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Availability of Carbohydrates in the Root Stubble of a High-Yielding Rice Cultivar, “Hokuriku 193”
2025 Volume 72 Issue 3 Article ID: 7203201
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2025 Volume 72 Issue 3 Article ID: 7203201
Root stubble represents a “hidden” (unrecovered) biomass in agriculture. This study investigated its potential as a source of fermentable sugars using the high-yielding rice cultivar Hokuriku 193. The ripened whole plant was disassembled into panicle, straw, and root stubble. The root stubble was further divided into the aboveground part (AP), basal part (BP), and root part (RP), with AP and BP together accounting for 81.8 % (w/w) of the root stubble. In comparison with the straw, AP contained more starch- and β-1,3-1,4-glucan; BP had more starch and ash; RP had more lignin and ash. The total amount of glucan and xylan in root stubble is equivalent to 61.8 % of that found in straw. Following gas-phase HCl pretreatment and the subsequent enzymatic saccharification, sugar yields from AP and BP exceeded 70 %. These results demonstrate that root stubble has strong potential as a new feedstock for saccharification substituting straw.
AP, aboveground part; BP, basal part; RP, root part; DAC, direct air capture; GrAAS, grass upcycling by activated acid into the sugar pool.
As the impacts of climate change on the global environment and living spaces become increasingly severe, it is essential to mitigate the emission of greenhouse gases resulting from large-scale consumption of fossil resources (United Nations Framework Convention on Climate Change secretariat [Internet]. Bonn: Japan's Nationally Determined Contribution; c2025 [cited 2025 Jun 24]. Available from: https://unfccc.int/sites/default/files/2025-02/Japans%202035-2040%20NDC.pdf). Carbohydrate-rich biomass can be converted into ethanol as a gasoline substitute, and further processed into chemical feedstock, sustainable aviation fuel, and other products via ethylene (Integrated Innovation Strategy Promotion Council Decision [Internet]. Tokyo: Bioeconomy Strategy; c2024 [cited 2025 Jun 24]. Available from: https://www8.cao.go.jp/cstp/bio/bio_economy_en.pdf). Therefore, securing a sustainable supply system of domestic biomass has become an urgent priority for achieving carbon neutrality and advancing a circular economy [1]. In this context, technological advancements have been promoted to enable ethanol production from carbohydrates, such as cellulose and xylan utilizing lignocellulosic resources, such as straw and husk, which can be sustainably sourced from agriculture as raw materials [2].
Rice is the most widely cultivated crop in Japan (United States Department of Agriculture [Internet]. Washington, D.C.: An Overview of Japan Agriculture Production; c2023 [cited 2025 Jun 24]. Available from: https://ipad.fas.usda.gov/highlights/2023/10/Japan/index.pdf). The stems and leaves, which are conventionally used as livestock feed or plowed back into the ground, are now being recognized as a significant domestic feedstock for saccharification and ethanol fermentation that does not compete with food production. The ongoing “DAC (direct air capture) Agriculture” project aims to enhance CO2 absorption by applying genome editing to develop “Super DAC Rice” from the high-biomass-accumulating cultivar Hokuriku 193 [3], offering a new feedstock for ethanol production (DAC Agriculture Project [Internet]. Tsukuba: Development of DAC Technology in Agriculture; c2023 [cited 2025 Jun 24]. Available from: https://www.agri-dac.naro.go.jp/eng/).
During rice cultivation, carbon dioxide is fixed from the atmosphere and incorporated into organic compounds in brown rice grains, rice husks, straw (composed of stems and leaves), and root stubble. The first three of these are easily available aboveground during harvest, and resource utilization technologies have been developed to use them as the main product or a by-product. In contrast, root stubble, consisting of the remaining straw above ground and the underground parts with roots, is typically plowed into the soil. The incorporation of the belowground part is considered a contributing factor to methane emissions during rice cultivation [4]. Therefore, collecting and using root stubble might have potential benefits, but its variability as a feedstock for saccharification has not been extensively evaluated.
In this study, we used the whole plant body of the cultivar Hokuriku 193 to analyze the weights and sugar composition of straw and root stubble. Moreover, each part was subjected to an enzymatic saccharification test following pretreatment with the vapor-phase hydrochloric acid method, grass upcycling by activated acid into the sugar pool (GrAAS) process [5], to examine the recovery characteristics of free sugars.
Rice plants (Oryza sativa L. cv. Hokuriku 193) were cultivated in the experimental paddy field of the Institute of Crop Science, National Agriculture, and Food Research Organization, Tsukuba, Japan (36°1′N, 140°6′E) in 2024. The basal application consisted of 5.6 g N m−2, 15.6 g P2O5 m−2, and 5.6 g K2O m−2. Seeds were soaked in water at 30 °C for 2 days, sown in nursery trays on April 24, and then incubated in the dark at 30 °C for 2 days. Seedlings were then grown in a paddy field for approximately a month and then transplanted on May 24. The planting density was 18.5 hills m−2, with 18 cm spacing between hills and 30 cm between rows. The heading date, defined as the date when panicles were visible in more than half of the plants, was August 7. The plant was collected at the late ripening stage on October 4. In this study, each rice plant was cut 15 cm above ground level. The lower part was defined as rice stubble (Fig. 1A). The upper part of the rice plant was divided into panicles (with grains) and straw. The rice stubble was further divided into three parts: the aboveground part (AP, Fig. 1B), basal part (BP, Fig. 1C), and root part (RP, Fig. 1C). BP and RP were washed with tap water to remove soil and small pebbles manually. All parts were dried at 65 ºC until reaching constant weight (Table 1). Subsequently, one sample of rice plant (panicles, 33.6 g; straw, 33.6 g; AP, 11.8 g; BP, 11.2 g; RP, 3.88 g) was used for further analysis. The straw, AP, BP, and RP, with moisture contents of 5.66 %, 6.65 %, 4.26 %, and 4.25 %, respectively, were ground using a food processor (Power Blender SPB-650, Conair Japan G.K., Tokyo, Japan) for 1 min and then powdered using a Wiley mill (MF 10.1 Grinding Mill, IKA Japan K.K., Osaka, Japan) to pass through a 4-mm mesh screen. Powders (3 g) from each sample were pulverized using a ball mill (Mixer Mill MM400, Verder Scientific Co., Ltd., Tokyo, Japan) for 4 min for component analysis (Table 2). The contents of glucans (total glucans, starch, sucrose, and free glucose), xylan, Klason lignin, acid-soluble lignin, and ash per absolute dried weight of each sample were measured as previously described [5]. β-1,3-1,4-Glucan content was measured using a β-Glucan Assay Kit (Mixed Linkage) (BIOCON Japan Ltd., Nagoya, Japan).
In this study, the rice stem and leaves were horizontally cut at a height of 15 cm from the ground level, and the lower part was defined as the rice stubble. AP, aboveground part. The underground part was further divided into two parts: the basal part (BP) and the root part (RP).
Table 1. Dry weights of disassembled parts of the rice plant (Cv. Hokuriku 193).
| Disassembled parts | Weight (g) | Ratio (%)＊ |
| Panicle | 35.7 ± 1.9 | 36.4 |
| Straw | 36.5 ± 4.1 | 37.2 |
| Root stubble | 25.9 ± 1.9 | 26.4 |
| (AP) | (11.8 ± 0.1) | (12.0) |
| (BP) | (9.4 ± 2.1) | (9.6) |
| (RP) | (4.8 ± 0.8) | (4.9) |
AP, aboveground part; BP, basal part; RP, root part. The average of three rice plant samples and the standard deviation (n = 3) are shown.
＊The weight ratio of each disassembled part to the total weight of the corresponding rice plant is indicated on a dry basis (dried at 65 ºC).
Table 2. Main components in dissected parts of the rice plant (Cv. Hokuriku 193).
| Parts | Components (%, per dry weight) | |||||||||
| Glucans | Xylan | Klason lignin | ASL ＊＊ | Ash | ||||||
| (Total) | Cellulose ＊ | Starch | β-1,3-1,4-Glucan | Sucrose | Free glucose | |||||
| Straw | 37.7 ± 0.5 | 27.2 ± 0.4 | 6.5 ± 0.2 | 1.2 ± 0.0 | 2.9 ± 0.3 | 1.7 ± 0.0 | 14.1 ± 0.2 | 13.1 ± 0.8 | 2.9 ± 0.0 | 10.0 ± 0.4 |
| Root stubble (AP) | 42.7 ± 0.3 | 25.2 ± 0.2 | 12.1 ± 0.1 | 2.7 ± 0.0 | 2.7 ± 0.1 | 1.5 ± 0.1 | 12.1 ± 0.1 | 9.6 ± 0.0 | 2.3 ± 0.1 | 11.2 ± 0.0 |
| Root stubble (BP) | 27.7 ± 0.5 | 10.6 ± 0.6 | 14.1 ± 0.1 | 1.0 ± 0.0 | 2.7 ± 0.1 | 0.8 ± 0.0 | 7.4 ± 0.3 | 9.3 ± 0.0 | 1.9 ± 0.1 | 37.1 ± 0.1 |
| Root stubble (RP) | 24.9 ± 0.0 | 23.5 ± 0.1 | 0.4 ± 0.0 | 0.1 ± 0.0 | 1.4 ± 0.1 | 0.3 ± 0.0 | 15.2 ± 0.1 | 17.8 ± 0.0 | 2.3 ± 0.0 | 20.8 ± 0.8 |
AP, aboveground part; BP, basal part; RP, root part. The average and standard deviation (n = 2) are shown.
＊The amount of cellulose was calculated by subtracting the amounts of glucose residues in starch, β-1,3-1,4-glucan, sucrose, and free glucose from that of the total glucans.
＊＊Acid-soluble lignin.
Next, each Wiley-milled powder sample was used for the hydrogen chloride pretreatment for enzymatic saccharification as previously described [5]. In brief, each sample (50 mg of dry matter) was placed at the bottom of a 2-mL plastic tube. A solution of 4 M CaCl2 and 1 M HCl in distilled water (0.1 mL) was carefully placed apart from the powdered sample in the tube. The tube was then closed with a screw cap and incubated at 40 ºC for 18 h. After the reaction, the solution was removed, and 0.95 mL of distilled water and zirconium beads (one bead, 5 mm in diameter (YTZ-06) plus 500 mg of smaller beads (YTZ-5), Nikkato Corporation, Sakai, Japan) were added to the tube for homogenization. The homogenization was performed six times using a bead mill (Micro Smash MS-100R, Tomy Digital Biology Co., Ltd., Tokyo, Japan). The resultant slurry in each tube was transferred to a 15-mL plastic tube and adjusted to a final volume of 5.5 mL with distilled water and a 50 mM (final) sodium acetate buffer (pH 5.0). The solution also contained 0.6 Filter Paper Unit [6] (final) of Cellic CTec2 (Novozymes Japan Ltd., Chiba, Japan), 0.82 mg (as protein) of Cellic HTec2 (Novozymes, the enzyme solution was dialyzed with 10 mM sodium phosphate buffer (pH 7.0) beforehand), 0.6 Cellobiase Unit [6] (final) of Novozyme 188 (Novozymes), 0.5 g/L (final) sodium azide, 0.05 g/L (final) chloramphenicol, and 0.05 g/L (final) tetracycline. The protein concentration of dialyzed Cellic HTec2 was measured using the RC DC™ Protein Assay Kit with BSA as the standard (Bio-Rad Laboratories, Inc. Japan, Tokyo, Japan). The reaction mixture was shaken at 80 rpm, 50 ºC for 48 h, and the amounts of liberated glucose and xylose residues in the solution were calculated as previously described [5].
The root stubble contains significant amounts of glucan and xylan, with the total content of these two sugar residues equivalent to 61.8 % of that in the straw, as calculated from the data in Tables 1 and 2. The AP showed a higher glucan concentration than the straw, which can be partly attributed to starch accumulation (Table 2). The starch concentration is also high in the BP, suggesting that starch storage in the root stubble is important for the growth of subsequent ratoon shoots for harvesting additional rice crops [7]. In contrast, the RP contained only a small amount of starch, indicating a limited role in temporary sugar storage. The amount of β-1,3-1,4-glucan was high in the aboveground parts (straw and AP). This glucan plays a role in temporary sugar storage for elongation in young vegetative tissues and is accumulated in mature stems, which might contribute to the mechanical strength of the entire plant [8].
The ratios of cellulose to xylose in the straw, AP, BP, and RP were 1.93, 2.08, 1.43, and 1.54, respectively, while ratios of cellulose to lignin were 1.70, 2.11, 0.96, and 1.16, respectively. Straw and AP exhibited higher ratios than BP and RP. Further, the amount of cellulose in rice tissues is significantly positively correlated with xylose and monolignol residues [9]. The high ratios observed in straw and AP in this study suggest that the cellulose content might be overestimated, possibly due to the inclusion of unextracted β-1,3-1,4-glucan, xyloglucan, callose, and other glucans, or due to the presence of various types of cell walls with different ratios of cellulose to other components in individual cells, such as in sclerenchyma cells, parenchyma cells, and root cells. The elevated Klason lignin content in RP might be attributed to a specific tissue differentiation involving suberin deposition in the endodermis, which helps protect the plant against physical and chemical stresses and biological attacks [10]. The high ash content in BP and RP could reflect the residual soil after washing and manual removal, although the unique functions of the root tissue might also contribute in part to these values. This tissue can oxidize and deposit iron at its surface [11], and polysaccharides such as pectin or mucilage capture aluminum and cadmium, thereby protecting the whole plant from toxicity [12, 13].
Next, the milled samples of straw, AP, BP, and RP were subjected to a gas-phase hydrochloric acid pretreatment followed by enzymatic saccharification. As shown in Fig. 2, AP and BP demonstrated strong potential as substrates yielding glucose and xylose recoveries comparable to those from straw. Based on the weights and sugar contents of AP and BP in the root stubble (Tables 1 and 2), efficient sugar recovery from the root stubble can be anticipated. Glucan recoveries of 30-40 % were also observed in the untreated controls (SC, APC, and BPC), likely due to the presence of readily degradable sugars such as starch and β-1,3-1,4-glucan (Table 2). The pretreatment process (GrAAS process) is an acid treatment where heat for acid hydrolysis is generated when the diffused hydrogen chloride gas dissolves in the water-containing components of the biomass. This gas-phase process was selected for pretreatment because, in a liquid-phase process with an acid or alkaline reagent, inorganic salts in BP and RP may dissolve or react and reduce the titer of the reagent. The poor RP values might be due to the unique cell wall structure rich in lignin, suggesting that further analysis and optimization of the pretreatment conditions will be necessary.
SS, straw with pretreatment; SC, straw without pretreatment; APS, aboveground part of the root stubble with pretreatment; APC, aboveground part of the root stubble without pretreatment; BPS, basal part with pretreatment; BPC, basal part without pretreatment; RPS, root part with pretreatment; RPC, root part without pretreatment.
The average of two samples and standard deviation (n = 2) are shown.
Thus, the quantitative and qualitative usefulness of root stubble as a new feedstock for sugar recovery has been demonstrated. However, at least two issues need to be addressed. First, there is a trade-off between maintaining or enhancing soil fertility and contributing to carbon storage through conventional root plowing in the field. It has been reported that the addition of roots to the soil does not increase the microbial inorganic N uptake capacity, but it does contribute to the increase in organic carbon content [14]. In practice, the root stubble is crushed and plowed into the soil as a whole, whereas some parts of the stubble might exhibit characteristics similar to rice straw (Table 2, AP and BP) when plowed. Furthermore, it has been reported that the addition of straw to soil increases microbial inorganic N uptake and the uptake/transformation of N2 by soil microorganisms [14]. In contrast, a considerable proportion of organic compounds in the plowed rice stubble, which are not converted into microbial biomass or carbon storage, could be fully decomposed in the soil; some parts are aerobically converted into CO2 without industrial utilization, while others anaerobically converted into CH4, which has a much severe impact on global warming than CO2 [4]. Therefore, extracting organic carbon from root stubble and converting it into valuable materials, instead of generating greenhouse gases from the soil, could be a more sustainable approach. It would be more beneficial for sustainable agriculture if the residue from enzymatic saccharification, mainly composed of hard-to-decompose organic compounds, is returned to the soil for improving fertility and carbon storage.
The second issue to address is the method of digging and collecting the root stubble. In conservation tillage, a novel agricultural technology aimed to achieve environmentally friendly management by minimizing the frequency or intensity of tillage operations, crushing and clearing root stubble with minimal soil disturbance is crucial to reduce clogging during sowing [15]. Mechanized treatment technologies, such as passive or active crushing and side throwing of root stubble, have been developed for this purpose. Research has also been conducted on the recovery and valorization of root stubble. For example, Zeng et al. reported a new roller-mill mechanism in a maize stubble harvester that collects stubble and separates it from soil for biofuel production [16]. If root stubble is recognized as valuable, further research should focus not only on its recovery and sustainable utilization but also on breeding and agronomic practices to enhance the quality and quantity of carbohydrates in root stubble. These efforts could contribute significantly to advancing the circular bioeconomy in agriculture [17].
The authors declare no conflict of interest.
This paper is partly based on results obtained from a project, JPNP18016, commissioned by the New Energy and Industrial Technology Development Organization (NEDO). We are grateful to H. Yamada and K. Hiramoto for their excellent technical assistance. The authors would like to thank Enago (www.enago.jp) for the English language review.
