*3.4. Nitrogen Uptake by Miscanthus* × *Giganteus*

Nitrogen fertilization caused a significant increase in the nitrogen uptake in all the examined parts of plants (*p* = 0.0000). For the control object, the nitrogen uptake by rhizomes decreased until July, whereas in fertilized plots it decreased until August (*p* = 0.0118) (Table 7). The highest uptake of nitrogen in rhizomes was found in December, while in whole plants it was found in November. Therefore, it can be presumed that rhizomes can be a nitrogen reserve for shoots. In the initial vegetation period, the nitrogen uptake in leaves was higher than that in stems. The accumulation of nitrogen in stems was found to be higher than in leaves starting in August (Figure 9). The highest nitrogen uptake was found in the case of whole plants, with an increasing tendency from July to September, where the differences became insignificant (Figure 10). The fastest increase in the N uptake by rhizomes was observed from October to November (Figure 10). In the case of the aboveground parts of plants, the nitrogen uptake increased from June to September and then decreased (Figure 10).


**Table 7.** Nitrogen uptake of *Miscanthus* <sup>×</sup> *giganteus* (kg m<sup>−</sup>2) (average for years 2014–2016).

**Figure 9.** Nitrogen uptake by leaves and stems during the vegetation period in the years 2014–2016 (average for years).

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**Figure 10.** Nitrogen uptake by the whole plants (average for years).

#### **4. Discussion**

Nitrogen fertilization is important for biomass production and its components. The results provided statistical evidence to prove that the number of shoots responded positively to N fertilization. Other studies have also shown an increase in the number of shoots after applying N [53–55]. The water

concentration in rhizomes and stems, the yield of dry mass leaves, and the nitrogen uptake was dependent on the level of nitrogen fertilization. Higher water content promoted metabolic processes and faster dry mass accumulation [56]. Therefore, research has been undertaken to determine the influence of nitrogen fertilization on the dynamics of the water content changes in rhizomes during the whole vegetation period. According to Drazic et al. (2017) [25], the number of stems per rhizome depended strongly on the soil type and was in strong positive correlation with the yield in all years. In our own research, the number of shoots were not significantly different during the experimental years.

In our research, the application of nitrogen stimulated the number of shoots. The plant height was also increased by N fertilization in various terms of harvesting. The plant height increased after the application of N, which was also reported by Cosentino et al. (2007) [54] and Finnan and Burke (2014) [39].

There have been conflicting results concerning the yield response of *Miscanthus* × *giganteus* to nitrogen fertilization and its yield components. Our positive responses to nitrogen fertilization were in agreement with Arundale et al. 2014 [57]. Moreover, Greef, J.M. (1995) [35] and Lee and Boe (2005) [26] obtained similar results when applying a 60 kg ha−<sup>1</sup> N dose as appropriate for proper rhizome development and *Miscanthus* × *giganteus* yield increase. In the research of Dierking et al. (2017) [17], a dose of 75 kg ha−<sup>1</sup> N contributed to the increase in the *Miscanthus* biomass yield, and this amount was applied annually. In the research of Lee and Boe (2005) [26], the dry matter yield visibly increased when the nitrogen fertilization increased up to 60 kg ha−<sup>1</sup> N. However, increasing the nitrogen dose further did not contribute to an increase in the *Miscanthus* yields. The Miscanthus dry matter yields obtained in this research were 2.55 and 2.49 kg m−<sup>2</sup> for 60 and 120 kg ha−<sup>1</sup> N, while in the control plant it was 1.3 kg m−2. Schwarz et al., 1994 [34], conducted an experiment involving nitrogen fertilization that did not have a significant impact on the *Miscanthus* yield. In their second year of cultivation, they obtained a yield of 0.8 kg m−2, and in the third year they obtained 2.2 kg m−2. Moreover, many other studies have shown that nitrogen fertilization is not required to obtain high yields of *Miscanthus* × *giganteus* biomass [58]. Christian et al. (2008) [33] did not find any answer to the applied N in 14 consecutive harvests. This result is supported by other studies that showed no response to N fertilization. However, some experiments have been concerned with soils featuring a large N content [13,21,25,34]. No reaction to nitrogen was found during the first two years after planting. Maughan et al. 2012 [21] reported a small positive reaction in a dose of 100 kg ha−<sup>1</sup> N of fertilizer. According to Kering et al. (2012) [13], Himken et al. (1997) [58], and Miquez et al. (2008) [21], *Miscanthus* yields are not dependent on the level of nitrogen fertilization, as they determined 2.5–3.0 kg m−<sup>2</sup> of D.M. and even 3.8 kg m−<sup>2</sup> of D.M. In our research, the dry matter yield with the nitrogen fertilization of all examined plants was insignificantly higher compared to the control. Only the leaf yields of D.M. depended on nitrogen fertilization.

The ambiguous response to nitrogen fertilization results from several reasons:


Precipitation is the most important factor that directly and indirectly affects the biomass yield of *Miscanthus* × *giganteus*. Plant biomass production reacts positively to annual rainfall [60], and the seasonal distribution of rainfall is a key factor that determines the formation of perennial grasses and biomass yield [26,60]. In this experiment, the precipitation was variable during the 3-year study period, with much less precipitation than 2015. In our research, the most favorable year with a high and evenly distributed precipitation was in 2016; however, this did not translate into dry matter yields but rather translated to the water content in all the examined plant parts. According to Heaton et al. (2004) [46], the biomass yield may be affected by rainfall during the growing season from April to September.

The nitrogen uptake was significantly affected by the analyzed factors—nitrogen fertilization and the term of harvesting. According to Roncucci et al. (2014) [14], the time of harvest is the most relevant factor in influencing the miscanthus nutrient uptakes. Late harvesting (W) led to a reduction in the nitrogen uptake of about 80% in the aboveground biomass. This nitrogen uptake is observed to be lower than the literature data. In 10 years of research in the UK, Christian et al. (2008) [33] reported that the N is 76 and 6 kg ha−<sup>1</sup> N. According to Roncucci et al. (2014) [14], N fertilization affected the nutrient uptake mainly in autumn, with no differences in winter. These results are in agreement with those of Himken et al. (1997) [58], who observed a higher N uptake with higher N fertilization rates in November, which is confirmed by our results. Nitrogen fertilization in the fertilizer treatments significantly affected the nitrogen uptake by all plant parts, which is confirmed by Strullu et al. (2011) [30].

Slightly higher results relating to the nitrogen uptake under various N doses in the harvest biomass of giant miscanthus were found in Christian et al. (2008) [33]. In Beale et al. (1997) [29], the rhizome nitrogen uptake decreased until July and then increased until December. Similar conclusions were presented in our research.

#### **5. Conclusions**

Nitrogen fertilization did not contribute to the increase in all the examined yield components. The proposed dose caused an increase in all the components of features and the dry matter yield. However, the differences were mostly insignificant. Only the dry mass of leaves increased significantly in the experiment. The water content in the rhizomes and stems increased under nitrogen fertilization. Therefore, we can assume that rhizomes, because of their significant nitrogen uptake, can constitute a nitrogen reserve for elements in the initial growth and development stages of plants. The results coming from our 3-year field experiment suggest that N fertilization is unnecessary for sustainable biomass production.

**Author Contributions:** Conceptualization, I.G.-B., W.H., and A.K.; data curation, M.K.; formal analysis, W.H. and A.J.-R.; investigation, I.G.-B., W.H., and M.K.; methodology, I.G.-B. and A.K.; project administration, I.G.-B.; resources, A.K. and A.J.-R.; software, M.K.; supervision, M.K.; visualization, M.K. and A.J.-R.; writing—original draft, I.G.-B. and W.H.; writing—review and editing, A.K. and A.J.-R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**


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