Effects of sorghum biomass quality on ensilability and methane yield

Sorghum is currently being introduced in the temperate regions of Europe. It is characterized by good digestibility and high biomass yields, which make it a useful crop for anaerobic digestion. In this study, six commercial sorghum varieties comprising four different cultivars of Sorghum bicolor L. Moench and two interspecific sorghum hybrids (Sorghum sudanense L. × S. bicolor L. Moench) were harvested on two different dates during the years 2016 and 2017 at two diverse soil‐climate sites in Germany. The fresh harvested material and silages were analyzed to examine the ensilability of the different varieties with contrasting maturity characteristics. Subsequently, methane production experiments were performed to determine the specific methane yield (SMY) of the samples. The sorghum fresh matter (FM) varied among the sorghum types, including the parameters total solids (TS; 22.69%–46.93%FM), water‐soluble carbohydrates (2.68%–11.38%TS), and nitrates (0%–0.35%TS). The excellent ensiling ability of all the sorghum types analyzed was confirmed by evaluating the fermentation profile (pH range of 3.7–4.6; dominant presence of lactic acid [LA]; acetic acid [AA] in the range of 0.70%–2.38%TS; insignificant amount of butyric acid). The SMY ranged between 231.25 and 321.31 Ln kg−1 VS and tended to decrease with the increasing harvest time and maturity. LA and AA were positively correlated with the SMY, while the neutral detergent fiber content was negatively correlated with it. The SMY—a key parameter reflecting the crop biomass quality for biogas production—was slightly higher for S. bicolor than for the sorghum hybrids. However, the results of this study confirmed that if the final purpose is biomethanation, the ensilability of different sorghum types imposes no restriction. Furthermore, different sorghum types offer a wide harvest window, which can be useful for cropping schemes, ensiling, and methane production.


| INTRODUCTION
Sorghum (Sorghum bicolor L. Moench)-an annual C4 plant of tropical origin-is currently being introduced in temperate regions of Europe. Recently, various breeding achievements have been made that are related to the cold tolerance and early maturity of sorghum. New sorghum varieties together with agricultural alterations due to climate change, such as warming and longer growing seasons, facilitate the cultivation of sorghum as feed for livestock or for bioenergy applications (Kanbar et al., 2020;Schaffasz et al., 2019;Windpassinger, 2016). Furthermore, including other (possibly new) crop species and cultivars into crop rotations promotes diversification and enhances the sustainability of crop management (Hufnagel et al., 2020;Strauß et al., 2019). Sorghum and sorghum hybrids (Sorghum sudanense L. × S. bicolor L. Moench) are generally characterized by an excellent biomass yield potential, high nitrogen and water use efficiency, pronounced abiotic stress tolerance, and no specific soil requirements (Strauß et al., 2019;Tari et al., 2013;Wannasek et al., 2017). The inherent robustness of sorghum is complemented by the ability to break disease, parasite, and weed cycles (Głąb et al., 2017;Ratnadass et al., 2012). Sorghums' Diabrotica resistance (Diabrotica virgifera virgifera LeConte; Western Corn Rootworm) is particularly beneficial for the infested European maize production areas (Windpassinger et al., 2015).
Commonly, crops for biogas production are harvested seasonally as whole plants. Subsequently, wet biomass is conserved by ensiling to maintain feedstock quality for yearround availability to biogas plants (Herrmann et al., 2011;Teixeira Franco et al., 2016;Villa et al., 2020). During the fermentation process, the microflora-principally lactic acid (LA) bacteria-convert plant sugars (water-soluble carbohydrates, WSCs) into organic acids (such as LA and acetic acid [AA]), reducing the pH. The rapid decline in the pH prevents undesired microbial activity and hence spoilage of crop material. The fermentation quality of the prepared silage is mainly determined by the biomass composition during ensiling and by the bacteria species dominating the fermentation process. Inappropriate ensiling conditions may degrade the silage quality and incur large losses in yield or nutritional content (Borreani et al., 2018).
Various studies have demonstrated that sorghum is characterized by good digestibility and high biomass yields, which make it a feasible crop for anaerobic digestion (Herrmann et al., 2011;Herrmann, Idler, et al., 2016;Windpassinger et al., 2015). For this reason, in Europe, the implementation of forage sorghum for crop rotations is increasing (Sorghum (ID, 2020)). Beneficial in this process is sorghum's high adaptability, which allows to easily modify current practices (timing of cultivation, regular recurring sequence of crops) for integrating them into crop rotations, as well as to develop alternative cropping schemes, such as double cropping systems (Goff et al., 2010;Strauß et al., 2019;Wannasek et al., 2019). However, the multiplicity of management options for maximizing the methane yield per acre presents a considerable challenge for farmers. Figure 1 shows an overview of the impact factors and interactions affecting the methane yields of biogas crops. The supply of herbaceous feedstock for anaerobic digestion aims at the highest possible methane yield per unit area (m 3 ha −1 ). This area-specific methane yield (SMY) is determined by the biomass yield (t ha −1 ) and the feedstock-SMY (energy density) per dry matter unit (m 3 t −1 ). Both these parameters are affected by numerous factors. The feedstock-SMY depends on the feedstock quality, pretreatment method, digestion technology, and process control. The

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PASTERIS ET Al. feedstock quality depends on the crop species, variety type, and cropping system selected and can be affected by natural site-specific conditions, crop management, and conservation. The harvest date plays a key role, because the decision to harvest for silage preparation not only affects the biomass yield but also has a significant impact on the biomass quality, that is, the ensilability, and thus on the SMY.
The nutrient and fiber composition of the crop biomass changes with plant maturity. In sorghum, the WSC content is high during the vegetative stage, but as grain filling progresses, the WSC content decreases, and the starch content increases (Piltz & Kaiser, 2004). The amount of cell wall components (cellulose [CEL], hemicellulose [HCEL], and lignin) increases with plant growth, negatively affecting degradability and biomethanation (Herrmann et al., 2011;Herrmann, Idler, et al., 2016). In contrast, less mature crops are characterized by a higher moisture content, which limits their suitability for ensiling. A low dry matter content leads to effluent production and subsequent energy and nutrient losses (Borreani et al., 2018). Clostridia-microorganisms that cause spoilage of silages-are less sensitive to pH reduction at higher water activity levels (McDonald et al., 1991). Thus, a lower pH and elevated LA concentration within the silage is needed for the inhibition of spoilage microorganisms at a low dry matter content. Hence, farmers must balance the maximum biomass yield and optimal feedstock quality for methane production. A few related studies involving regional settings and the availability of sorghum varieties were recently performed (Hassan et al., 2019;Morozova et al., 2020;Wannasek et al., 2017Wannasek et al., , 2019. However, to the best of our knowledge, no studies have focused on the impact of the sorghum biomass quality on the ensilability with regard to the SMY. In the one study on this topic, the common interaction of decreasing methane yields with increasing maturity stages could not be confirmed (Herrmann, Plogsties, et al., 2016). The presented data regarding sorghum silages indicate notable differences in the development of dry matter and the lignin content depending on the maturity stage at harvest. The nature of the correlation remains unclear (Herrmann, Plogsties, et al., 2016). Thus, there is a need for knowledge that can assist in determining the best harvest time of forage sorghum for ensiling and subsequent use as feedstock for biogas production. The objective of this study was to investigate the effects of different maturity characteristics of sorghum and different harvest dates on its ensilability and the subsequent SMY of silages. To achieve this, six commercial sorghum varieties-four varieties of Sorghum bicolor L. Moench and two sorghum interspecific hybrids (Sorghum sudanense L. × S. bicolor L. Moench)-were harvested on two different dates during the years 2016 and 2017 at two diverse soil-climate sites in Germany, to assess the fresh matter (FM) quality, fermentation profile of the silages, and SMY obtained using the silages as a substrate.

| Crop material
The study was conducted using four different variety types of Sorghum bicolor L. Moench, which were optimized for biomass yield (low starch content and almost no grains), that is, Amiggo, Herkules, KWS Zerberus, and RGT Gguepard, as well as two sorghum hybrids (Sorghum sudanense L. × S. bicolor L. Moench), that is, KWS Freya and Lussi (Table 1). The selected varieties covered a wide range of sorghum types and maturity tendencies, facilitating a comprehensive overview of the differences among them.

| Field sites
The cultivation experiments were carried out during the years 2016 and 2017 at two experimental field sites, which were selected to represent distinct soil-climate conditions of field cropping in Germany: in the north-east, Marquardt (MQ; 52°28′01.7″N 12°57′37.2″E; 42 m above sea level; soil type: low loamy sand; average annual rainfall: 585.8 mm; annual mean temperature: 9.3°C) and in the south, Straubing (ST; 48°51′48.3″N 12°36′56.4″E; 339 m above sea level; soil type: strong clayey silt; average annual rainfall: 757 mm; annual mean temperature: 8.6°C). The weather in Marquardt (2016) was characterized by dry phases during May, August, and September. Temporarily, in May, June, and September, the average temperatures increased to 4°C above the long-term average values. Overall, compared with the long-term average, 2016 was 1.2°C warmer and had less precipitation (77.6 mm). In 2017, there was above-average precipitation in June, July, and August. Overall, the year was very rainy; the precipitation of 750 mm exceeded the long-term average by approximately 160 mm. The temperatures were close to the long-term average, exhibiting slight increases in May (1.2°C) and June (1.6°C). At the Straubing site, the weather in 2017 was characterized by a humid spring and a dry summer. Only August precipitation reached the long-term average. The months of June and August were significantly warmer-2.5°C and 2°C, respectively, above the long-term average (DWD, 2017(DWD, , 2018. The investigations conducted followed a uniform, standardized protocol. The trials were established as a randomized block with four replicates of each variety and plot. Each harvest plot was divided into five rows and had a size of 1.5 × 9 m 2 , with a distance of 26.6 cm between the single rows, resulting in densities of 25 (S. bicolor) and 35 plants per m 2 (S. bicolor × S. sudanense). The sowing time for all the experiments and years was set for the middle of May (Table 2). Site-specific crop management (fertilization, crop protection measures) was applied. Sorghum varieties were harvested at two different harvesting dates (HD1 and HD2) and phenological growth stages (BBCH code), which were determined according to TFZ (2012; Table 2). Whole plants were chopped using a mounter forage harvester (Hege 212; Wintersteiger AG) with a Kemper Champion 1200 (Kemper GmbH & Co. KG) to a particle-size range of 5-7 mm.

| Silage preparation
Ensiling was conducted immediately after the chopping of the sorghum, by using 2-L glass jars (J. WECK GmbH u. Co. KG) as laboratory-scale silos. Chopped sorghum samples were compressed manually using a special pressing device that ensured identical conditions for all the samples. Laboratory silos were filled completely; thus, no headspace remained within the glass jars. A glass lid, rubber ring, and four metal clamps were used to close the silos for preventing air infiltration but allowed the escape of gases formed during ensiling. The storage temperature was set at 25°C for the process duration of 90 days. The ensiling was conducted without silage additives. The silage preparation was performed in duplicates for each variant. The conservation losses during the ensiling (FM losses [FML]) were determined by weighing the silos after filling and after storage via the method of Weißbach (2005), as described by Herrmann et al. (2015).

| Chemical analyses
Crop samples of fresh material and silages were stored at -18°C directly after harvest and feed-out from the laboratory-scale silos, respectively, for further analysis of chemical composition and methane production. For characterization of the harvested parental matter before ensiling, the WSCs were determined to be monomeric and dimeric sugars, as well as fructans, in water extracts with the addition of HgCl via high-performance liquid chromatography (HPLC; UltiMate 3000 UHPLC system, Thermo Fisher Scientific Inc.) according to Hoedtke and Zeyner (2010). The nitrate content was determined in water extracts of plant material dried at 60°C via ion chromatography (ICS-1000, Thermo Fisher Scientific Inc.), as described by VDLUFA (2006). Samples from FM and silages were dried at 105°C until reaching a constant weight to determine their total solids (TS) content. The volatile solids (VS) contents of the silages were calculated by determining the ash contents of samples in a muffle furnace at 550°C, via the method of VDLUFA (2006). As described by Weißbach and Kuhla (1995), the TS content of the ensiled material was corrected to account for the losses of organic acids and alcohols (ALCs) that occurred during oven drying. All the parameters referred to as the percentage of TS are based on the corrected TS value. A Sen Tix 41 electrode (WTW) was employed to measure the pH values of the silages. Cold water extracts from sorghum silage samples were prepared for determination of the contents of LA, volatile fatty acids (VFAs), and ALC. The LA content was determined via HPLC (Dionex) with a Eurokat H column (Knauer). Gas chromatography was performed to quantify the contents of the following: AA, which included both acetic and propionic acid; butyric acid (BA), which included butyric, isobutyric, caproic, valeric, and isovaleric acid; and ALC, which included ethanol, propanol, 1,2-propanediol, and 2,3-butanediol. Further details are available in Herrmann, Idler, et al. (2016). The contents of elemental carbon and nitrogen were measured using the DUMAS catalytic combustion methodology described by VDLUFA (2006). These values were used to calculate the C/N ratio. The crude protein (CP) content was calculated by multiplying the N content by 6.25. Furthermore, fiber analyses (neutral detergent fiber [NDF], acid detergent fiber [ADF], and acid detergent lignin [ADL]) were performed using an Ankom 2000 analyzer system and a fiber filter bag (Ankom Technology Corp.), as described by Herrmann et al. (2011). The CEL content was calculated as the difference between the ADF and ADL contents, and the HCEL content was obtained by subtracting the ADF content from the NDF content. Gravimetric determination was performed to measure the portion of crude fat (CF) in silages; hydrolysis was conducted with 3 N hydrochloric acid, followed by partitioning with petroleum ether for 1 h at 90°C using an Ankom XT10 extractor (Ankom Technology Corp.). The content of non-fibrous carbohydrates (NFC) was determined by subtracting the CP, CF, NDF, and crude ash contents from 100%TS.

| Batch anaerobic digestion tests
The SMYs from the sorghum silage samples were examined by performing batch anaerobic digestion tests in 2-L glass reactors. To obtain a VS substrate /VS inoculum ratio of 0.4-0.5, 1.5 L of inoculum and 50 g of silage were added to each reactor. The inocula (average chemical characteristics: pH, 8.04; TS content, 5.17%; VS, 72.39%; N, 4.5 g kg −1 ; NH 4 -N, 2.87 g kg −1 ; and organic acids, 0.3 g L −1 ) were obtained | 807 PASTERIS ET Al.
T A B L E 2 Description of the field trial regarding the principle growth stage at harvest for the six sorghum types at two different harvest dates, sites, and years HD from previous laboratory anaerobic digestion experiments.
A water bath at a mesophilic temperature (37°C) was used for reactor incubation. Batch tests were performed for approximately 30 days (or until the daily rate of biogas during three successive days was <0.5% of the total biogas obtained up to that time [VDI 4630, 2016]). The SMYs of all variants are presented as 30-day values. A scale gas meter was used to collect the produced biogas. An acidified saturated NaCl solution was employed as a barrier solution to determine the volume of gas obtained, using the barrier solution displacement method. The biogas volumes obtained from the silages were corrected via subtraction of the biogas volume produced exclusively by the inocula. The corrected volumes were normalized to standard conditions (dry gas, 0°C, 1013 hPa). A mobile gas analyzer equipped with infrared sensors (BM5000, Geotechnical Instruments Ltd.) was used to determine the amount of methane (CH 4 ) in the biogas produced. The SMY of each sample was calculated as the sum of methane generated during the batch test period, in terms of the VS contents of the silages added to the reactors. Further details are available in Herrmann, Idler, et al. (2016).

| Statistical analyses
To determine the significant effects of cultivation and harvest conditions on sorghum silage quality parameters, the chemical characteristics of the silages, and the SMYs, an analysis of variance (ANOVA) was conducted by setting the harvest date, sorghum type, and harvest date × sorghum type interactions as fixed effects. The analyses were conducted separately for the two experimental field sites and harvest years (only MQ). A multiple comparison of means of the SMYs was performed to determine differences between sorghum types for each harvest date. The level of significance (α) was set as 0.05. The software SAS 9.4 (SAS Institute Inc.) and the PROC MIXED procedure were used for statistical analyses. Multiple comparisons of means were conducted using the SIMULATE test procedure.

| Chemical characteristics of raw materials
The harvested fresh material of the six different sorghum types revealed different chemical compositions between the two harvest dates at sites MQ16, MQ17, and ST17 (Table 3). The TS contents obtained from the samples reflected the maturity tendency of each of the six sorghum types analyzed ( Figure 2). For all the samples, the TS content was higher with a later harvest date, and this trend was observed at all the sites studied. Nevertheless, the majority of the samples analyzed exhibited TS values within the optimum range for ensiling reported by Amon et al. (2007;Figure 2 Table 3, the WSC content differed significantly among the sorghum varieties. In . The shadowed area indicates the optimum TS range for ensiling according to Amon et al. (2007) general, S. bicolor × S. sudanense hybrids exhibited lower WSC contents than S. bicolor varieties. In almost all cases, a decreasing tendency was observed from HD1 to HD2 for the S. bicolor × S. sudanense hybrids, with the exception of the ST17 samples. In contrast, the WSC content of the S. bicolor types increased with maturity. In MQ16 and ST17, Sorghum type SB3 exhibited the highest concentration of WSC on HD2, whereas in MQ17, SB1 on HD2 ranked first (Table 3).

| Silage fermentation characteristics
A typical silage fermentation profile was determined for all the samples ( Table 4). The pH values oscillated between 3.7 and 4.6, and the BA concentrations were insignificant in all cases, indicating adequate material conservation and the absence of secondary fermentation. According to the data obtained, the LA content was the highest among the fermentation acids; it was in the range of 1.5%-6.4%TS for all cases. Only for Marquardt, sorghum variety had a statistically significant effect on the LA concentration (Table 4). Additionally, in MQ16, the results depended significantly on the HD and the effect HD × T. In contrast, the LA content in Straubing was unaffected by the factors studied. In the majority of cases, the AA concentration exhibited a slight increment from HD1 to HD2, with the exception of the S. bicolor × S. sudanense hybrids in MQ16. As indicated by this table, the AA contents differed significantly among the sorghum types (p < 0.001), with the middle-late maturity varieties S. bicolor containing higher concentrations than S. bicolor × S. sudanense. Moreover, in MQ17 and ST17, the AA content depended significantly on the HD, but this effect was not observed in MQ16. The ALC content varied widely among the samples (Table 4). In MQ16 and ST17, significant effects of HD, T, and HD × T were observed. In contrast, the results for MQ17 indicated no significant differences for the factors analyzed and remained relatively constant below 0.84%TS for all the cases (Table 4). The results for the FML differed significantly among the varieties. Nevertheless, this parameter exhibited no significant difference between the effects examined, the only exception being the HD effect in MQ16.

| Nutrient and fiber compositions of crop silages
Chemical characterization of all six sorghum variety types after ensiling was performed for each site, year, and HD (Tables 5 and 6). The HD and T significantly affected almost all the parameters in Table 5. The TS content after ensiling was higher for S. bicolor × S. sudanense than for the S. bicolor types. Statistical analyses indicated that the TS and VS results depended significantly on the type of sorghum, HD, and the interaction of the HD and sorghum type, regardless of the location. The HD affected the CP content for the MQ16 and ST17 samples but not the MQ17 samples. Generally, S. bicolor × S. sudanense exhibited higher CP contents than the S. bicolor variants, but the effect of T was substantial only for the samples collected in Marquardt. The same tendency was observed for the CF content for both years in Marquardt, but again, no detectable differences for the Straubing samples were observed. The NFC composition was significantly affected by the HD in all the cases, but there was also a significant effect of the sorghum type on the NFC in MQ17 and ST17. The C:N ratios were always >45. The lowest value for this parameter was observed in MQ17 for the sorghum type SBSS1 harvested on HD1 (45; Table 5), and the highest value corresponded to SB1 in ST17 harvested on HD2 (76 ;  Table 5). Nevertheless, only in Marquardt was the sorghum type a significant influencing factor. Additionally, the results indicated that the HD affected the C:N ratios for all the samples obtained at MQ16 and ST17. The variable HD had a significant impact on almost all the fiber components in the samples, with the exception of HCEL in MQ16 and ADL in MQ17 and ST17 (Table 6). Considering the T factor, there was a statistical difference in the CEL content for both the sites (whereas for the NDF and ADF contents, there was a significant difference only in MQ17 and ST17). Furthermore, the ADL content was significantly affected by the sorghum type in both harvest years in Marquardt. The ADL content was higher for the S. bicolor × S. sudanense hybrids than for S. bicolor. The effect of the HD × T interaction on the parameters was insignificant, with the exception of the ADF and ADL contents in MQ17 and HCEL in ST17 (Table 6).

| Specific methane yields of crop silages
The SMY was negatively affected by the HD in almost all the cases in this study ( Figure 3). This effect was clearly evident for the samples from Marquardt, where the values decreased from HD1 to HD2. The sorghum type significantly affected the SMY for all the Marquardt samples; however, for the Straubing samples, the sorghum type had no significant effect. Nevertheless, a trend was observed for the majority of the cases: earlier-maturing S. bicolor × S. sudanense hybrids had an inferior SMY (ranging from 231.25 to 294.98 L n kg −1 VS) than S. bicolor (ranging from 262.15 to 321.31 L n kg −1 VS). For instance, the hybrids SBSS1 and SBSS2 had the lowest SMYs at both HDs, and the SB1, SB3, and SB4 types had the highest SMYs.

| Effects of chemical composition on methane formation
The Pearson's correlations among all the traits are presented in Table 7. Of major interest were the interactions that       correlated with the solids content (TS and VS), but moderately negatively correlated with the pH and ADL content. These last two parameters (pH and ADL) exhibited multiple moderate correlations with other factors, particularly the fiber fractions. Figure 4 shows the relationship between the SMY and the TS content (associated with sorghum variety: hybrids S. bicolor × S. sudanense [SBSS] or S. bicolor [SB]) and the specific BBCH code number that characterized each phenological stage of the sorghum types harvested in MQ16, MQ17, and ST17. A slight correlation is observed, indicating that the SMY decreased with crop maturity. In the diagram presented, this tendency is explained not only from the TS perspective but also from the perspective of BBCH classification, which reflects the plant's current development stage. The Sorghum bicolor hybrids exhibited SMYs of >250 L n kg −1 VS in all cases, even at the higher BBCH stages, with a maximum value of 314 L n kg −1 VS (for SB3 in ST17). Furthermore, the TS contents of all the S. bicolor samples remained below 35%FM. In contrast, the S. bicolor × S. sudanense hybrids exhibited BBCH numbers of >70 (development of fruit) even at the first HD, leading to higher TS contents and lower SMYs (<300 L n kg −1 VS for all cases).

| DISCUSSION
This study shows the ensiling ability of sorghum varieties with contrasting maturity characteristics (early, mediumlate, and late). Additionally, it revealed that the silage quality varied among different sorghum types, harvest dates, and locations. Nonetheless, in all the cases examined, it was possible to produce adequate amounts of methane per unit of biomass. A discussion of the harvested biomass quality, silage fermentation profile, and methane production for all six sorghum types in MQ16, MQ17, and ST17 is presented in the following sections.

| Ensilability of raw material
One of the most important factors defining a successful silage process is the TS content of the fresh material. It is well known that an optimum TS content, along with other parameters such as the WSC content and buffer capacity of biomasses, yields favorable conditions for the desired silage fermentation (Kung et al., 2018;Teixeira Franco et al., 2016). As expected, for all six sorghum types studied, the TS content increased with a later harvest, revealing the differences between their maturity characteristics. Wannasek et al. (2017) found that four different S. bicolor sorghum types and one S. bicolor × S. sudanense, with diverse maturity tendencies, exhibited different TS fractions when testing the effects of climate, sorghum variety, and time of harvest on plant development and biomass yields. The results of the present study indicate that the early sorghum hybrids reached the optimal maturity stage at HD1 for both sites and both years (optimum TS content range is 27%-35% according to Wannasek et al., 2017). In contrast, medium(-late) S. bicolor types reached the optimum TS content at HD2. Only for HD2 in MQ16 was the TS content of S. bicolor × S. sudanense types higher than the optimum. This was confirmed by the phenological development stage at harvest (BBCH code; senescence), which was characteristic of these samples (Table  2). Nevertheless, these attributes were not observed for any sorghum type in 2017, suggesting a possible effect of the year on the results. During May, June, and July 2016, temperatures higher than the long-term average were registered at Marquardt. These favorable conditions may have positively affected crop development, particularly for the earlier maturity sorghum types. In agreement with our results, Mahmood F I G U R E 4 Specific methane yield (SMYs) of Sorghum bicolor × Sorghum sudanense (SBSS) and S. bicolor (SB) types and the TS content with respect to the growth stage at harvest (BBCH code according to TFZ, 2012 and Honermeier (2012) found that the S. bicolor × S. sudanense sorghum type "Bovital" exhibited a higher TS content than the S. bicolor types "Aron" and "Rona 1" when comparing the effects of row spacing on chemical composition and methane yield between different sorghum species.
In addition to the TS content, the WSC content is important during ensiling, as WSCs are the central compounds transformed into organic acids by LA bacteria (McDonald et al., 1991). Many authors specified an optimum WSC range of approximately 60-80 g kg −1 TS to obtain high-quality silages (Villa et al., 2020;Woolford, 1984). The results obtained were within this range in most cases, but some S. bicolor × S. sudanense hybrids were below the limit, and some S. bicolor hybrids were above the limit (Table 3). Amer et al. (2012) found that large amounts of WSCs improved ensiling characteristics when performing experiments using two different varieties of sorghum and forage millet. In relation to these findings, S. bicolor types at HD2 presented the best conditions as a pre-ensiled material for both sites and years, with WSC contents reaching 11%DM in some cases. Rodrigues et al. (2020) reported a similar WSC average (10.7%DM) for S. bicolor types. McDonald et al. (1991) reported that the optimum nitrate content is in the range of 0.6%-1.0%TS, and the results for fresh materials were in all cases below this range. During ensiling, nitrate is usually reduced to nitrite and nitric oxide-compounds that can suppress clostridial growth, avoiding silage spoilage (McDonald et al., 1991). Nevertheless, if other ensiling parameters are favorable, nitrate is not absolutely needed, and high silage quality can be achieved.
Overall, the different maturity-tendency sorghum type biomasses differed in their TS and WSC contents at different harvest dates, but these differences did not affect the success of the ensiling process (see Section 4.2).

| Fermentation quality
It is generally accepted that high-quality silage is characterized by low pH, high LA content, and insignificant amounts of BA (Kung et al., 2018;Teixeira Franco et al., 2016;Villa et al., 2020). As expected, according to the favorable ensilability of the sorghum samples, silages of all the variants analyzed in this study fulfilled these requirements and exhibited adequate silage fermentation quality. Neither the HD nor the sorghum type significantly affected the final silage quality, although these two factors significantly affected individual silage fermentation parameters, such as the AA and ALC contents (Table 4).
In a more detailed analysis, the pH values lay within the common range of 3.7-5 reported by Villa et al. (2020). Among all the products of silage fermentation, LA represented the largest proportion, indicating typical LA silage fermentation (Buxton & O'Kiely, 2003). Herrmann, Idler, et al. (2016) reported LA ranges of 2.5%-12%TS and 5.2%-13%TS for sorghum S. bicolor × S. sudanense and S. bicolor × S. bicolor silages, respectively. The results of the present study are consistent with these values, with the exception of the samples from MQ16 (HD2), which exhibited lower LA concentrations. The ANOVA results (Table 4) indicate that the sorghum type affected the LA content of the samples obtained at Marquardt. Generally, later-maturity sorghum types exhibited larger amounts of LA. This effect was not significant for the ST17 samples.
However, there were statistical differences in the AA content among the sorghum types from the different sites. This acid is typically found to be the second-highest parameter in terms of the concentration in silages, with values between 1% and 3%TS (Kung et al., 2018). The predominant number of silages analyzed in the present study was within this range. The AA amounts differed significantly between HD at MQ17 and ST17, with a visible increasing trend from HD1 to HD2. The increment of this parameter can be beneficial for the silage aerobic stability owing to the antifungal properties of AA (Kung et al., 2018).
The absence or minor amounts of BA existing in the samples indicate that clostridial metabolism did not dominate silage fermentation. The presence of BA typically results in energy losses and low-quality silages for animal feeding (McDonald et al., 1991;Pahlow et al., 2003), but Villa et al. (2020) argued that biogas-purpose silages can contain larger amounts of BA, which can lead to greater SMY production. Nonetheless, even with some BA, the FML of the samples were insignificant for the majority of the cases studied (Table 4).
The presence of ALCs is also evident in silages, with ethanol being the most common compound found (Kung et al., 2018). Researchers have evaluated the ethanol concentrations in different S. bicolor silages and obtained values between 1% and 3.4%TS (Miron et al., 2005). Additionally, Herrmann, Idler, et al. (2016) reported ALC contents ranging from 0.5% to 4.3%TS and from 0.6% to 4.1%TS for S. bicolor × S. sudanense and S. bicolor × S. bicolor hybrids, respectively. In agreement with the literature, the ALC content range in the present study was between 0.3% and 6.12%TS (Table 4). However, it was impossible to establish trends, owing to the high variability among the results. Given that ethanol can be produced by different microorganisms (such as heterolactic acid bacteria, enterobacteria, and yeast), this inconsistency may have been caused by the activity of diverse epiphytic microflora (Kung et al., 2018;McDonald et al., 1991).

| Silage nutrient and fiber characteristics
The average CP content obtained for the silages studied is consistent with values reported by other authors for