Nitrogen Immobilisation and Microbial Biomass Build-Up Induced by Miscanthus x giganteus L. Based Fertilisers

: Cultivation of Miscanthus x giganteus L. ( Mis ) with annual harvest of biomass could provide an additional C source for farmers. To test the potential of Mis -C for immobilizing inorganic N from slurry or manure and as a C source for soil organic matter build-up in comparison to wheat ( Triticum aestivum L.) straw (WS), a greenhouse experiment was performed. Pot experiments with ryegrass ( Lolium perenne L.) were set up to investigate the N dynamics of two organic fertilisers based on Mis at Campus Klein-Altendorf, Germany. The two fertilisers, a mixture of cattle slurry and Mis as well as cattle manure from Mis -bedding material resulted in a slightly higher N immobilisation. Especially at the 1st and 2nd harvest, they were partly signiﬁcantly different compared with the WS treatments. The fertilisers based on Mis resulted in a slightly higher microbial biomass C and microbial biomass N and thus can be identiﬁed as an additional C source to prevent nitrogen losses and for the build-up of soil organic matter (SOM) in the long-term. by the respective N content of the and by extrapolation to the amount of soil in the upper 30 cm of a hectare. Statistical analyses were performed using IBM SPSS Statistics 27. Normal distribution of data was tested using the Shapiro–Wilk test. Levene’s test based on means was used to verify homogeneity of variances. To identify treatment differences between three treatments, a one-way analysis of variance (ANOVA), following by a post hoc Tukey’s HSD (honest signiﬁcance difference) test were used. To identify differences between two


Introduction
Technological developments, as well as economic conditions (agricultural subsidies, world market trade), have reduced the production costs in agriculture in the last decades. This has changed production methods resulting in nutrient access and pollution of the environment, especially in areas with high livestock density and slurry application and that, ultimately, threaten the long-term stability of agricultural production [1,2].
Inadequate soil management in arable farming can lead to soil degradation with negative effects on crop production being compensated by, for example, increased fertilisation; but more intensive treatments often lead to negative effects on the environment [3,4]. Along with agricultural intensification, increased nitrogen (N) use resulted in lower N use efficiency (NUE) [5,6], to an accumulation of N in soil and to nitrate leaching into ground and surface waters, resulting in eutrophication. Furthermore, the risk of NH 3 emissions with toxic effects on the respiratory system of mammals and humans and N 2 O emissions, which is a potent greenhouse gas, increased with enhanced N inputs [7,8].
In addition, changed production practices, like the replacement of cereals with root crops and fodder crops with a lower C/N ratio [9] and past land use changes by conversion of grassland to cropland [10][11][12][13] all led to a decrease in soil organic carbon (SOC) in many cases. Climate change with rising temperatures increases the decomposition of organic matter further and results in SOC losses [14]. Therefore, it is essential to use organic fertilisers and other C sources in a way that retains N and C in the crop-livestock-soil system  The experiment aimed to stimulate microbial growth by adding an additional agricultural C source to immobilise inorganic N and enhance SOC in the soil. Therefore, the biomass of Mis grown on another field was used for two utilization pathways, mixed with Cattle Slurry (CS) or used as bedding material creating Cattle Manure (CM). The Mis biomass was harvested in April 2017 (exp. 1,2)  In one pathway of Mis use, CS was mixed with Mis (Cattle Slurry mixed with Miscanthus = CS-Mis) and, as a complementary treatment, CS was mixed with WS (Cattle Slurry mixed with Wheat Straw = CS-WS) and both converted into spreadable substrates. This has the objective to bind odorous compounds of the cattle slurry (CS), to keep nutrients in the topsoil for a longer time against precipitation, to reduce gaseous N emissions and to slow down the nitrification. It also aims to achieve a slower and longer lasting N mineralization of the mixtures. For the determination of the best possible mixing ratio of both mixed treatments (CS-Mis, CS-WS) concerning maximum absorption of CS to Mis and of CS to WS biomass, different amounts of CS (from 1 to 10 kg of CS in steps of 0.5 kg CS) were mixed with 1 kg of Mis or WS. As a result of this pre-test, a complete absorption (no excess liquid visible) of the liquid fraction of CS to Mis and WS, respectively, over seven days, was achieved at a ratio of 5 kg of CS to 1 kg of Mis and at a ratio of 8.5 kg of CS to 1 kg of WS. After mixing, the two mixture treatments were stored for five weeks on a manure slab and covered with a silage film to prevent precipitation intrusion and allow for N immobilisation.
The other option to use Mis on a farm was the use of Mis as bedding material in livestock. For this purpose, cattle were bedded with Mis (Cattle Manure from Miscanthus = CM-Mis) and, as a reference, cattle were bedded with WS (Cattle Manure from Wheat Straw = CM-WS) according to standard farm practice and mucked out after about six weeks. As a reference treatment for the two mixtures, a pure CS was tested in the experiment. In addition, two further treatments were tested, this was a mineral N-fertilisation (Urea Ammonium Nitrate solution = UAN) as well as a treatment without any N applied (No Nitrogen applied = NoN). All abbreviations of the fertiliser products and fertiliser feedstocks are listed in Table 2. The application rates of the tested treatments were 120 kg N ha −1 (experiment 1) and 170 kg N ha −1 (experiments 2 and 3). The nutrient content of the applied fertilisers (Tables 3  and 4) was determined by a certified laboratory following the requirements of the Fertiliser Ordinance 2017 of Germany [62]. Table 3. Nutrient contents of the used treatments for experiment 1 (120 kg total N ha −1 ) and experiment 2 (170 kg total N ha   %  45  32  31  3  1  50  5  3  C/N  ratio  10  29  20  21  10  -288  73 Indication of the nutrient content in: 1 kg m −3 ; 2 kg t −1 . Indication of the nutrient content in: 1 kg m −3 ; 2 kg t −1 . The tested treatments were mixed with 6.2 kg dry matter soil and with additional plant macro-and micronutrients applied in inorganic form as shown in Table 5. These nutrients were also applied after the second and fourth harvest to avoid nutrient deficiency effects other than N. The soil was then filled into Kick-Brauckmann pots (with closed drainage) and German ryegrass (Lolium perenne L., Valerio) was sown at a sowing rate of 0.15 g per pot (sowing rate of 40 kg ha −1 ). Each of the three experiments was set up as a completely randomized block design with five replicates per treatment (35 pots per experiment, 105 pots for the three experiments). To ensure ideal growth conditions for plants and soil microorganisms, all pots were adjusted to 60 to 70% of the maximum water holding capacity (WHC) by applying distilled water regularly. For this, pots were weighed twice to thrice a week, depending on temperature conditions and then irrigated. Table 5. Form of supply and amounts of macro-and micronutrients to each pot supplied at the start of the experiment, after the second and after the fourth grass harvest. Nitrogen was only supplied once via the test materials at the start of the experiment.

Plant and Soil Analyses
In each experiment, plants were cut six times with scissors to 0.03 m height. The thermal time (cumulative day degrees from 6 to 22 o'clock) and the day after sowing of the respective harvests is shown in Table 6. The obtained biomass was dried to a constant weight at 60 • C to calculate the dry matter yield. The dried biomass was ground by using a disk mill (TS 250, Siebtechnik GmbH, Mülheim an der Ruhr, Germany) and 6 mg ± 0.2 mg of each ground sample was weighed into tin cartridges. The C and N concentrations of each harvest-biomass was analyzed by using an elemental analyzer (EA 3000 series, HEKAtech GmbH, Wegberg, Germany). Plant N uptake was calculated by using dry matter yield and N concentration. It was extrapolated to one hectare, assuming a soil bulk density of 1.32 g cm −3 (Ah horizon up to 30 cm depth).
At the end of the experiments, a soil aliquot of each pot was used to analyze inorganic N (N min = NH 4 + + NO 3 − ; NO 2 − was not detectable), soil microbial biomass C (MBC) and soil microbial biomass N (MBN). For this, soil samples were sieved at 2 mm and all visible roots were removed. For the analysis of inorganic N, 25 g of the field-fresh soil was weighed into PE bottles, mixed with 100 cm 3 of 1% K 2 SO 4 and placed on an overhead shaker at 22 rpm for 60 min. After shaking, all extracts were filtered (VWR 305; particle retention: 2-3 µm). The first 10 cm 3 of the filtrate were discarded to obtain the purest possible extract. The extract was filled into plastic cuvettes, then stored until further analysis at −18 • C. The inorganic N content was determined with the AutoAnalyzer 3 from Bran + Luebbe GmbH Norderstedt, Germany.
MBC and MBN were analyzed by chloroform fumigation-extraction [63,64]. Therefore, two portions of 10 g of moist soil were weighed into PE bottles. One sample was for fumigation-and the other one for non-fumigation-extraction. The fumigation was carried out in a vacuum desiccator at 25 • C using ethanol-free chloroform (CHCl 3 ) for 24 h in the dark. The fumigated and non-fumigated samples were then extracted with 40 cm 3 of 0.5 M K 2 SO 4 and placed on a horizontal shaker at 180 rpm for 30 min. After shaking, all extracts were filtered (VWR 305; particle retention: 2-3 µm) and stored until analysis at −18 • C to avoid microbial transformation processes. Just before starting the analyses, extracts were defrosted rapidly to room temperature. In all extracts, organic C and total N were detected after combustion at 800 • C by using a Multi N/C 2100S (Analytic Jena, Jena, Germany). MBC was calculated as the ratio of extractable C (EC) and k EC . EC is the difference between organic C extracted from fumigated soils and non-fumigated soils, whereas k EC is a coefficient with the value of 0.45 [65] and represents the fraction of microbial C released in 24 h of fumigation. MBN gets calculated as the ratio of extractable N (EN) and k EN . EN is the difference in organic N extracted from fumigated soils and non-fumigated soils, where k EN is a coefficient with the value of 0.54 [32,63] and represents the fraction of microbial N.
Plant N uptake, inorganic N, MBC and MBN were extrapolated to one hectare, assuming a soil bulk density of 1.32 g cm −3 (Ah horizon up to 30 cm depth). The amount of N mineralized from applied fertilisers was estimated by subtracting the sum of plant N uptake of NoN treatments from the N uptake of the fertilized ryegrass. The total inorganic N (N min ) value at the start and end of the test was included in the calculation by subtracting these differences from the N uptake of each treatment. This calculation does not take into account N losses in the form of ammonia and nitrous oxide. These are assumed to be minimal because the organic fertilisers were incorporated into the soil immediately and the soil moisture was around 60% WHC.

Statistical Analyses
Dry matter yields are shown as arithmetic means (n = 5). Nitrogen uptake was calculated on a pot basis by multiplying the dry matter plant yield by the respective N content of the biomass and by extrapolation to the amount of soil in the upper 30 cm of a hectare. Statistical analyses were performed using IBM SPSS Statistics 27. Normal distribution of data was tested using the Shapiro-Wilk test. Levene's test based on means was used to verify homogeneity of variances. To identify treatment differences between three treatments, a one-way analysis of variance (ANOVA), following by a post hoc Tukey's HSD (honest significance difference) test were used. To identify differences between two Agronomy 2021, 11, 1386 7 of 17 treatments, t-test was used. Tukey's HSD and t-test were performed separately for each experiment. When data were not normally distributed or no homogeneity of variance were detected, Welch test and Games-Howell test were used to identify differences for three or more treatments, Mann-Whitney-U test was used to identify differences between two treatments. p-values of 0.05 were used as threshold for significant interactions.

Plant N-Uptake
The plant N uptake indicates clear differences in the N availability of the applied N fertilisers. Ryegrass fertilized with mineral N (UAN) showed the highest N uptake at the 1st harvest, but already at the 3rd harvest, no differences in N uptake was detected between the mineral treatment and the treatment without N addition (NoN). When ryegrass was fertilized with cattle slurry (CS), lower amounts of N were plant-available especially at the 1st harvest as compared to mineral fertilisation ( Figure 1). (honest significance difference) test were used. To identify differences between two treatments, t-test was used. Tukey's HSD and t-test were performed separately for each experiment. When data were not normally distributed or no homogeneity of variance were detected, Welch test and Games-Howell test were used to identify differences for three or more treatments, Mann-Whitney-U test was used to identify differences between two treatments. p-values of 0.05 were used as threshold for significant interactions.

Plant N-Uptake
The plant N uptake indicates clear differences in the N availability of the applied N fertilisers. Ryegrass fertilized with mineral N (UAN) showed the highest N uptake at the 1st harvest, but already at the 3rd harvest, no differences in N uptake was detected between the mineral treatment and the treatment without N addition (NoN). When ryegrass was fertilized with cattle slurry (CS), lower amounts of N were plant-available especially at the 1st harvest as compared to mineral fertilisation ( Figure 1). The cumulated N uptake of ryegrass was significantly lower (7% to 24%) compared to pure CS when Mis or WS were mixed with CS and then applied as C-rich organic N fertiliser (Table 7). Compared to pure CS fertilisation, the addition of WS to CS induced a 7% to 17% reduction in plant N uptake and the addition of Mis induced a slightly stronger reduction with plant N uptake being reduced by 12% to 24% (Table 7). The cumulated N uptake of ryegrass was significantly lower (7% to 24%) compared to pure CS when Mis or WS were mixed with CS and then applied as C-rich organic N fertiliser (Table 7). Compared to pure CS fertilisation, the addition of WS to CS induced a 7% to 17% reduction in plant N uptake and the addition of Mis induced a slightly stronger reduction with plant N uptake being reduced by 12% to 24% (Table 7). Table 7. Percentage of N uptake of the two mixtures (CS-Mis = Cattle Slurry-Miscanthus (5 kg to 1 kg); CS-WS = Cattle Slurry-Wheat Straw (8.5 kg to 1 kg)) to N uptake of cattle slurry (CS), at the time of each harvest and cumulatively (cum). Listed for experiment (exp.) 1,2 and 3, respectively. Different letters within a column and within each experiment number show significant differences. One-way ANOVA; p < 0.05; ns = not significant; n = 5. Especially in the period after application until the 1st harvest, the addition of Mis or WS to CS caused a significant reduction in plant N uptake. The addition of WS to CS significantly reduced plant N uptake by 50% compared to CS fertilisation only. The addition of Mis to CS caused an even greater reduction in plant N uptake (53% to 61%), which was statistically significant in exp. 1 and 2, compared to WS addition (48% to 50%) ( Table 7).

Harvest
At the 2nd and 3rd harvest, N uptake of ryegrass, fertilized with mixtures of Mis or WS and CS slightly increased and the N uptake of ryegrass fertilized with pure CS slightly decreased, compared to the 1st harvest, however, only to a small extent (Figure 1). Therefore, in exp. 1 and 2, at the 2nd harvest, N uptake in ryegrass fertilized with CS-Mis is only 4% to 7% lower than that of the ryegrass fertilized with CS only.
Moreover, at the 2nd harvest, N uptake of ryegrass fertilized with CS-WS increased so that it was identical (exp. 1) or even higher than plant N uptake after pure CS fertilisation (exp. 2). In exp. 3, at the 2nd harvest, only 70% (CS-Mis) to 80% (CS-WS) of the N was taken up by plants compared to fertilisation with pure CS (Table 7). From the 3rd harvest until the end of the experiment (temperature sum of more than 3000 • C), the plant N uptake in ryegrass fertilized with the mixtures (CS-Mis, CS-WS) was in most cases higher than that after CS fertilisation only ( Figure 1, Table 7).
Plant N uptake after CS-Mis fertilisation was only slightly lower than that after CS-WS application ( Figure 1, Table 7).
When ryegrass was fertilized with the two cattle manure types, with Mis or WS, there was no significant difference in the cumulative plant N uptake, but rather in the dynamics of the relative N uptake (Table 8). Until the 1st harvest, the unfertilized ryegrass (NoN) took up the same amount of N as the ryegrass fertilized with CM-WS or CM-Mis, respectively ( Figure 1). In exp. 1 and 2, at the 2nd harvest, N uptake was slightly higher when CM-Mis was applied, but decreased in the further development mainly to a lower N uptake level compared to CM-WS (Table 8). Exp. 3 showed larger differences in the dynamics of plant N uptake between ryegrass fertilized with the two manure types. Here, ryegrass fertilized with CM-Mis took up more N (13% to 19%) at the 3rd and especially at the 4th harvests, whereas uptake was lower at the 1st and 2nd harvest (Table 8). Table 8. Percentage of the new type of cattle manure from Miscanthus shredded bedding (CM-Mis) to N uptake of conventional cattle manure from wheat straw bedding (CM-WS), at the time of each harvest and cumulatively (cum). Listed for experiment (exp.) 1, 2 and 3, respectively. Different letters within a column and within each experiment number show significant differences between the two types of manure (p < 0.05, t-test); ns = not significant; n = 5.

Microbial Mineralisation-Immobilisation as Affected by Added Miscanthus Straw
The fraction of mineralised N was significantly reduced after adding organic C in the form of Mis or WS to cattle slurry in each experiment ( Figure 2A). Thereby, the addition of Mis resulted in a lower mineralized N fraction compared to the WS addition, in all 3 experiments (Figure 2A). In exp. 2 and 3, the difference was statistically significant. In exp. 1 and 3, Mis addition even resulted in no additional N mineralization compared to unfertilized ryegrass (Figure 2A). In exp. 2, 13% of the fertilized N from CS-Mis became plant available as inorganic N. In contrast, more N was mineralized in CS-WS, which was 6% in exp. 1, 20% in exp. 2 and 17% in exp. 3. These differences result mainly from the different N release patterns after application to the soil, as shown by plant N uptake especially at the 1st harvest (Table 7). This reduced N uptake, as a result of Mis or WS addition to CS, may indicate lower N release from CS through mineralization or increased N immobilisation by soil microorganisms facilitated by easily available C added with CS-Mis or CS-WS ( Table 7).
The microbial biomass C (MBC) and N (MBN) were both not significantly affected by adding C to CS either as Mis or WS (Figures 3 and 4). However, the mean of MBC was slightly higher in CS-Mis compared to CS-WS, moderately in all experiments ( Figure 3A). The MBN indicated the same tendency of increasing after addition of CS-Mis compared to fertilisation with CS-WS ( Figure 4A). Thus, the lower N mineralization of CS-Mis compared to CS-WS (Table 7, Figure 2A) is generally reflected in a slightly higher microbial biomass ( Figures 3A and 4A). In CS-Mis, MBN was 23 kg ha −1 to 60 kg ha −1 higher and in CS-WS, MBN was 19 kg ha −1 to 51 kg ha −1 higher than MBN in the non-fertilised treatment (Table 9). Apparently, when Mis was used for mixing with CS, soil microorganisms were able to immobilize more N as compared to WS.  tilized ryegrass (Figure 2A). In exp. 2, 13% of the fertilized N from CS-Mis became plant available as inorganic N. In contrast, more N was mineralized in CS-WS, which was 6% in exp. 1, 20% in exp. 2 and 17% in exp. 3. These differences result mainly from the different N release patterns after application to the soil, as shown by plant N uptake especially at the 1st harvest (Table 7). This reduced N uptake, as a result of Mis or WS addition to CS, may indicate lower N release from CS through mineralization or increased N immobilisation by soil microorganisms facilitated by easily available C added with CS-Mis or CS-WS (Table 7).  The microbial biomass C (MBC) and N (MBN) were both not significantly affected by adding C to CS either as Mis or WS (Figures 3 and 4). However, the mean of MBC was slightly higher in CS-Mis compared to CS-WS, moderately in all experiments ( Figure 3A). The MBN indicated the same tendency of increasing after addition of CS-Mis compared to fertilisation with CS-WS ( Figure 4A). Thus, the lower N mineralization of CS-Mis compared to CS-WS (Table 7, Figure 2A) is generally reflected in a slightly higher microbial biomass ( Figures 3A and 4A). In CS-Mis, MBN was 23 kg ha −1 to 60 kg ha −1 higher and in CS-WS, MBN was 19 kg ha −1 to 51 kg ha −1 higher than MBN in the non-fertilised treatment (Table 9). Apparently, when Mis was used for mixing with CS, soil microorganisms were able to immobilize more N as compared to WS.     1  34  60  51  80  15  21  2  23  48  25  123  50  40  3  26  23  19  46  −16  46 When cattle manure (CM) from Mis as well as from WS were used as organic fertiliser, in exp. 2 and 3, a lower fraction, although not statistically significant, of CM-Mis was mineralized than of CM-WS ( Figure 2B). In exp. 1, the same amount of N was mineralized as became plant available from the soil N pool in the unfertilized ryegrass. Consequently, no additional N was mineralised of both CM-Mis and CM-WS ( Figure 2B). The tendency for lower N mineralization after CM-Mis fertilisation compared to CM-WS fertilisation was accompanied by a higher MBN in all experiments. In exp. 1 and 2, the difference was slightly lower, in exp. 3 it was obvious and significant for both, MBN and MBC ( Figures  3B and 4B). In CM-Mis, MBN was higher (46 kg ha −1 to 123 kg ha −1 ) and in CM-WS, MBN was mostly higher compared to the non-fertilised treatment (−16 kg ha −1 to 50 kg ha −1 ) ( Table 9).

Exp. CS CS-Mis CS-WS CM-Mis
After application of UAN and in the treatment without any N addition (NoN), the MBC did not differ and the MBN predominantly did not differ statistically significantly from the treatment with organic fertilisation (data not shown). MBC was slightly higher When cattle manure (CM) from Mis as well as from WS were used as organic fertiliser, in exp. 2 and 3, a lower fraction, although not statistically significant, of CM-Mis was mineralized than of CM-WS ( Figure 2B). In exp. 1, the same amount of N was mineralized as became plant available from the soil N pool in the unfertilized ryegrass. Consequently, no additional N was mineralised of both CM-Mis and CM-WS ( Figure 2B). The tendency for lower N mineralization after CM-Mis fertilisation compared to CM-WS fertilisation was accompanied by a higher MBN in all experiments. In exp. 1 and 2, the difference was slightly lower, in exp. 3 it was obvious and significant for both, MBN and MBC ( Figures 3B  and 4B). In CM-Mis, MBN was higher (46 kg ha −1 to 123 kg ha −1 ) and in CM-WS, MBN was mostly higher compared to the non-fertilised treatment (−16 kg ha −1 to 50 kg ha −1 ) ( Table 9).
After application of UAN and in the treatment without any N addition (NoN), the MBC did not differ and the MBN predominantly did not differ statistically significantly from the treatment with organic fertilisation (data not shown). MBC was slightly higher after UAN fertilisation (UAN: exp. 1 = mean 1096 kg ha −1 ± SD 104; exp. 2 = 1269 kg ha −1 ± 116; exp. 3 = 1254 kg ha −1 ± 184) than in the non-fertilized treatment (NoN: exp.

Miscanthus-Induced N Immobilisation
As an organic C source, we tested the utilisation of Mis and WS concerning N immobilisation and MB build-up which can yield in C sequestration. We demonstrate that Mis is at least as good as WS as a utilizable C source facilitating N immobilisation and microbial growth eventually contributing to the formation of microbial necromass and thus SOC. Nevertheless, the increase in MB is low, which is mainly caused by the large MB background, as caused by grassland conversion to arable land in 2013, overriding the effects of organic fertilisers. Thus, we expect clearer effects in soils with lower SOM.
The process of microbial N mineralisation-immobilisation depends on the biochemical composition of the substrate. In general, these processes are characterized by the NH 4 + content, the C/N ratio and the holocellulose and lignin contents [36,[66][67][68]. For WS, the holocellulose content is estimated to be 68% to 76% and the lignin content is estimated to be between 8% and 25% [36,[69][70][71][72]. For Mis, the holocellulose content is given as 70% and the lignin content as between 14% to 19% [71,73], being in the same range as WS. In contrast, the C/N ratio of WS was clearly lower at 73 (exp. 1,2) and 115 (exp. 3) compared to that of Mis at 166 (exp. 1,2) and 288 (exp. 3). Additionally, C availability was enhanced by lower mixing ratio of CS-Mis (5:1) compared to CS-WS (8.5:1) (Tables 3 and 4), suggesting a higher microbially available C derived from CS in the Mis-based fertiliser (CS-Mis, CM-Mis). In contrast, the NH 4 + content of both mixtures was almost identical. Thus, the higher microbially available C input in the form of Mis appears to have caused greater microbial N immobilisation, especially by the time of the 1st harvest, which is confirmed by a higher MBN in CS-Mis treatment ( Figure 4A). The addition of Mis as bedding material also resulted in a higher C/N ratio of CM-Mis compared to CM-WS (Tables 3 and 4) and thus also (like CS-Mis already) resulted in lower N uptake at 1st harvest ( Figure 1). For CM-Mis, we also assume the reason for a stronger N immobilisation being higher easily available C (as already for CS-Mis) compared to CM-WS. Another influence on the higher N immobilisation of the Mis treatments and for the MBN tending to be higher, might be the smaller particle size of Mis or a difference in the surface structure as compared to WS, which accelerates and facilitates microbial degradation processes [74]. This, however, needs to be verified in future studies. Eiland et al. (2001a) [71] and Eiland et al. (2001b) [75] tested the addition of Mis to pig manure to produce a growth medium for plants via composting processes. They reported a clear reduction in nitrification at a C/N of 35 compared to a C/N of 11, we observed N immobilisation at a C/N ratio of less than 30 (CS-Mis, CM-Mis). However, the two experimental settings cannot be compared because, unlike Eiland et al. (2001a) [71] and Eiland et al. (2001b) [75], we incorporated our treatments into the soil and there is a likelihood of NH 4 + released by microorganisms being fixed at negatively charged sites of clay minerals and SOM. Moreover, soil potassium (K) status, K + saturation, moisture conditions and the cation exchange capacity of the soil influence the amount of NH 4 + that can be fixed and thus reduce its nitrification and plant availability [76]. Jensen et al. (2001) [77] and Leth et al. (2001) [78] also conducted composting experiments of Mis with pig slurry and other N sources and observed high microbial activities respectively. Like our experiments, these results indicate a high amount of C in Mis that can be easily degraded by microorganisms, provided that a sufficient amount of available N is accessible.
Some other studies showed promotion of MB and thus N immobilisation after Mis biomass incorporation into the soil or application to the soil surface [79][80][81]. In contrast, Schimmelpfennig et al. (2015) [82], detected no initial N immobilisation after addition of Mis and slurry, successively, possibly because more N was added than became immobilised. Rex et al., (2015) [83] determined a decrease of fungal biomass, but an increase in bacterial biomass, when Mis biomass and pig slurry were applied together compared to pig slurry alone.
Especially on agricultural fields with a high potential of pedogenic N-mineralization, an additional C-input can reduce the risk for N-losses by induced microbial N-immobilisa-tion [35,36], causing N to be assimilated into the microbial cells and thus decreasing the inorganic N of the soil [74]. Reichel et al., (2018) [35] and Wei et al., (2020) [36] induced N immobilisation by the application of high carbon amendments, such as wheat straw and spruce sawdust and mention the holocellulose/lignin ratio as a future tool to prevent N losses. Our experiments, suggesting a tendency for microbial N immobilisation to be slightly higher when Mis is used compared to WS, each in conjunction with excreta ( Figure 1, Table 7), lead us to assume that Mis may find suitability for N immobilisation even without the addition of excreta as a high carbon amendment for N immobilisation.
The relative data of N uptake of the two mixtures (CS-Mis, CS-WS) to N uptake of CS (Table 7) as well as the percentage of N uptake of the new type of cattle manure from Miscanthus shredded bedding (CM-Mis) to N uptake of conventional cattle manure from wheat straw bedding (CM-WS) ( Table 8) demonstrate to the farmer how these fertilisers can be estimated and applied in comparison to the well-known fertilisers.

Miscanthus as C Source for Microbial-Derived C Sequestration and SOM Build-Up
For build-up of SOC, N compounds are essential [30,31]. In many areas, high organic N amounts are already formed by excretions in animal farming. These have a high potential for humus build-up, which, however, cannot be exploited without sufficient C availability and the humus build-up can only be insufficiently formed, resulting in a higher risk of N losses instead. In contrast, sufficient C availability with simultaneous N supply, as provided by CS-Mis and CM-Mis, could contribute to the formation of microbial necromass. Especially necromass has an essential role in the formation, conservation and stability of SOM and is thus a key component of C sequestration in soil [30,31,84,85]. Thus, the increase of SOM as C and N storage improves soil fertility. This would result in a reduction of N losses and in future could increase the NUE of organic N fertilisers. Future studies need to verify the role of Mis in microbial necromass formation to better understand its contribution to SOC build-up.
The cultivation of Mis enables farmers to develop an additional C source regionally [52]. The process of mixing CS with Mis, compared to the cascade utilisation as bedding material, does require an additional working step. However, both options contribute an increase in the SOM by promoting MB [30,31]. The additional source of Mis-C in areas with high livestock numbers and thus high demand for bedding and fodder material in form of cereal straw may also buffer the demand for cereal-based C in arable regions [86], leaving more C in arable regions to conserve SOC and thus counteract the continuous SOM losses [13]. Additional C production through Mis cultivation also counteracts dependence on external C sources such as imported organic fertilisers like slurry and farm manure in predominantly arable farming areas. Furthermore, Mis can fulfill and compensate the increasing demand for bedding materials in livestock production [53], which is also a result of societal requests for animal welfare conditions as well as the rising demand for cereal straw as a feed component.
In addition to Mis as an accredited crop of ecological compensation conservation areas [48], other greening measures, such as hedges or trees in agroforestry systems also provide an alternative C source that could be used for induced N immobilisation and SOC build-up. Removal of vegetation material from buffer strips, erosion strips and riparian strips as C-source would result in nutrient removal and thus nutrient reduction of the greening part, which benefits biodiversity at these sites. This would be a positive development regarding nature conservation (increase of plant diversity, habitat for insects and other animals). The useful utilisation as a C-source could ensure the removal of mown material, but requires tests concerning the effectiveness on N immobilisation and the effects on the MB.
If organic N-fertilisers containing Mis are applied, where N is directly required, yield deficits due to microbial N immobilisation are to be expected, if they are applied exclusively. In this case, the N demand can be supplied by the application of additional ready available N fertilisers and the residual effects can be included in subsequent vegetation periods, as is also common practice with the application of other organic fertilisers [42]. An estimation of the amount of accounting for subsequent growing seasons is not possible due to missing data from field trials. Earlier application of organic farm manures in the winter months in order to expect higher N mineralisation in spring is also not recommended due to missing data from field trials and due to the risk of N losses to ecosystems.

Conclusions
The specific characteristics of Miscanthus (Mis), such as the higher C/N ratio compared to WS, were reflected in a slightly higher N immobilisation. Especially at the 1st and 2nd harvest, CS-Mis and CM-Mis were partly significantly different from the comparative treatments CS-WS and CM-WS. The Mis-C resulted in a slightly higher MBC and MBN and thus can contribute as an additional C source to prevent N losses and for the maintenance or build-up of SOM on agricultural farms. We assume that high background values of SOM and thus a high starting content of MB, as caused by grassland conversion to arable land, overrode the effects.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.