Morphological composition and fiber partitioning along regrowth in elephant grass CT115 intended for ethanol production

Leaf share, plant age and growth season are often overlooked as modifiers of the biomass quality in energy crops. The current work studied the effect of the given factors on the biomass yield and the biomass quality in Elephant grass CT115, intended for bioethanol production, in Veracruz, Mexico. Two seasons per year, 5 months each, were tracked on a 2-weeks basis. The climate is warm wet with summer rains, 1,142 mm of annual rainfall, and 26 °C monthly temperature. From day 56 of the wet season or from day 84 of the dry season, stems accumulated 12 or 6 Mg ha−1, respectively, while green leaves increased only 1 Mg. Higher biomass quality was recorded for the leaf fraction, or for the wet season regrowth. For instance, lignin contained in stems meant twice that of leaves, whereas stems recorded 20% less lignin in the wet season as compared to the dry season. Despite holocellulose being similar between fractions or seasons, hemicellulose and cellulose showed inverse correlation, while lignin and cellulose contents were directly correlated in stems. Increasing the annual harvest of green leaves will improve biomass quality, which is known to increase biodegradability and might improve the annual ethanol yield.


Scientific RepoRtS
| (2020) 10:15118 | https://doi.org/10.1038/s41598-020-72169-2 www.nature.com/scientificreports/ stem elongation driven by intraspecific competition 9 , so that green leaf accumulation may grow slower, which might limit the ethanol yield. For instance, in elephant grass, an increase of 35 Mg ha −1 in overall yield occurs at unvarying green leaf mass; therefore, green leaf accumulation has a biologic limit far smaller than that of stem 6 . Elephant grass is far from being a unique raw material whose biomass quality remains constant. Apart from the within variety broad variation, which is the reason we undertook the current research, elephant grass has an enormous genetic diversity. Some attributes that have served the purpose of identifying and discriminating among genotypes involve those related to the chemical composition 11 , as well as some morphological features such as plant height, and number of tillers 12 .
Analytical methods used in the field of ruminant nutrition have allowed to understand both the chemical composition and the biodegradability of energy crops intended for bioethanol production 13 . In such approach, the fiber content is measured as the fraction of feedstocks which is insoluble in neutral detergent (such fraction named NDF). The combined content of cellulose and lignin corresponds to the fraction recovered after diluting a sample in acid detergent (such fraction named ADF) 14 , and the lignin content (named ADL) is assessed as the remnant from dilution in sulfuric acid 15 . In addition, hemicellulose and cellulose contents are estimated by subtracting ADF from NDF, or ADL from ADF, respectively.
Bioethanol yield is directly correlated to in vitro digestibility of the dry matter and inversely correlated to ADF and ADL contents 5 . Furthermore, the content of lignin, inherent to stem aging, has been proposed as the main factor limiting fiber digestibility 16 . Accordingly, higher digestibility 17 and lower lignin content 18 , both leading to higher ethanol yield, converge in the leaf fraction 5 . In elephant grass, the content of most fiber components increase as the plant ages 19 , whereas such content may differ within 19 and between 7 growth seasons.
Variations in biomass quality due to plant age, plant composition, and season of regrowth are often overlooked in research works dealing with conversion of grass crops for bioethanol production. For instance, they merely mention the grass species 20 or the fraction 21 . In fact, most studies on morphological and chemical composition of elephant grass deal with few age classes, a target growth height, or a fixed cutting frequency. In order to fill that gap of knowledge, the present study closely tracks the accumulation pattern and the fiber partition in both leaves and stems of elephant grass CT115, throughout five months of undisturbed regrowth, during the wet and dry seasons.

Results and discussion
Morphological composition. Yield is presented by season, fraction and regrowth age in Fig. 1. Overall biomass yield corresponds to the upper limit of the piled graphic. Leaf yield is shown at the base of the figure, in order to draw attention to the low relative variability in leaf yield across both seasons. When regrowth occurred under limiting weather conditions, leaf yield did not surpass 4 Mg ha −1 . However, during the wet season, when higher soil moisture and higher temperature were available to promote regrowth, leaf yield reached 5 Mg ha −1 . In growth cycles 154 days long, despite the leaf accumulation showing a biologic limit, stem accumulated 16 Mg ha −1 in the wet season or 10 Mg ha −1 in the dry season, whereas leaf proportion meant only 20% of the available biomass by day 154 in either season. Similar data for leaf proportion and leaf yield have been reported for elephant grass subjected to a single harvest per year 22 . However, management under long growth cycles implies reducing the annual harvest of green leaves across the year, as noticed in a previous work 23 .
Decisions on the utilization of elephant grass CT115 intended for ethanol production should focus on increasing the annual harvest of green leaves, in order to improve the yearly harvest of ethanol from a given field 17,23 . Cutting intervals under 70 days of regrowth might be established in order to prevent excessive stem accumulation. That in turn, according to a previous work, might increase both the leaf yield per harvest as well as the biomass yield per year 23 . The continuous stem accumulation, at relatively unvarying offer of green leaves, coincides with a previous study in which elephant grass is kept under undisturbed growth 6 . Furthermore, higher annual biomass yield has been reported for cutting intervals under three months, which also achieved a higher harvest of green leaves through the year 23 . www.nature.com/scientificreports/ Cutting intervals around 70 days might prevent useless stem accumulation and reduce the fiber content of the harvested biomass, therefore promoting a higher biodegradability 24 . Longer cutting intervals have been associated to a higher stem growth, a higher plant lignification, and lower biodegradability 16 . Fiber partition as affected by season and fraction. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents are shown in Fig. 2, whereas cellulose, hemicellulose and acid detergent lignin (ADL) contents are shown in Fig. 3, both organized by morphological component and season. Across season, the season-fraction interaction was not significant for NDF (P = 0.99), ADF (P = 0.94), hemicellulose (P = 0.97), cellulose (P = 0.42), holocellulose (P = 0.71), ADL (P = 0.17) or ashes (P = 0.92) contents. In consequence, differences between seasons remain true within each fraction and differences between fractions remain true within each season. Leaf had 34 ± 10.8 g kg −1 less NDF (mean ± sed; P = 0.002), 85 ± 9.5 g kg −1 less ADF (P < 0.0001), 51 ± 7.1 g kg −1 more hemicellulose, 61 ± 5.1 g kg −1 less cellulose, 24 ± 2.7 g kg −1 less ADL, and 28 ± 5.9 g kg −1 more ashes, as compared to stem (P < 0.0001). In addition, the wet season regrowth showed similar NDF (P = 0.31) and ashes (P = 0.40) contents, but 71 ± 6.2 g kg −1 less ADF (P < 0.001), 60 ± 7.1 g kg −1 more hemicellulose, 66 ± 5.1 g kg −1 less cellulose (P < 0.0001) and 5.3 ± 2.7 g kg −1 less ADL (P < 0.05) than the dry season regrowth. For information about the adjustment and significance of each explicative variable on the model, refer to Supplementary Tables.
Green leaf meant higher hemicellulose, but lower cellulose and lignin contents than stems. In fact, a high biodigestibility of the dry matter has been reported for leaves, as compared to stems, for the grasses Cynodon sp., Arundo donax, and Cenchrus purpureus 5 . In addition, higher digestibility and higher protein content have been reported for the leaves of Andropogon gayanus 18 . A higher hemicellulose concurs with a lower lignification  www.nature.com/scientificreports/ and higher content of non-fiber soluble components, which could be converted to ethanol. Furthermore, a great number of research works had been addressed to the conversion of hemicellulose to ethanol 25 . The higher content of biodegradable compounds as well as the lower cellulose and lignin contents recorded for the wet season regrowth, coincides with a previous study where elephant grass was managed at a cutting interval of 8 weeks throughout the year 8 . In addition, a higher in vitro digestibility, which is related to higher ethanol production 5 , was reported for the grass Andropogon gayanus grown in the wet season, as compared to the dry season regrowth 18 , which might imply a lower cell wall content (NDF). On the other hand, a study about variations in the chemical constitution of elephant grass between seasons found higher quality for the dry season regrowth 7 . This finding, which diverges from the current study, may be due to the important differences in the rainfall distribution throughout the year, since Indonesia is located in the Equator, and rainfall occurs to some extent in every month.
Holocellulose content was similar for the leaf and stem fractions (663 ± 7.1 and 673 ± 7.5, P = 0.35). Likewise, it was similar for the wet and dry seasons (665 ± 7.3, 671 ± 7.3, P = 0.58). The given similarities occurred despite the wide inverse variations in cellulose and hemicellulose contents both between fractions and between seasons (Fig. 3). The leaf from the wet season averaged 138 g kg −1 more hemicellulose than cellulose, and the stem from the dry season showed 100 g kg −1 more cellulose than hemicellulose. Surprisingly, hemicellulose and cellulose contents were similar between the stem from the wet season and the leaf from the dry season (Fig. 3).  Table 1, by morphological fraction: leaf and stem. In both seasons leaf fraction recorded less ADF since day 42 (except contents were alike on day 56 of the wet season), less ADL since day 70, and more ashes since day 56 or 42 of the wet and dry seasons, respectively. During the first 98 days of regrowth, NDF and ADF contents followed increasing trends in either season or either fraction; while they remained constant afterwards.   Fig. 4, ordered by season, morphological fraction and age. Actual means and statistical differences for the visual information of such figure, are presented in Table 2. The higher hemicellulose and lower cellulose contents recorded for the leaf fraction across each season (Fig. 3) remained true virtually on every age in either season.
For the leaf fraction, hemicellulose content increased through day 70 of the wet season or through day 98 of the dry season, whereas the cellulose content increased through day 42 for the leaf fraction, in either season, and kept on similar records from then onwards. Regarding stem fraction, hemicellulose content increased only during the wet season, through day 56, then decreased, but it reached a second maximum on day 126. Cellulose content was relatively constant in either season for the stem fraction, but reached a maximum on day 98 in both seasons.
The similar holocellulose content found between leaf and stem fractions on average across seasons (Fig. 3), remained true in ten out of the eleven ages, in either season. This was especially interesting, given that hemicellulose and cellulose contents differed between leaf and stem, virtually on every age class ( Table 2).
A higher hemicellulose content for the leaf of elephant grass has been previously reported for the dry season, while a higher cellulose content has only been reported for the wet season; both results in a study in Thailand 26 , as an average for eight varieties of elephant grass. Climate and variety differences explain the discrepancies with the current work.
Published data are consistent with the fact that grass age and the content of most of the fiber constituents are directly related 19,27,28 ; nonetheless, just a few age classes are usually included. Hemicellulose content has been reported to decrease for the whole plant of elephant grass in long-lasting growth cycles 19 . The current study gives rationale for such fact, since along regrowth, an increment of the stem proportion (Fig. 1), whose hemicellulose content was lower (Table 2), will lead to a lower hemicellulose content for the whole plant (see Supplementary  Tables).
All seven variables describing chemical constitution in the current research work showed similar records from day 98 onwards, in each season and for each morphologic fraction.
Recommendations. Biomass quality of elephant grass CT115 can be improved, by means of increasing both the share of green leaves and the share of the wet season regrowth, in the biomass harvested along the year. A higher biomass quality, will in turn increase the annual yield of ethanol per area unit.
Strategies to accomplish a higher quality of the harvested biomass, as proposed above, may involve (1) cutting intervals of around 56 days during the wet season or around day 70 during the dry season, and (2) reduce cutting intensity. The latter implies cutting to a greater height, so that the fodder left uncut in the field will facilitate conclusions Elephant grass CT115 must be harvested by day 56 of the wet season or by day 70 of the dry season, in order to increase both the share of green leaves per harvest and the annual yield of green leaves. Green leaves recorded more hemicellulose, less cellulose and less lignin than stems, whereas the regrowth from the wet season recorded more hemicellulose, less cellulose and less lignin, than that from the dry season. Holocellulose content was similar between leaves and stems of elephant grass, as well as between the regrowth from the wet season and that from the dry season.
When elephant grass is cultivated as energy crop for conversion to ethanol, leaves should be preferred over stems, whereas the wet season regrowth should be preferred over that from the dry season.
Age is the main factor affecting the chemical composition of elephant grass. Cutting intervals around 56 days for the wet season or around 70 days for the dry season provide an acceptable yield-quality balance. Longer intervals would sacrifice biomass quality, while shorter ones would sacrifice yield. The lower biomass quality of the late regrowth is explained in terms of both higher stem share and higher stem lignification.
Hemicellulose and cellulose contents were inversely correlated. In addition, ADL and hemicellulose contents were inversely correlated for the leaf fraction, while ADL and cellulose contents were directly correlated for the stem fraction.

Methods
Location and weather. The field assay was conducted at the Papaloapan Site of the Mexican Institute for Forestry, Agricultural and Livestock Research (INIFAP), Veracruz, Mexico. Climate is warm-wet with a summer rainy season, 1,142 mm of annual rainfall, and 25.8 °C of monthly temperature 29 . Chemical determinations were Table 2. Hemicellulose, cellulose and holocellulose contents in leaf and stem of elephant grass CT115, across 154 days of undisturbed regrowth, for the wet and dry seasons. se standard error, R 2 model adjustment. a,b,c…g Means in the same column with different lowercase letter are different (P < 0.05). A,B Means in the same row and variable, with different uppercase letter are different (P < 0.05). Hemicellulose content = NDF -ADF, cellulose content = ADF -ADL, and holocellulose = hemicellulose + cellulose. (1) Y ijk = µ + S i + F j + SF ij + ε ijk (2) Y ijk = µ + A i + F j + AF ij + ε ijk