*2.1. Study Site and Fertilization Treatments of Miscanthus* × *Giganteus*

An investigation of *Miscanthus* × *giganteus* and nitrogen fertilization was conducted after 10 years of establishing crops (2014–2016) at the Experimental Station of Wroclaw University of Environmental and Life Sciences, Pawlowice (geographical location 17◦7 E and 51◦08 N in the Lower Silesian Voivodship, Wrocław, Poland). Pawlowice is characterized by a vegetation period (March–November) that lasts 223–230 days, with an average temperature during the growing season of 14.5 ◦C and an annual rainfall ranging between 500 and 600 mm (around 350 mm during the growing season). The soil conditions were defined as alluvial soil, very light on loose sand, and sandy gravel (V grade) (soil classification used in Poland). These soils are weak with a low humus level, and are poor in organic matter. The fifth class of soil quality (6 classes of soil quality: I class—the best arable land; VI—the weakest arable soil) comprises weak arable soils [47].

Plowing was carried out in 2003 at the depth of 20–25 cm, followed by rotary harrowing before planting. Miscanthus rhizomes (10 cm long with 3–6 nodes) were planted in a row spaced 75 cm apart and another row spaced 48 cm apart (on 1 ha–27,777 rhizomes). *Miscanthus* × *giganteus* was planted in 2004. Plantation was fertilized annually from the year 2004 to 2013 at the beginning of the growing season using the following doses: 40 kg ha−<sup>1</sup> of N ammonium nitrate 32%, 17.5 kg ha−<sup>1</sup> of P 40% enriched superphosphate, and 50 kg ha−<sup>1</sup> of K potassium salt. The plots were separated by a distance of 1.0 m, and all measurements (non-destructive and destructive) were taken at least 0.2 m from the edge of the plot in the years 2014–2016. The dimension of the plot was around 20 m2. Nitrogen treatments of 0 and 60 kg/ha were applied in March/April during each of the 3 years (17/3/2014, 18/3/2015, 17/4/2016) after pulling out the bedding. Fertilization was annually (from 2014 to 2016) applied during the field experiment, where the following doses were used: 17.5 kg ha−<sup>1</sup> of P 40% enriched superphosphate, 50 kg ha−<sup>1</sup> of K potassium salt. After fertilization, the mulch was placed in its original position.

Fertilization was applied via a hand broadcast at the beginning of the vegetation period.

No significant pests and weeds were found in the *Miscanthus* cultivation during the experiment, so the use of herbicides was not necessary.

#### *2.2. Plant Growth Measurement*

Miscanthus sampling started from the 30th day of the vegetation period and every 30 days until the end of vegetation period (June, July, August, September, October, November, and December) in the years 2014–2016. At each date of sampling, a plant sample of the aboveground part of the plant and rhizomes was sampled from an area of 0.25 m2. The fresh mass of the rhizomes and the aboveground part was determined. Additionally, 10 randomly selected shoots were sampled from each replication to perform measurements on plant material—the height of the upper leaf, the diameter measured 10 cm from the soil surface, and the number of leaves per one stem. All the measurements (except the number of shoots) were made on 10 shoots per plot. The number of shoots was counted from a unit of 0.25 m<sup>2</sup> from each replication. Both white and yellow rhizomes were sampled.

Terminal (from outer rows) plants from the external rows were not included in the analysis because of the so-called edge effect. After the end of the vegetation period, *Miscanthus* was harvested at 10–15 cm using a circular saw. Harvested crops were weighed and the percentage of dry matter was determined. The dry biomass weight was determined by drying samples (specific weight, 500 g) to 60 ◦C for up to 48 h, then drying them at 105 ◦C for 4 h. Further, the harvested crops were weighed and the fresh mass yield was determined. The dry biomass weight was determined by drying samples (specific weight, 500 g) to 60 ◦C for up to 48 h, then drying them at 105 ◦C for 4 h. On this basis, the dry biomass yield per 1 m2 in a given year was calculated.

Water concentration was calculated according to the Formula (1):

Water concentration (%) = (100 × (FM − DM))/FM. (1)

FM—fresh mass. DM—dry mass.

#### *2.3. Soil and Weather Conditions*

Tables 1 and 2 summarize the soil conditions for the *Miscanthus* plantation in this trial. Soil samples were twice taken (April, July) during the vegetation period and after its end (November) each year. These dates were presented as annual mean values. Soil samples were taken from the experimental field at a 0–20 cm soil depth and were thoroughly mixed to make a representative composite soil sample. The analysis was comprised of pH, humus, C, N, P, K, S, and micronutrients. Analyses were performed according to the following methods: the soil reaction (pH/KCl (potassium chloride)) was found using the potentiometric method; the total organic carbon was found using Tiurin's method [48]; the total nitrogen (classical distillation) content was found using the Kjehdal method both in soil and plant material [48]; the available forms of potassium and phosphorus were found using the Egner–Rhiem method; magnesium was found using the Schachtschabel method [49]; the total carbon content (TOC) was found via oxidimetric titration [50]; sulfur in the extract was found using the Johnson–Nishita procedure [51]; humic substances (HS) were found using the short fractionation method [52]; and the total contents of Fe, Mn, Zn, and Cu were found using an atomic absorption spectrophotometer (ASA) after mineralization with a concentrated mixture of acids using atomic-absorbent flame spectrophotometry Varian spectra AA 200 [52].

**Table 1.** The content of organic matter and soil abundance in macronutrients for a depth of 0–20 cm in 2014–2016.



**Table 2.** Soil abundance in the micronutrients at the depth of 0–20 cm in 2014–2016.

The soil's carbon stock was typical for light alluvial soils, and the C: N ratio was on average 10.6:1, which indicates the appropriate process of the organic decomposition (Table 1). In the experimental years, the soil reaction ranged from 4.8 to 5.0 (acidic), which was favorable for *Miscanthus* cultivation, and the arable layer's richness in nutrients was as follows: P—very high; K—medium; Mg—low; S—medium; Fe—low; Mn—medium; Zn—high; and Cu—low (Tables 1 and 2). The assessment of the soil's nutrient content was determined by limit numbers to assess the content of elements developed by the Polish Institute of Soil and Plant Cultivation in Puławy [47].

Monthly data on the temperature and precipitation in the years 2014–2016 are presented in Table 3. The temperatures in the years 2014–2016 oscillated between ±9 ◦C in IV through to an average of ±17 ◦C from V to VIII. During the experimental years, the thermal conditions were favorable for the development of *Miscanthus*, with mild winters characterized by positive temperatures. The highest temperatures were recorded in 2015, while the lowest were in 2016 (Table 3).

The optimal amount of rainfall for *Miscanthus* × *giganteus* depends on many factors, including the air temperature, soil type, and groundwater level; however, 600 mm was sufficient for the development of *Miscanthus* [14,26]. The year with the lowest rainfall was 2015. Despite the lack of rainfall, there were no reduction in the yield. The highest rainfall during the growing season was recorded in 2016 (Table 3).

**Table 3.** Weather conditions during 2014–2016 with a 30-year average for Wroclaw, Lower Silesia (Poland).


#### *2.4. Statistical Analysis*

The experiment was conducted with a randomized block design in four replications to test the effects of N fertilization on the morphological traits and yield of *Mischanthus.* The analysis of variance (ANOVA) and the mixed model with repeated measurements was used. The doses of nitrogen fertilizers were assumed to be a fixed factor, while the years were random. The results of the biometric measurements of the *Mischanthus* were analyzed via ANOVA in the Statistica program (13.1 StatSoft, Kraków, Poland).

#### **3. Results**
