Does Soil Microbial Community Respond to Moderately Elevated Nitrogen Deposition? A Correlation Analysis in a Cool Temperate Forest Surrounded by Pasture Grasslands in Northern Japan

We analyzed relationships between nitrogen deposition (deposition of nitrate and ammonium ions) and soil microbial properties, which were spatially varied in a cool temperate forest surrounded by normally fertilized pasture grasslands in northern Japan. The aim of the present study was to gain the primary information on soil microbial response to moderately elevated nitrogen deposition (< 10 kg N ha −1 y −1 ). We established three experimental plots in the forest edge adjacent to the grasslands and other three plots in the forest interior at least 700 m away from the grasslands. During May to November 2018, nitrogen deposition in each plot was measured. In August 2018, litter and soil (0−5 cm depth) samples were collected from all plots to measure net nitrogen mineralization and nitrication rates as indicators of microbial activity, and microbial biomass carbon and nitrogen and various gene abundances (i.e. bacterial 16S rRNA, fungal ITS, bacterial amoA, and archaeal amoA genes) as indicators of microbial abundance. Nitrogen deposition in the forest edge was 1.4-fold greater than that in the forest interior, even while the maximum deposition was 3.7 kg N ha −1 . Nitrogen deposition was signicantly correlated to the net nitrogen mineralization and nitrication rates and the 16S rRNA and bacterial amoA gene abundances. Microbial community structures in litter and soil samples were also analyzed using a high throughput DNA sequencer for the bacterial 16S rRNA and fungal ITS gene amplicons. Microbial community structures were different between litter and soil samples but were similar between the forest edge and interior. Signicant correlations of nitrogen deposition to the soil carbon-to-nitrogen ratio, and the nitrate and ammonium contents were also observed. Thus, our results show that moderately elevated nitrogen deposition in nitrogen-limited forest edges likely stimulate microbial activities and abundances in soils.

Therefore, we need to know more about the effects of moderately elevated nitrogen deposition (less than 10 kg N ha − 1 y − 1 ) on nitrogen-limited forest ecosystems, in order to obtain reliable responses of forest ecosystems against increased nitrogen deposition and their feedbacks to climate change through the changing biogeochemical and energy dynamics.
Soil microbial community is one of the fundamental components sensitive to changes in nitrogen deposition and availability (Janssens et  (2009), fungal species isolated from boreal forest soils responded nonlinearly but parabolically to nitrogen addition from 0 to 200 µg N which are equivalent to only 0.1% or less of the amount of organic substrates in the soils. Thus, soil microbial community can be activated rather than restricted by moderately elevated nitrogen deposition.
In the present study, we focus on the relationship between spatially varied nitrogen deposition and soil microbial properties within a cool temperate forest in eastern area of Hokkaido, Japan. The eastern Hokkaido including the investigated forest is receiving relatively low nitrogen deposition from the atmosphere (Chiwa et al. 2015; EANET, https://monitoring.eanet.asia/document/public/index), while the boundary area of the forest (i.e. forest edge) is possibly receiving more nitrogen deposition than the interior area of the forest (forest interior) owing to nitrogen fertilization in the surrounding pasture grasslands (Reinmann and Hutyra 2017; Remy et al. 2016Remy et al. , 2017Remy et al. , 2018aRemy et al. , 2018b. These grasslands have been fertilized with nitrogen in normal extent as a normal agricultural management practice since the land reclamation from forest to grassland in 1950s. Therefore, investigating the relationship between nitrogen deposition and soil microbial properties in this forest ecosystem would provide the primary information on soil microbial responses to moderately elevated nitrogen deposition for over 60 years, rather than those responses to manipulationally and extremely elevated nitrogen deposition in short-term.

Methods/experimental
Site description

Establishment of experimental plots
In May 2018, we established six experimental plots in the forest (Fig. 1a). Three of the plots were located in the forest edge (Edge 1 to 3), a boundary between the forest and adjacent pasture grasslands, while the other three were located in the forest interior (Interior 1 to 3) at least 700 m away from the grasslands (Fig. 1a)

Nitrogen deposition observation
The amounts of nitrogen deposition in the six experimental plots were measured during the period of May 9th to November 20th, 2018, by continuously collecting throughfall water from the atmosphere to ground through the canopy vegetation. Seven of shaded plastic buckets equipped with collecting tubes and funnels (21 cm in diameter) were randomly put on each of the experimental plots. Throughfall water was collected by the buckets at an almost bi-weekly interval and then ltrated using a 0.45 µm pore sized membrane lter (ADVANTEC 25CS045AN, Toyo Roshi Kaisya LTD., Tokyo, Japan). Then, the concentrations of NO 3 − and NH 4 + were analyzed using an ion chromatography (Dionex-Integrion, Thermo Fisher Scienti c, MA, USA). The amount of nitrogen deposition for the individual collection interval was quanti ed by multiplying the ion concentrations in the collected water sample and the amount of throughfall. Then, the total amount of nitrogen deposition during the six-month observation period was quanti ed by summing up the nitrogen deposition for all collection intervals.

Litter and soil sampling
Litter and surface mineral soil (0-5 cm depth) samples were collected in August 10th, 2018. Three sets of litter and soil samples were collected from each of the experimental plots to obtain the representative mean and the interspatial variation of soil microbial properties within a plot. Here, we determined the locations of litter and soil sampling avoiding the area directly below trees to reduce the possibility of speci c effects from roots and rhizospheres on collected samples. Litter samples were collected by gloved hands from an area of 30 cm × 30 cm randomly selected within each of the plots, and soil samples were then collected using a shovel. Collected litter and soil samples were cooled and transferred to the laboratory within a day. Soil samples were gently passed through a 4-mm sieve to remove gravel and plant tissues, while litter samples were pieced into a smaller size (ca. less than 2 mm × 2 mm) to obtain a homogenized sample. Both the litter and soil samples were immediately used for further analysis of soil microbial properties. Portions of the samples were air-dried and analyzed for total carbon and nitrogen contents (Koarashi et al. 2018) and pH (H 2 O), as presented in Table 1. Ammonium and nitrates contents in fresh litter and soil samples are also measured (Urakawa et al. 2014(Urakawa et al. , 2016) and presented in Table 1. All data of soil properties in this study are presented with the unit per area after the conversion with measured bulk density in Table S1. Table 1 Chemical properties of litter and soil (0-5 cm) samples a) and signi cance of their correlations to nitrogen deposition b) .  Table S2.
Microbial community structures were also evaluated using a high throughput DNA sequencer (MiSeq, Illumina) for the bacterial 16S rRNA and fungal ITS gene amplicons in litter and soil samples. DNA samples extracted from Edge 2 and Interior 1 plots were used in this evaluation to capture the difference in the microbial community structure between these two contrasting plots.

Summary of nitrogen deposition
Cumulative nitrogen deposition via throughfall for the six-month period (from May to November 2018) was ranged from 2.2 to 3.7 kg N ha − 1 in the six experimental plots (Fig. 1). The maximum and minimum amounts of nitrogen deposition were observed in the most northern plot of the forest edge (Edge 3; Fig. 1a) and in the most northern plot of the forest interior (Interior 3), respectively. In summary, mean nitrogen deposition for three plots of the forest edge was 3.5 ± 0.9 kg N ha − 1 , which was 1.4-fold higher than that of the forest interior (2.5 ± 0.7 kg N ha − 1 ). A large proportion (> 76%) of the nitrogen deposition was in the form of NH4 + form in the study area.

Soil microbial properties vs. nitrogen deposition
Both of the net mineralization and nitri cation rates showed positive correlations to nitrogen deposition (Fig. 2). These positive correlations were statistically signi cant without any signi cant interactive effects from combinations of soil layer and nitrogen deposition (p > 0.05).
The abundances of 16S rRNA and bacterial amoA genes showed positive correlations to nitrogen deposition (Fig. 3). The positive correlation between 16S rRNA gene abundance and nitrogen deposition was statistically signi cant without signi cant interactive effects from the combinations of soil layer and nitrogen deposition (p > 0.05). The positive correlation between bacterial amoA gene abundance and nitrogen deposition was also statistically signi cant, while there was signi cant interactive effect from the combinations of soil layer and nitrogen deposition. The slope value for the relationship between nitrogen deposition and amoA gene abundance in the surface mineral soil was 3.5-fold greater than that in the litter layer. There was no signi cant correlation between other microbial properties and nitrogen deposition (p > 0.05). There was also no signi cant difference in the microbial species composition between Edge 2 and Interior 1, while the microbial composition was signi cantly different between the litter and soil layers (Fig. 4).

Environmental factors vs. nitrogen deposition
There was no apparent relationship between nitrogen deposition and environment factors (i.e. temperature and soil water content) (Fig. 5). Comparing mean values of these environmental factors between the forest edge and interior, the differences were only 0.2 °C in temperature and 1% in soil water content.

Soil chemical properties vs. nitrogen deposition
In contrast to the environmental factors, some soil chemical properties were found to be signi cantly correlated with nitrogen deposition (Table 1). Carbon-to-nitrogen ratios of the litter and soil samples showed negative correlations to nitrogen deposition. In soil samples, NO 3 − content showed a positive correlation to nitrogen deposition, while NH 4 + content showed a negative correlation to nitrogen deposition. This should include the ndings of the study including, if appropriate, results of statistical analysis which must be included either in the text or as tables and gures.

Discussion
Soil microbial activity vs. nitrogen deposition Nitrogen disposition was greater in the forest edge than in the forest interior (Fig. 1). A signi cant contribution of nitrogen fertilizer applied to surrounding pasture grasslands to the forest was suggested because more than 76% of the deposited nitrogen was observed to be in the form of NH 4 + . However, the amount of nitrogen deposition in this forest was less than half of threshold amount to cause adverse The moderately elevated nitrogen deposition likely enhanced soil microbial activity in the forest edge (Fig. 2). This microbial response is different from observations of the reduction in soil microbial CO 2 release in forests under extensively elevated nitrogen deposition (Janssens et al. 2010;Zhang et al. 2018). In our forest, enhancement of soil organic matter decomposition may be possible due to the enhancement of microbial activity at the edge sites; this is partly supported by the observations of the negative correlations between soil carbon-to-nitrogen ratio and nitrogen deposition ( Table 5). The relative abundance of carbon to nitrogen in organic matter generally decreases with the progress of microbial decomposition where organic carbon is mineralized and released as CO 2 while nitrogen is retained and reutilized by soil microbial community (Koarashi et al. 2014;Kramer et al. 2017). Correlations between nitrogen deposition and individual content of soil inorganic nitrogen species (Table 1) were probably resulted from the enhanced consumption of NH 4 + and production of NO 3 − through nitri cation under the moderately elevated nitrogen deposition (Fig. 2).
Such an enhancement of soil microbial activity under moderately elevated nitrogen deposition (Fig. 2) can contribute to increasing CO 2 production through decomposition of soil organic matter, and thus to increasing atmospheric CO 2 concentration. Moreover, taking into account for the previously-known

Soil microbial abundances vs. nitrogen deposition
There was a remarkable difference between bacterial and fungal abundances in the terms of their correlations to nitrogen deposition (Fig. 3). This difference between bacteria and fungi is considered to re ect the different nitrogen demand between these two different types of microbes (Strickland and Rousk 2010). In general, bacterial biomass is relatively enriched in nitrogen compared with fungal biomass, suggesting a higher nitrogen demand of bacterial body (Strickland and Rousk 2010). Therefore, the observed linkage between the bacterial abundance and nitrogen deposition can be reliable when a high sensitivity of bacteria to changing nitrogen availability is assumed. The different responses to nitrogen deposition between bacteria and fungi in our forest can also be inferred from the lower ratio of microbial biomass carbon to nitrogen and the lower ratio of fungal to bacterial gene abundances at the edge sites than at the interior sites (Fig. 3), while the differences in these ratios between the edge and interior sites were not statistically signi cant (p > 0.05).
The bacterial amoA gene abundance appeared to respond to nitrogen deposition, but the archaeal amoA gene abundance was not (Fig. 3). In the investigated forest, Isobe et al. (2018) also found the synchronous temporal change in gross nitri cation rate and bacterial amoA gene abundance during the wintertime. This was somewhat different from previous suggestion that archaeal ammonia oxidizer plays an important role in soil nitri cation process in temperate forest and agricultural upland soils in Europe (Leininger et al. 2006). One of the possible interpretations of this discrepancy between European and Japanese forest soils is that soil conditions of our forest is preferable for bacterial ammonia oxidizers which have larger-sized cell body and higher cell-speci c-unit activity compared with archaea (Jia and Conrad et al. 2009). Then, the speci c dependence of bacterial ammonia oxidizer on autotrophic growth while contrary dependence of archaeal oxidizers on autotrophic growth and heterotrophic growth as well (Jia and Conrad et al. 2009) might result in the dominant contribution of bacterial community to nitri cation in our forest soils.
Moreover, this speci c sensitivity of bacterial ammonia oxidizers might be associated with the changes in species compositions of those bacteria (Isobe et al. 2020). While the overall compositions of bacterial and fungal communities were less sensitive to moderately elevated nitrogen deposition in the investigated forest (Fig. 5)

Conclusions
In a Japanese forest surrounded by pasture grasslands, we found that soil microbial activities and their abundances were increased along with spatial gradients of nitrogen deposition between forest interior and boundary edge area, nevertheless elevated level of nitrogen deposition in forest edges was not extreme, but moderately (< 10 kg N ha − 1 year − 1 ). Our nding was different from the most of previous to SRX7906320 as the SRA experiment accession numbers). Other data that support the ndings of this study are available from the corresponding author upon reasonable request. The codes that process the data of this study are also available from the corresponding author upon reasonable request.

Figure 1
Locations of six experimental plots in a Japanese cool temperate forest (a), and nitrogen deposition via throughfall for 6 months (May 9th to November 20th, 2018) in each experimental plot (b). Purple arrows in the top panel represent major wind ow which transport fertilizer from pasture grasslands to our forest.
Error bars in bottom panel represent standard deviations (n = 7).

Figure 2
Correlations of net nitrogen mineralization and nitri cation rates to nitrogen deposition. The probability level (p value) for statistically signi cance examined by two-way ANCOVA was presented above panels.
The two-way ANCOVA was applied to the correlation and the difference in correlations between litter and soil samples (see text for details). Arrows represent correlations with p < 0.05.

Figure 3
Correlations of microbial biomass carbon and nitrogen and various gene contents, such as bacterial 16S rRNA, fungal ITS, bacterial amoA, and archaeal amoA genes, to nitrogen deposition. Statistical analysis of correlation was conducted in the same manner as Figure 2 (see text for details). Correlations of microbial biomass carbon and nitrogen and various gene contents, such as bacterial 16S rRNA, fungal ITS, bacterial amoA, and archaeal amoA genes, to nitrogen deposition. Statistical analysis of correlation was conducted in the same manner as Figure 2 (see text for details).