E ﬀ ect of Pasture Management System Change on In-Season Inorganic Nitrogen Pools and Heterotrophic Microbial Communities

: It has been assumed that the system of long-term pasture management exerts a signiﬁcant impact on the soil microorganisms count, subsequently a ﬀ ecting the availability of mineral nitrogen (N min ). This hypothesis was tested in a three-year experiment on a long-term pasture with two distinct systems of grass sward management, i.e., grazing and mowing. Mowing signiﬁcantly increased the microorganisms count by 13%, 28%, 86%, and 2% for eubacteria (EU), actinobacteria (AC), molds (MO), and Azotobacter (AZ), respectively. The main reason was drought in 2006, which resulted in the domination of Dactylis glomerata L. in the grass sward, instead of Lolimum perenne L. and Poa pratensis L. The content of N min decreased through the vegetative growing season, reaching its lowest value after the 3rd grazing cycle. The impact of microorganisms on the N min pools increased in the order: molds < eubacteria < actinobacteria. The count of actinobacteria in the alkaline organic soil increased in response to drought, contribution of Dactylis glomerata L. in the sward, and the shortage of available phosphorus. The sound pasture management system is possible by introducing alternate grazing and mowing cycles. The core of sustainability is the enhanced activity of actinobacteria after changing the system from grazed into mowed.


Introduction
Grasslands are a natural source of high quality forage, rich in energy, proteins, and nutrients [1]. Pasture positively affects the condition of grazing animals and significantly reduces the costs of milk production. Study of the relationship between grazing animals and sward quality and its productivity is one of the fundamental research areas in countries with a high level of animal husbandry [2,3]. The productivity of natural or extensively fertilized grasslands depends on the availability of plant nutrients within the consecutive mowing or grazing cycle. Under these conditions, nutrient availability, especially nitrogen (N) to the sward plants, depends to a great extent on the intensity of organic matter decomposition. Hence, composition and activity of soil microbial communities seem to be a decisive factor for controlling, or even enhancing grassland productivity [4].
Pastures are the main source of feed for ruminants over the world. The intensification of dairy cow production, especially in leading countries in Europe, decreases, but does not eliminate, the need for feed supply from pastures [29]. The natural methods of pasture sward use are periodical grazing/resting cycles. During the grazing period, animals expel excreta, which are rich in all plant nutrients, but especially in N, directly onto the pasture sward. The expected production effect of N in excreta is its direct impact on growing plants and indirect influence on soil N pools. A high N concentration, especially in urine patches, results in ammonia volatilization (N-NH 3 ) directly into the atmosphere [30]. The second source of easily mineralized N, left on a sward after a grazing period, are sward litters. The transformation pathways of these N pools in the sward soil and their subsequent impact on soil N pools are very complex [31]. Easily mineralizable N significantly impacts the activity of soil microoganisms. As a consequence, the stable organic carbon pool is enriched in organic compounds of microbial origin [32]. The narrowed C:N ratio, especially in organic soil, results in an enhanced increase of N mineral pools, i.e., ammonia (N-NH 4 ), and nitrate (N-NO 3 ). During the vegetative season, both pools are a direct source of N to the growing plants. However, during winter or early spring, nitrates undergo leaching, subsequently disturbing the functioning of water ecosystems [33,34].
The management concept known as sustainable intensification of agriculture seems to be an adequate approach to a sound use of pastures. The key advantage of the grazing system is the relatively low cost of milk or meat productivity. The main disadvantage of this system is the great threat of nitrate leaching to water ecosystems [35]. A short-term change in the grazing system by temporally implementing the mowing seems to be the simplest way to exploite easily available N pools, while maintaining the previous production level of feed for animals, concomitant with the simultaneous protection of the neighboring ecosystems.
The main objective of this study was to evaluate the impact of heterotrophic microbial communities, taking into account variable environmental conditions (weather) in two management systems of long-term pasture, i.e., grazing and mowing, on the in-season variability of inorganic nitrogen pools and the availability of other plant nutrients.

Site, Soil, and Climate
The research was conducted at Brody, an experimental station belonging to Poznań University of Life Sciences. The farm is located about 50 km west of Poznań. From the northwest, it borders the Bolesława Papi Reserve with lake Zgierzynieckie. The natural grassland covers 160 ha, of which 40 ha is used as a pasture that is divided into 12 quarters. The research was carried out in 2006,2007, and 2008 on quarter No. 2. The sward of this quater before the experiment was dominated by three groups of species, representing grasses (61%), legumes (9%), forbs (10%), and weeds (quack grass-9% and dicotyledonous species-11%). The key grass species, i.e., perennial ryegrass and common meadow-grass covered 60% of the sward area. The geographical location of the studied pasture was: entry to the accommodation unit-N 52 • 11.67". The investigated pasture is classified as medium wet grassland (bonitation class IV) [36]. It was established on muck soil originated from low peat, dark gray (0-40 cm). The second horizon occupies low peat very strongly decomposed, almost black (40-65 cm). Beneath the organic layers, silty clay occurs. Prior to the initiation of the study, the soil contained 35% of organic matter in the 0-30 cm layer, and had moderate drainage.
The local climate, classified as intermediate between atlantic and continental regions, is seasonally variable, particularly during the summer [37]. Analysis of average air temperatures during the research showed a significant difference in annual mean temperatures as compared to the long-term average (1961-2005) of 13.4 • C during the vegetative growing season (months 4-10) (Figure 1). The mean temperature during each grazing cycle was 15.9, 14.6, and 15.1 • C for 2006, 2007, and 2008, respectively. The average temperature for the consecutive grazing cycle showed a typical annual variation (Table 1; Figure 1). The warmest grazing cycle was summer 2006, when the average temperature reached almost 23 • C; concomitant with a shortage of precipitation which resulted in severe drought. These two courses of meteorological events led to a grass "burnout", finally resulting in a significant change in the grass species composition (Table A1). The total sum of precipitation was highly variable, especially during the summer months (Table 1; Figure 1). 2, 3, and 4 cycles, see Table 1 for detailed dates). The second factor was the sward management system, i.e., grazing or mowing. Grazing duration was determined, depending on the grass yield, based on the yearly stocking density (SD) of 5.94 ha −1 Livestock Unit (LSU). Total SD was constant within each grazing event and between years, reaching on average 72 SD per 3 ha per day. The grazing period differed in the length, and lasted 1.0 to 5.0 days in 2006; 2.5-4.0 days in 2007, and 3.0-4.0 days in 2008. The daily length of the grazing event was four hours, lasting from 7.30 to 11.30 a.m.. Sward was mowed by lawn mower Pasquali (100 cm single blade) at the end of each grazing event. Four replicated experimental plots of 30 m 2 were established for each management treatment along a transect. The distance between each plot from its centre was 35 m. This distribution was chosen due to the random movement of animals during grazing ( Figure 2). The study on the content of Nmin, soil available nutrients, and the soil microbiome composition was carried out on specially established subplots (four, 1 × 2 m in size) within each main plot.

Experimental Design
The study was conducted on a rectangular pasture quarter of 100 × 300 m, 3.0 ha in size. The experiment was arranged as a two-factorial design. The first factor was the number of grazing/mowing (acronym Gr/Mo) cycles during each growth season (from May to October, i.e., 1, 2, 3, and 4 cycles, see Table 1 for detailed dates). The second factor was the sward management system, i.e., grazing or mowing. Grazing duration was determined, depending on the grass yield, based on the yearly stocking density (SD) of 5.94 ha −1 Livestock Unit (LSU). Total SD was constant within each grazing event and between years, reaching on average 72 SD per 3 ha per day. The grazing The daily length of the grazing event was four hours, lasting from 7.30 to 11.30 a.m. Sward was mowed by lawn mower Pasquali (100 cm single blade) at the end of each grazing event. Four replicated experimental plots of 30 m 2 were established for each management treatment along a transect. The distance between each plot from its centre was 35 m. This distribution was chosen due to the random movement of animals during grazing ( Figure 2). The study on the content of N min , soil available nutrients, and the soil microbiome composition was carried out on specially established subplots (four, 1 × 2 m in size) within each main plot. The annual rates of fertilizer N as ammonium nitrate were 35 kg ha −1 , applied before the 1 st and the 3 rd grazing cycle (total 70 kg ha −1 ). Phosphorus fertilizer was not applied. Potassium was applied at the beginning of the vegetative growing season in the form of 40% K2O fertilizer and a dose of 40 kg K2O ha −1 .

Soil Sampling and Analysis
Soil was sampled four times per year, one after each Gr/Mo event. (Table 1). Twenty 1.5-cmdiameter individual soil cores were collected per each subplot of the main plot at 0-15-cm depth and then composited. Samples were sieved to < 2 mm and dry stored prior to analysis. Subsamples (100 g) for mineral nitrogen determination were deep-frozen (−20 °C). For Nmin determination, 20 grams of each soil sample was shaken for 1 h with 100 mL of 0.01 M CaCl2 solution (soil/solution ratio 5:1; m/v).
Concentrations of NH4-N, NO3-N were determined with the colorimetric method using flow The annual rates of fertilizer N as ammonium nitrate were 35 kg ha −1 , applied before the 1st and the 3rd grazing cycle (total 70 kg ha −1 ). Phosphorus fertilizer was not applied. Potassium was applied at the beginning of the vegetative growing season in the form of 40% K 2 O fertilizer and a dose of 40 kg K 2 O ha −1 .

Soil Sampling and Analysis
Soil was sampled four times per year, one after each Gr/Mo event. (Table 1). Twenty 1.5-cmdiameter individual soil cores were collected per each subplot of the main plot at 0-15-cm depth and then composited.
Samples were sieved to <2 mm and dry stored prior to analysis. Subsamples (100 g) for mineral nitrogen determination were deep-frozen (−20 • C). For N min determination, 20 g of each soil sample was shaken for 1 h with 100 mL of 0.01 M CaCl 2 solution (soil/solution ratio 5:1; m/v).
Concentrations of NH 4 -N, NO 3 -N were determined with the colorimetric method using flow injection analyses [38]. Soil samples for determination of available nutrients (P, K, Mg, Na) were air-dried and crushed to pass a 2-mm mesh size. The extractable nutrients were determined using 0.5 M HCl solution [39]. The content of available P in the extract was determined calorimetrically [40], while contents of K, Mg, and Na were determined using a flame type atomic absorption spectrometer [41].

Soil Microbiome Determination
Total count of bacteria (including of Azotobacter genus), actinobacteria, and molds were determined according to the plate method. The groups of microorganisms were cultured on solid substrates using appropriate dilutions of soil solutions, expressed as CFU·g −1 of soil dry matter. Counts of heterotrophic bacteria were determined on the Merck standard agar medium (peptone from casein 5 g·L −1 , meat extract 3 g·L −1 , NaCl 5 g·L −1 , agar 12 g·L −1 ) following 5 to 6-day incubation at temperature of 28 • C [42].

Statistical Analyses
The collected data were subjected to a conventional analysis of variance using STATISTICA ® 10 (StatSoft, Krakow, Poland). Homogeneous subsets of means were identified with Tukey's test at a significance level of p < 0.05. The Pearson correlation coefficient was used to quantify the strength of the relationships between the number of microorganisms and the contents of soil available nutrients. In the second step, Principal Component Analysis (PCA) was used to illustrate the dependence between the count of microorganisms and soil chemical properties of soil as well as weather conditions.

Results
Yields of sward in consecutive years of study were affected by the management system (Table A2). However, significant differences were only recorded in 2007 (+33%) and in 2008 (+22%). As a rule, irrespective of the study year, the highest yield was the attribute of the 1st grazing/mowing cycle. Its share in the total yield, depending on a particular year, varied from about 30% in 2007 and 2008 to 42% in 2006. The sward yields of consecutive grazing cycles were significantly lower, but as a rule significantly higher in the mowing system.

Microbial Population
The composition of the soil microbial community in the long-term pasture underwent a significant variability in response to two management systems, i.e., grazing and mowing (Table 2, Figure 1). The greatest impact on the microbial composition was exerted by the weather in consecutive years. The coefficient of variation for the microorganisms count increased in the order: In 2006, a strong difference was observed between the soil microbial community composition in their response to the sward management system in consecutive grazing cycles. Only Azotobacter count responded significantly to a change in the type of pasture management, decreasing in the mowed sward. This trend underwent changes in consecutive Gr/Mo cycles, showing at the end of the 2006 vegetative growing season a significantly higher population density in soil under mowed sward. This proved to be a dominnat trend of Azotobacter population response to the management system, in spite of fluctuation, during consecutive years of study.
In spite of a high year-to-year variability, the molds population showed in general, a constant trend in response to the sward management system. In 2006, the first response of molds to the management system was recorded after the 3rd Gr/Mo cycle. The molds count in soil under mowed sward increased 5-fold as compared to the grazed sward. The same trend was observed in other years. The eubacteria population, for most of the studied grazing cycles, irrespectively of the weather course in consecutive years, showed a relatively low sensitivity to the sward management system. A higher bacteria count in soil under grazing was only significant in two of the 12 Gr/Mo cycles. Actinobacteria showed the strongest response to the interaction of all experimental factors ( Figure 3). As in the case of bacteria, the maximum count of these microorganisms was recorded after the 3rd Gr/Mo cycle, i.e., in August. The effect of pasture management on the actinobacteria number was the most remarkable in dry years. No significant advantage for either of the management systems was observed for the first Gr/Mo cycle. In dry years, in the full summer and the early autumn, i.e., after the 3rd and 4th Gr/Mo cycle, the mowing system had a significant, even huge impact on the actinobacteria population compared to the grazed system. Year * Cut * System 6 59.6 *** 82.9 *** 48.5 *** 57.0 18.5 *** Gr-grazed, Mo-mowed pasture sward; 1 Eubacteria, 2 Actinobacteria, 3 Molds, 4 Azotobacter, 5 Microbiological index; a the same letter indicates a lack of significant differences within the treatment; ***, **, * indicate significance at p < 0.001, < 0.01, and <0.05. respectively. Figure 3. Effect of the grassland management in consecutive cuts during the growing season on the actinobacteria count. a The same letter indicates a lack of significant differences within the treatment (p < 0.05).

Mineral Nitrogen
The content of Nmin, as the key factor affecting grass sward productivity, showed high sensitivity to the interaction of all the studied factors (Table 3). Distinct differences were recorded between trends of nitrate (N-NO3) and ammonium (N-NH4) content variability (Figures 4 and 5).
The N-NO3 content, averaged over years, was the highest in 2008, and the lowest in the wet 2007. An evaluation of the effect of consecutive Gr/Mo cycles on Nmin content variability during the growing season requires a detailed analysis, including the impact of the management systems ( Figure 4). The lowest content of N-NO3 was recorded after the first Gr/Mo cycle. On average, the N-NO3 content decreased up to the 3rd grazing cycle ( Figure 4). In dry years, the N-NO3 content after the 3rd cycle was higher in the soil under mowed sward. After the 4th Gr/Mo cycle, the significant impact of the mowing system was revealed in 2008. A completely different trend was observed in the wet 2007. The highest N-NO3 content was recorded just after the 1st Gr/Mo cycle. For subsequent cycles, it was low, especially in soil under the grazing system.
The average content of ammonium (N-NH4), averaged for Gr/Mo cycles and management systems, was much lower compared to the N-NO3 content (Table 3, Figure 5). The lowest content of N-NH4, constituting only 17% of N-NO3, was recorded in 2008. In other years, the content of this N Figure 3. Effect of the grassland management in consecutive cuts during the growing season on the actinobacteria count. a The same letter indicates a lack of significant differences within the treatment (p < 0.05).

Mineral Nitrogen
The content of N min , as the key factor affecting grass sward productivity, showed high sensitivity to the interaction of all the studied factors (Table 3). Distinct differences were recorded between trends of nitrate (N-NO 3 ) and ammonium (N-NH 4 ) content variability (Figures 4 and 5).
The N-NO 3 content, averaged over years, was the highest in 2008, and the lowest in the wet 2007. An evaluation of the effect of consecutive Gr/Mo cycles on N min content variability during the growing season requires a detailed analysis, including the impact of the management systems ( Figure 4). The lowest content of N-NO 3 was recorded after the first Gr/Mo cycle. On average, the N-NO 3 content decreased up to the 3rd grazing cycle (Figure 4). In dry years, the N-NO 3 content after the 3rd cycle was higher in the soil under mowed sward. After the 4th Gr/Mo cycle, the significant impact of the mowing system was revealed in 2008. A completely different trend was observed in the wet 2007. The highest N-NO 3 content was recorded just after the 1st Gr/Mo cycle. For subsequent cycles, it was low, especially in soil under the grazing system.
The average content of ammonium (N-NH 4 ), averaged for Gr/Mo cycles and management systems, was much lower compared to the N-NO 3 content (Table 3, Figure 5). The lowest content of N-NH 4 , constituting only 17% of N-NO 3 , was recorded in 2008. In other years, the content of this N form was only slightly higher. The trend of the N-NH 4 content was highly specific. Its highest content was recorded after the 1st Gr/Mo cycle. In 2006, it was 4-and 9-fold higher with respect to the amount of N-NH 4 recorded in 2007 and 2008. As a rule, it declined in the subsequent two grazing cycles, reaching its lowest value after the 3rd cut. A recovery in the N-NH 4 content was recorded after the 4th Gr/Mo cycle, with the exception of 2006. Gr-grazed, Mo-mowed pasture sward; pH-soil reaction, N-NH 4 -Ammonium ion, N-NO 3 -Nitrate ion, N min -Mineral nitrogen, Mg-Magnesium, P-Phosphorus, K-Potassium, Na-Sodium; a the same letter indicates a lack of significant differences within the treatment; ***, **, * indicate significance at p < 0.001, <0.01, and <0.05, respectively.    Effect of the grassland management in consecutive cuts in the growing season on the nitrate content. a The same letter indicates a lack of significant differences within the treatment (p < 0.05).

Figure 5.
Effect of the grassland management in consecutive cut on ammonium content. a The same letter indicates a lack of significant differences within the treatment (p < 0.05). Figure 5. Effect of the grassland management in consecutive cut on ammonium content. a The same letter indicates a lack of significant differences within the treatment (p < 0.05).

Soil pH and Available Nutrients
The pH showed very low variability during the study period, being in the alkaline range (Table 3). Its changes during the vegetative growing season were significantly affected by weather. The highest variability of pH, up to 0.4 units (7.2-7.6), was recorded in the very dry 2006, whereas in the other two years, it ranged to over 7.5.
Phosphorus (P) content was very stable both within each vegetative growing season and between years. As a rule, the highest P content occurred during the 1st Gr/Mo cycle, decreasing along the growth season. The content of available P showed a distinct decrease in consecutive years of study. It decreased from about 250 to 150 mg kg −1 in 2006 and 2008, respectively. The main reason for its steep decline was the lack of P fertilizer application. Its only source, but only in the grazing system, was animal excrement. The advantages of grazing over mowing system was significant in five cases, and the opposite trend applied in only one case (4th Gr/Mo cycle in 2007). The relationship between the P content and the microorganisms population was negative, as has been proved for actinobacteria (AC) (Tables A2 and A3).
The impact of grazing on the content of soil available potassium (K) was very stable. The advantage of grazing over mowing ranged from 2-fold to 12-fold after the 4th Gr/Mo cycle in 2008. The variability in sodium (Na) content was much lower. The content of magnesium (Mg) was, in general, high and stable both within each season and between years. As a rule, a higher content of Mg was recorded in soil under mowed sward. The most decided advantage of mowed sward was observed after the 4th Gr/Mo cycle.

Relationships between Microbes Count with Available Nutrients
In order to evaluate the relationships between counts of tested microbes groups and the content of inorganic nitrogen with respect to the contrastive systems of long-term pasture sward management, a principal component analysis (PCA) was performed. The analysis clearly reveals a distinctive strength of relationships between the size of each microbes group and the contents of both inorganic N forms in dependence on the management system and weather (Figures 6 and 7).  3 Molds, 4 Azotobacter, 5 Microbiological index; 6 temperature; 7 precipitation; pH, N-NH4-Ammonium ion, N-NO3-Nitrate ion, Nmin-Mineral nitrogen, Mg-Magnesium, P-Phosphorus, K-Potassium, Na-Sodium.

Discussion
To explain the in-season variability in the microbes population and community composition requires an accurate knowledge of weather conditions during a respective grazing cycle [23,47]. The most spectacular phenomenon, which greatly impacted the composition of the soil microorganisms For the grazed sward, PC1 was significantly correlated with 8 of 15 analyzed characteristics (Table A2). The highest, and, at the same time, positive coefficients of correlation (for R 2 > 0. 50) were recorded for EU, AC, X-index, pH and T, and negative coefficient for N-NH 4 , P (Table 4). PC2 significantly but negatively correlated with N-NO 3 , and consequently with N min and Na. PC3 was significantly, but negatively associated with Azotobacter count, and PC4 positively with the molds population. The eigenvectors for the examined variables were broadly scattered on the two first PC axes. The closest to absolute of 1 were N-NH 4 and P. These two variables were negatively correlated with PC1, but significantly correlated with each other (Table A3). The content of N-NH 4 was negatively correlated with the actinobacteria count and showed a negative trend with the bacteria count, and the content of N-NO 3 . The second group of variables, as results from the distance of eigenvectors to the absolute of 1, were the EB, X-index, AC and Mg, and also T (Figure 6a). The location of the eigenfactor for N-NO 3 , being much closer to the absolute of 0 than of 1, clearly indicates its much lower importance with an explanation of the 1st and 2nd PC axes variation.
For the moved sward, PC1 was significantly correlated with 9 of 15 analyzed soil characteristics (Figure 6b, Table A4). The highest and, at the same time, positive coefficients of correlation were recorded for N-NO 3 and AC (r > 0.9). The other significant variables showed much lower values of the coefficient of correlation (positive for EU, X-index, N min , Mg, and negative for P and N-NH 4 ( Table 4). PC2 was significantly, but negatively associated with Na. PC3 was significantly, and positively associated with molds, and PC4 negatively with Azotobacter count.
For the first group of variables, the closest eigenvector to absolute 1 were the X-index, EU, AC, and N-NO 3 (Figure 6b). The strongest relationship between this set of variables was recorded for AC and N-NO 3 (r = 0.86 ***). A much lower, but significant relationship was found for AC and N-NH 4 (r = −0.59 *) ( Table 4). This set of relationships is identical to that as found in soil under grazed sward.

Discussion
To explain the in-season variability in the microbes population and community composition requires an accurate knowledge of weather conditions during a respective grazing cycle [23,47]. The most spectacular phenomenon, which greatly impacted the composition of the soil microorganisms community, was drought during the 2nd grazing cycle in 2006. The shortage of water at the onset of summer (June/July) resulted in complete change in the grass species composition of the mowed sward (Table A1). At the start of the experiment, the grazed pasture was dominated by two species, i.e., perennial ryegrass (33%) and common meadow-grass (30%). There was no change due to drought in the grass species composition of the grazed sward. In the mowed sward, the contribution of orchard-grass in this year increased during the period, extending from the 2nd to the 4th Gr/Mo cycle in 2006 from 3.5% to 39%. All eight subsequent grazing cycles increased step-by-step from 40% to 54%. This grass is more tolerant to water stress than other grass species due to a higher photosynthesis rate, and the same time a lower transpiration rates under water stress [48].
This tremendous shift in the grass species composition resulted in an increase in the population of soil microorganisms by 13%, 28%, 86%, and 2% for bacteria, actinobacteria, fungi, and Azotobacter, respectively. This can be explained by significant differences in the impact of the dominant grass species on the propagation of microorganisms present in the root rhizosphere or bulk soil [2,[49][50][51], the most remarkable being the increase of the molds count in response to the expansion of orchard-grass in the mowed sward. This phenomenon can be explained by the drying-rewetting process, which creates subsequently favorable conditions for mold propagation [23,52]. The 2nd responsive microbial group to the increased share of orchard-grass were actinobacteria. The effect was the most pronounced in the grazing cycle directly following drought (3rd cycle in 2006, and 2008). The mowed sward had a significant, even huge impact on the actinobacteria population compared to the grazed system. The observed phenomenon results from the induction by actinobacteria under stress conditions, such as drought, alkaline soil pH, and recalcitrant plant organic matter, of several different survival strategies [53,54]. All these stress factors acted during this study.
The average content of soil ammonium (N-NH 4 ), averaged for grazing cycles and management systems, was much lower compared to the N-NO 3 content (Table 4, Figure 6a,b). In the soil/plant system, it fulfills dual functions, i.e., being a basic substrate for the nitrifying organisms, and a nitrogen source for plant growth [19]. In our study, the in-season trend of the N-NH 4 content was highly specific. Its highest content was recorded after the 1st grazing cycle. It then declined, reaching its lowest value after the 3rd grazing cycle. A N-NH 4 content recovery was, in general, recorded after the 4th grazing cycle. The trend obtained, in spite of year-to-year variability, is typical for natural grassland soils [53]. The impact of microbial community, i.e., eubacteria and actinobacteria, on the in-season trend of the N-NH 4 content was almost the same. However, the way of pasture sward management significantly impacted the N-NH 4 release from the soil resources. The N-NH 4 content in both management systems responded to the actinobacteria population in the same manner, i.e., fitting the quadrate regression model. The grazing system created more favorable conditions for N-NH 4 release from soil resources, as indicated by cardinal indices of the regression models developed (Figure 7). For the grazed sward, the optimum actinobacteria count of 53.6 cfu·10 4 resulted in the N-NH 4 content of 15.7 mg kg −1 soil. For the mowing system, the 74.4 cfu·10 4 resulted in the N-NH 4 content of 11.0 mg kg −1 soil. These two sets of data suggest a higher efficiency of actinobacteria in the grazed sward. This finding can be explained by their capiotrophy, resulting in a high potential to utilize animal excrement [49].
Grazing system: N-NH 4 = 0.018Ac 2 − 2.76Ac + 114.1 for n = 12, R 2 = 0.63, p ≤ 0.01 (2) Mowing system: N-NH 4 = 0.0096Ac 2 − 1.99Ac + 114.1 for n = 12, R 2 = 0.56, p ≤ 0.01 The optimum bacteria count for the minimum N-NH 4 content was 76.7cfu·10 4 and 103.6cfu·10 4 , whereas the minimum N-NH 4 content was 8.3 and 8.2 kg ha −1 , for the grazing and mowing systems, respectively. The recovery of the N-NH 4 content after the 4th cut content is a result of the decreased competition for nitrogen between plants and microbes. In autumn, the rate of the pasture sward growth lessens, but the rate of ammonium release is still high [53]. The presented models explicitly corroborate the higher potential of actinobacteria as compared to bacteria to release N-NH 4 from recalcitrant organic matter [54,55]. The positive relationship between P, K and N-NH 4 , taking into account the lack of P fertilizer application, indicates P soil content as the key factor limiting inorganic N release from soil resources (Figure 6a,b). This relationship was significant, however, only for the mowing system: P = −0.34AC + 118 for R 2 = 0.74, n = 12, P ≤ 0.01 (4) The model obtained clearly shows that the increasing population of actinobacteria in the soil under the moved sward resulted in the decreasing content of available P. The presented analysis showed that the effective P-fertility milieu for actinobacteria growth was soil poor in available P but at the same being within the alkaline range. The data obtained fully corroborates the hypothesis of Mander et al. [56] about the much stronger P solubility in response to bacteria action in soil with a low P pool. It has recently been documented that a high soil pH, concomitant with water stress, accelerates the rate of actinobacteria sporulation [57]. A study by Hashimoto et al. [58] showed that Ca 2+ ions present in the growth milieu of Streptomyces significantly enhance the stimulation of aerial mycelium. These two facts can explain the positive impact of alkaline pasture soil on actinobacteria activity with respect to inorganic N release from organic soil. The advantage of actinobacteria over eubacteria as a microbial agent responsible for inorganic N release from recalcitrant organic matter is a consequence of the response of this group to unfavorable weather conditions [50]. The data obtained explicitly corroborate the higher potential of actinobacteria as compared to bacteria to release N-NH 4 from recalcitrant organic matter [54].
The contents of both N forms were negatively correlated with each other, which was significant in the mowing system (Table 4, Table A4, Figure 6a,b). The lowest content of N-NO 3 , except 2007, was recorded after the first cut. On average, the N-NO 3 content increased after the 3rd and especially the 4th cut ( Figure 4). The same trend was observed for microbe count (Table 3). In the spring, nitrogen is effectively taken up by the fast-growing grasses [51]. As a consequence, a temporary shortage of available N can significantly decrease the rate of soil microorganisms propagation [25,52]. A shortage of N-NO 3 was found to be the key factor limiting the population of microorganisms, except Azotobacter (Table A4). The order of limitation based on the coefficient of correlation was as follows: The population of actinobacteria displayed the most resistance to the shortage of N-NO 3 . The advantage of this phylum over others can be explained by the ability of these microorganisms to use N from recalcitrant organic matter [55]. The strength of this relationship was much more significant in the moving system ( Figure 8). The same type of trend was recorded for bacteria, but their impact on the N-NO 3 content was slightly weaker as presented by the regression models developed: Grazing system: N-NO 3 = −0.026CFU 2 + 3.75CFU − 57.1 for n = 12, R 2 = 0.34, p ≤ 0.05 (5) Mowing system: N-NO 3 = 0.93CFU + 2.59 for n = 12, R 2 = 0.60, p ≤ 0.01 The first equation clearly indicates that the N-NO 3 content increased up to the bacteria count of 72.1 cfu 10 4 . The higher density of bacteria led to a decrease in N-NO 3 content. The course of the model developed corroborates the hypothesis by Schimel and Bennett [19] about the significant impact of the bacteria population on the soil N pool. A quite different model was obtained for the mowing systems. The N-NO 3 content increased in accordance with the increasing density of bacteria. The models obtained unambiguously show that the mowing system created more favorable conditions for nitrate release from soil resources. The study showed that grass yield of the mowed sward was significantly higher as compared to the net yield obtained on the grazed sward. There were, however, no differences in the total biomass (net sward yield on the grazed sward + sward litter) between treatments, irrespective of its management. The mowed sward was not enriched in nutrients by cows during the grazing cycle. However, it was potentially rich in both easily mineralized N and C of micorbial origin [32]. Consequently, the harvested yield depended only on the fertilizer N and its soil resources, left by animals before the management system change from grazing into mowing. However, past N resources were sufficiently high because, in autumn 2008, as shown in Figure 4, the N-NO3 content significantly exceeded that recorded in the grazed sward. The key reason for this relationship was actinobacteria activity which resulted in a strong release of N-NH4 from its organic pools ( Figure  7a). As recently documented by Zhou et al. [59], a chronic N supply to a permanent grassland results in an enhanced activity of actinobacteria, which leads to an enhanced rate of ammonia nitrification. The stronger impact of actinobacteria on the mineral N content can be also explained by a favorable pH, which has been latterly considered as the key driver of their activity [60]. The favorable growth milieu for actinobacteria, concomitant with their capiotrophy, and a high potential for ammonia oxidation resulted in a higher nitrate content in soil under the mowed sward as compared to the grazed one. The size of N mineral pools in 2008 indicates that the mowing system could be even prolonged for the next year, at least.

Conclusions
The 3-year studies showed the microbial community structure to be highly sensitive to the system of long-term pasture management. Grazing affected negatively as compared to mowing the number of fungi, actinobacteria, and bacteria. The content of inorganic N decreased during the growing season, reaching its lowest value after the 3rd Gr/Mo cycle. The impact of microorganisms on the release of inorganic N from soil resources increased in the order: Molds < Bacteria < Actinobacteria. The observed regularity in actinobacteria impacting the inorganic N pools, irrespective of the sward management system, clearly stresses their importance for N transformation in muck soil. The negative correlation between the content of N-NO3 and the The study showed that grass yield of the mowed sward was significantly higher as compared to the net yield obtained on the grazed sward. There were, however, no differences in the total biomass (net sward yield on the grazed sward + sward litter) between treatments, irrespective of its management. The mowed sward was not enriched in nutrients by cows during the grazing cycle. However, it was potentially rich in both easily mineralized N and C of micorbial origin [32]. Consequently, the harvested yield depended only on the fertilizer N and its soil resources, left by animals before the management system change from grazing into mowing. However, past N resources were sufficiently high because, in autumn 2008, as shown in Figure 4, the N-NO 3 content significantly exceeded that recorded in the grazed sward. The key reason for this relationship was actinobacteria activity which resulted in a strong release of N-NH 4 from its organic pools ( Figure 7a). As recently documented by Zhou et al. [59], a chronic N supply to a permanent grassland results in an enhanced activity of actinobacteria, which leads to an enhanced rate of ammonia nitrification. The stronger impact of actinobacteria on the mineral N content can be also explained by a favorable pH, which has been latterly considered as the key driver of their activity [60]. The favorable growth milieu for actinobacteria, concomitant with their capiotrophy, and a high potential for ammonia oxidation resulted in a higher nitrate content in soil under the mowed sward as compared to the grazed one. The size of N mineral pools in 2008 indicates that the mowing system could be even prolonged for the next year, at least.

Conclusions
The 3-year studies showed the microbial community structure to be highly sensitive to the system of long-term pasture management. Grazing affected negatively as compared to mowing the number of fungi, actinobacteria, and bacteria. The content of inorganic N decreased during the growing season, reaching its lowest value after the 3rd Gr/Mo cycle. The impact of microorganisms on the release of inorganic N from soil resources increased in the order: Molds < Bacteria < Actinobacteria. The observed regularity in actinobacteria impacting the inorganic N pools, irrespective of the sward management system, clearly stresses their importance for N transformation in muck soil. The negative correlation between the content of N-NO 3 and the contents of N-NH 4 and P clearly indicates that any increase in the first reduced the content of the remaining ones.
The advantage of actinobacteria over other groups was due to their better adaptation to moisture stress, and soil P availability, subsequently leading to a higher release of inorganic N from its organic soil pool. The mowing system of pasture management created much more favorable conditions for both actinobacteria propagation and growth and consequently resulted in a higher amount of released inorganic N. The key reason for the actinobacteria increase in the soil under mowed sward was water stress in summer 2006, which radically changed the grass species composition of sward. The increasing share of orchard-grass in the mowed sward created favorable conditions for actinobacteria growth, which significantly impacted soil mineral N release from its resources in organic soils. It can be finally concluded that the temporally introduced mowing system is an effective way to reach sustainability in long-term pasture management.