE, due to their insufficient selectivity (sensitivity), high labour intensity and time of capacity, cannot be recommended as methods for every day, routine analytical procedure in the soil health parameter determining in relation to nitrogen. The obtained results allow us to draw another significant conclusion. Only the "dynamics" of target microorganisms and/ or their direct metabolites can be used to assess the activity of processes and use such indicators for prognostic purposes.