Photoperiod Sensitive Energy Sorghum Responses to Environmental and Nitrogen Variabilities

Recently introduced photoperiod-sensitive (PS) biomass sorghum (Sorghum bicolor L. Moench) needs to be investigated for their yield potentials under different cultivation environments with reasonable nitrogen (N) inputs. The objectives of this study were to 1) evaluate the biomass yield and feedstock quality of four sorghum hybrids with different levels of PS ranging from very PS (VPS) hybrids and to moderate PS (MPS) hybrids, and 2) determine the optimal N inputs (0~168 kg N ha−1) under four environments: combinations of both temperate (Urbana, IL) and subtropical (College Station, TX) regions during 2018 and 2019. Compared to TX, the PS sorghums in central IL showed higher yield potential and steady feedstock production with an extended daylength and with less precipitation variability, especially for the VPS hybrids. The mean dry matter (DM) yields of VPS hybrids were 20.5 Mg DM ha−1 and 17.7 Mg DM ha−1 in IL and TX, respectively. The highest N use efficiency occurred at a low N rate of 56 kg N ha−1 by improving approximately 33 kg DM ha−1 per 1.0 kg N ha−1 input. Approximately 70% of the PS sorghum biomass can be utilized for biofuel production, consisting of 58-65% of the cell wall components and 4-11% of the soluble sugar. This study demonstrated that the rainfed temperate area (e.g., IL) has a great potential for the sustainable cultivation of PS energy sorghum due their observed high yield potential, stable production, and low N requirements.


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The summary of field operations was described in Table 1. In IL, the soybean stubble was disked prior to 135 planting, and sorghum was planted at 180,000 seeds ha -1 using an ALMACO four-row Kinze planter (Nevada, IA).

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In TX, the prior crop was cotton; fields are cultivated and prepared in the fall and sorghum was planted at 180,000 137 seeds ha -1 using an ALMACO four-row Max-emerge planter. Both locations had the same N fertility program, and 138 aqueous urea ammonium nitrate (UAN: 32-0-0) was knife-injected approximately a week after planting in IL and at 139 the same day after planting in TX. The biomass harvest timing was based on site observation, when 50% of sorghum  [32]. The NIRS analysis provides a more 156 detailed insight on biomass composition, including structural glucan, xylan, galactan, arabinan, acetyl, protein, and 8 applied unit of N fertilizer was defined as the N input efficiency (NIE), which is often referred to the agronomic 163 NUE and was calculated based on Eq. 2. The amount of N in crop biomass (or crop nitrogen removal) that was 164 originated from the applied N was defined as the N recovery efficiency (NRE) shown in Eq. 3.

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TX17500, and TX17800, respectively, in TX-2018. The two-way interaction between location and hybrid also 204 affected biomass yield (Table 4), and the 2-year average showed that TX08001, TX17500, and TX17800 hybrids 205 had higher yield potential in IL than TX except for TX176000 (Table 5). In IL, the TX08001, TX17500, and   206   TX17800 biomass yield averaged across 2018 and 2019 were significantly higher by 10%, 22%, and 15 %,   207 respectively, compared to the yield from TX. The average across the two years and four hybrids showed that the PS 208 sorghums had higher yield potential in IL (20.4 Mg DM ha -1 ) than TX (19.0 Mg DM ha -1 ) ( Table 5). The interaction 209 between N rate and other factors did not show any significant impact on biomass yield (Table 4). Increased N rate 210 generally increased the sorghum biomass yield (Table 4). However, the biomass yields of four hybrids showed 211 similar responses to the increased N rate, and there was no benefit of N rates above 112 kg ha -1 ( Table 6).

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The three-way interaction of location, year, and hybrids was also significant for tissue N, P, K 214 concentrations (Table 4). In each environment, all hybrids showed similar tissue N concentrations except for concentrations were generally higher in 2019 (8.7 g kg -1 ) than 2018 (7.0 g kg -1 ) in IL; by contrast, increased N 217 concentrations were observed in 2018 (8.7 g kg -1 ) compared to the concentration in 2019 (7.8 g kg -1 ) in TX. This 218 was likely due to the high N concentrations of the TX17500 (10.4 g kg -1 ) and TX17800 (9.8 g kg -1 ) hybrids in 2018 219 ( Fig. 3b). For P concentrations, the TX17500 hybrid tended to have higher P concentrations than other hybrids in 220 both locations, although differences were significant only in 2018. Across all environments, P concentrations in 221 TX17500 biomass were approximately 13% higher than the average of the other three sorghum hybrids. All hybrids 222 had higher tissue K concentrations in TX than in IL. The averages across all hybrids and the two years showed that 223 the PS sorghum grown in TX increased the tissue K concentrations by approximately 75% (  Table 8). The 4-way interaction of location, year, N-rate, and hybrid was not significant for the biomass 261 structural and soluble compositions; however, the 3-way interaction among location, year, and hybrid showed a 262 significant impact on the concentrations of structural and soluble components ( Table 8)

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Across two experimental years, the effect of the 2-way interaction between locations and hybrids on the biomass 268 structural and soluble components are shown in Table 9. For both IL and TX, the TX17800 hybrid consistently 269 contained higher concentrations of the structural glucan, xylan, and lignin than other hybrids; likewise, TX17800 270 tended to have higher concentrations for both biochemical-and thermochemical-processing interested components 12 (i.e., BIC and TIC). For instance, the BIC and TIC concentrations of the TX17800 hybrid was approximately 5 % 272 and 7 % higher, respectively, compared to other hybrids (Table 9). In contrast, TX17800 had the lowest 273 concentrations of soluble sucrose and other non-structural inorganics (e.g., ash). TX17600 had the highest sucrose 274 concentrations among hybrids by increases of approximately 34 % and 89 % in IL and TX, respectively (Table 9).

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For other structural components, the TX08001 (VPS hybrid) had the highest concentrations of galactan, and the 276 TX17800 (MPS hybrid) had the lowest arabinan among hybrids. The VPS hybrid TX17500 had the highest biomass 277 ash concentration among hybrids. Between two locations, the TX site generally had higher concentrations of the 278 BIC, TIC, acetyl, corresponding to the lower ash concentrations than observed at the IL site ( Table 9). The N effect 279 on feedstock composition showed that only a few chemical compositions, including lignin, acetyl, ash, and sucrose, 280 responded significantly to the N rate for all hybrids and no interaction of N rate x hybrid was observed (Table 8).

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The increased N rate increased lignin and ash concentrations but reduced acetyl and sucrose concentrations (Table   282 10  (Table 1); by contrast, the TX17800 hybrid showed higher yield potential in IL.
Weather variations also influenced biomass yield significantly [10]. The monthly temperature in IL and TX 299 were consistent in both the experiment years compared to the 30-year averages; however, the precipitation was 300 highly variable (Fig 2). The great variability in precipitation not only raises the difficulties for optimizing the field 301 management but also influences crop development as well as biomass productions [39][40][41]. In TX, for instance, the 302 seasonal precipitation was concentrated during the late growing season (Sept. and Oct.) in 2018; whereas, most of 303 the seasonal precipitation accumulated during the early season (May to July) in 2019 (Fig. 2). The biomass yields 304 across the two years showed more variation in TX than IL, likely resulting from the highly fluctuating precipitation 305 pattern (Fig. 3 slows the crop's early establishment and delays growth and yield [21,40]. In this study, the overall biomass yield of 309 the PS sorghum was similar to our previous results reported by Maughan et al. (2012) [10], but approximately 30% 310 lower than the other reports (>30 Mg DM ha -1 ) where the field trials were mostly in the long-growing season (> 150 311 days) areas [11][12][13]. The relatively low yield was presumably due to the short production seasons and can be 312 improved by prolonging the growing season, such as early planting [7].

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A substantial environmental impact on tissue nutrient concentrations shown in this study was also observed 314 in several studies [29,34,[42][43][44][45]. The decline in N concentrations was usually corresponding to the increase in 315 biomass yield, which was attributed to the effect of a growth dilution [42,45,46]. This dilution effect on P and K 316 concentrations, however, was not observed in this study, which was also reported by Maw et al. (2020) [45].

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Compared to IL, the higher soil K content in TX, showing approximately 4-fold soil K (  bioenergy materials, it is crucial to minimize the content of the non-structural components but water stress has been 328 reported to increase the biomass non-structural components for different bioenergy feedstocks, such as corn stover, 329 miscanthus, switchgrass, and mixed perennial grasses [48][49][50]. McKinley et al. (2018) [12] reported that the soluble 330 compounds in energy sorghum stem biomass were generally higher, along with increased sucrose, in rainfed systems 331 than irrigated systems. For these four hybrids, a significantly increased concentration of the soluble components was 332 observed in the TX-2019 regimes (Fig. 4).Dry conditions in the TX-2019 began in July as is typical compared to the 333 30-year benchmark (Fig. 2). Since the TX-2019 site received adequate rain early to facilitate crop establishment, the 334 following dry season (Jul. to Oct.) did not affect the biomass yield of the well-established sorghum due to a good 335 water stress resistance; however, the non-structural compounds did respond to the dry environment [50].

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Among four sorghum hybrids, no consistent yield trends were observed in IL. In TX, the TX17600 hybrid 338 consistently produced higher biomass yield than the other hybrids (Fig.3). The TX17600 hybrid also showed less  [31,42,43]. Although the four PS hybrids had similar N requirements, the TX17500 showed a higher stover P 349 concentration/requirement than the other hybrids. The high biomass sorghum hybrids with lower tissue nutrient 350 concentrations, usually along with lower nutrient removal, have more sustainable benefits for the cropping system 351 [45,51].

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The composition analysis ( Fig. 4 and Table 9) indicated that the TX17600 hybrid had a juicier stalk and 353 higher sucrose concentration than other hybrids, and its environmentally-insensitive and MPS characteristics are likely favorable for stable and high yield productions by growing TX17600 in the region with a more extended 355 harvest season (e.g., subtropical or tropical areas) [3]. Understanding the feedstock compositions are essential for 356 both qualities and quantities of the biofuel products, and different quality attributes can be used as indices based on 357 the conversion technology [15,52]. Among hybrids, the TX17800 hybrid consistently resulted in the highest 358 concentrations of the energy-rich components mainly contributed by the glucan, xylan, and lignin. In temperate 359 region such as IL, the TX17800 hybrids can be considered a good candidate for lignocellulosic energy crops using 360 either bio-or thermo-processes because of the excellent yield potential along with high feedstock quality [15]. In 361 tropical or subtropical regions of TX, the TX17600 produced steady yield with high carbohydrate-rich compounds 362 ( Table 9). The two VPS hybrids (TX08001 and TX17500) in IL did not show distinct differences in terms of yield 363 potential (Table 5); however, the TX08001 hybrid showed a higher feedstock quality than TX17500 by increasing 364 the energy potential (higher BIC and TIC concentrations) and lowering ash concentrations ( Table 9). The first-365 generation TX08001 energy sorghum hybrids have been consistently reported for both high yield and quality 366 potentials in several studies [7,12,31]. The acetic acid, derived from the acetyl functional group, can be produced 367 during the hydrolysis processes (e.g., the dilute acid or enzymatic hydrolysis pretreatment), and the increased acetic 368 acid likely inhibit the fermentation effectiveness (biological inhibitor) and corrode the processing pipelines [52,53].

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In both locations, no differences in acetyl concentration were observed among hybrids; however, the sorghum grown 370 in TX resulted in higher acetyl concentrations than IL (Table 9).

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Nitrogen, a primary nutrient for synthesizing amino acids, nucleic acids, or other essential organic 373 compounds, facilitates crop growth. Although the four hybrids and two locations showed similar yield responses to 374 different N inputs in this study, the increased N supply generally improved biomass yield and the standout N rate for 375 significant yield improvement was less than 120 kg-N ha -1 , which was also shown in other studies 376 [4,20,26,54,56,57]. This study showed that increased N input led to an increase in biomass N concentrations, which 377 was often reported in many studies [34,44,56,58]. The improved biomass yield due to N supply can dilute P and K 378 in plant tissue and lower their concentrations, which was only observed for tissue K concentrations in IL [44,45,56].

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The limited response of P and K concentrations to N input was presumably due to 1) the substantial environmental 380 impact on tissue nutrient concentrations, and 2) a sufficiency supply of P and K nutrients in the soil for plant uptake 381 [45,59]. For nutrient removal, plant N removal increased with more N input because of the increases in both biomass yield and N concentration [34,44,56]. Compared to TX, the increased N removal rate in IL (~0.5 kg N ha -1 uptake 383 per 1.0 kg N input) was due to higher yield and tissue concentration responses to the N fertilizer. The N removal in 384 TX (~0.2 kg N ha -1 uptake per 1.0 kg N input) was similar in several reports [29,34,56]. In TX, the increased K 385 removal with increasing N input was a result of the DM biomass accumulation [45]. The feedstock compositions 386 were less influenced by different N rates compared to the biomass yield, which is similar to several studies 387 [20,57,60,61]. The reduced sucrose concentrations occurring at a high N rate in this study (Table 10)  for optimizing N management [31,34,56,63]. Variations in the growing environment can be the main factor to 394 influence NUE as well as the associated management strategies for the energy sorghum cultivation [34,56]. In IL, 395 the increase in PNUE in 2018 was due to the improved DM yield corresponding to the lowered biomass N 396 concentrations, which was likely due to a favorably growing condition, such as a sufficient water supply [34].  [34,65,66]. Although the N effect on NIE and NRE was not 402 significant in this study, the better NIE and NRE likely occurred at the low N-rate [34,56,65,66,69]. Many studies 403 have shown that the biomass sorghum has a low N fertilizer requirement (mostly < 120 kg-N ha -1 ) for optimizing 404 biomass yield with desirable feedstock quality for biofuel productions [29,56,57,[67][68][69]. Moreover, the biomass 405 yield, PNUE, and NIE can be further improved by extending the growing season [11,12,31]. Even though the low N 406 input was favorable in this study, the adequate N supply could facilitate PS energy sorghum to achieve its maximum 407 yield potential and nutrient use efficiency by providing the best growing conditions (e.g., irrigation, early planting 408 for extending season length) for the optimum biomass production and N accumulation [4,7,12,35].

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This study found cultivation location, year, and hybrid significantly influences biomass yield of photoperiod-412 sensitive sorghum. The 2-year-averaged yield of two VPS sorghum hybrids (TX08001 and TX17500) grown in the 413 IL site was higher by 10% and 22%, respectively than the TX site. Compared to TX, the more consistent weather    Table 6. The significant 2-way interaction between location (IL and TX) and N-rate (0, 56, 112, and 168 kg-N ha -1 ) on biomass yields, nutrient concentration, and removal. Lowercase letters indicate mean separation α=0.05 organized highest to lowest value (no mean separations were applied if the location x N rate interaction was not significant).   biochemical-processing interested components based on the sum of the structural glucan, xylan, galactan, and arabinan TIC: thermochemical-processing interested components based on the sum of the BIC and structural lignin * Significant effect at the level of P < 0.05 Table 9. The significant effects of location (IL and TX) and hybrid (TX08001, TX17500, TX17600, and TX17800) on concentrations of feedstock components. Lowercase letters indicate mean separation α=0.05 organized highest to lowest value (no mean separations were applied if the variable effect was not significant).

Location
Hybrid Structural components (g kg -1 ) Non-structural (g kg -1 ) Arabinan BIC: biochemical-processing interested components based on the sum of the structural glucan, xylan, galactan, and arabinan TIC: thermochemical-processing interested components based on the sum of the BIC and structural lignin †: weak location x hybrid interaction effect (P = 0.0535) 2c Gal: Galactan Arab: Arabinan BIC: biochemical-processing interested components based on the sum of the structural glucan, xylan, galactan, and arabinan TIC: thermochemical-processing interested components based on the sum of the BIC and structural lignin