The Effect of Sodium Hydroxide Treatment of Rice Straw on In situ Disappearance of Hemicellulose and Lignin in its Cell Wall

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1 The Effect of Sodium Hydroxide Treatment of Rice Straw on In situ Disappearance of Hemicellulose and Lignin in its Cell Wall Chisato HARADA, Yoshiko SAITO, Yutaka NAKAMURA and Hajime MINATO Faculty of Agriculture, Ibaraki University, Ami-machi, Ibaraki-ken , Japan (Received August 3, 2000; Accepted November 13, 2000) Abstract In situ disappearance of cell wall constituents of the untreated and NaOH-treated rice straw was examined in detail. In situ cellulose digestion of rice straw was significantly improved by treatment with 4% NaOH. Hemicellulose in the cell walls of rice straw abundantly contained galactose and xylose, followed by arabinose, glucose, and uronic acids. In situ digestion of xylose residues in hemicellulose of the straw was inferior to that of other monosaccharides, but that of the straw was remarkably improved by the NaOH-treatment. The same amount of guaiacyl- and syringyllignins was released from the untreated rice straw during in situ digestion. On the other hand, more abundant syringyl-lignin than guaiacyl-lignin was released from NaOH-treated rice straw during the in situ digestion. In situ disappearance of ester-linked p-coumaric and ferulic acids from the untreated rice straw was larger than that of the ether-linked acids. While, in situ disappearance of ether-linked p-coumaric and ferulic acids from the NaOH-treated rice straw was larger than that of the ester-linked acids. The results obtained in this study suggest that the partial destruction of a network structure built up from hemicellulosic polysaccharides and lignin in rice straw cell walls by NaOH-treatment makes it easy for the access of cellulolytic microorganisms and their cellulases to cellulose and accelerates the in situ cellulose digestion of rice straw. Animal Science Journal 72 (1): 19 25, 2001 Key words: Rice straw, In situ digestion, Cell wall monosaccharides, Phenolic acids, Lignin. Rice straw of about 107 tons is produced every year in our country as a by-product of rice crop production. Although rice straw is attracting a special interest as a roughage resource recently, only 13% of the produced straw is used as a feedstuff for ruminants. Lower utilization of the rice straw as feedstuff is partly due to a low dry matter (DM) digestibility of rice straw. Cereal straws, including rice straw, are largely constituted of cell walls, which are mainly composed of cellulose, hemicellulose and lignin. The close physical and chemical association between lignin and cell wall polysaccharides would prevent accessibility of microorganisms to their substrates and limit the efficient utilization of the straws for ruminant feeding. Various physical and chemical treatments of cereal straws to improve their nutritive values for ruminants have been done11). Of cereal straw treatments with chemicals, sodium hydroxide is one of the most important chemical procedures. Several reports have shown that NaOH-treatment of cereal straws induced the partial solubilization of hemicellulose and in situ DM digestibility of the straws did increase2,4). Also, our previous works indicated that the treatment of rice straw with 4% NaOH solubilized a large amount of hemicellulose and lignin, and in situ DM digestibility of the straw was remarkably improved8), and that the content of esterified phenolic acids of the straw decreased by the NaOH-treatment9). In this study, to clarify some of the mechanism Corresponding: Hajime MINATO (fax: +81 (0) , hminato@ipc.ibaraki.ac.jp) Anim. Sci. J. 72 (1): 19-25, of

2 HARADA, SAITO, NAKAMURA and MINATO the observed improvement in DM digestibility of rice straw by treatment with NaOH, in situ disappearance of cell wall constituents of the untreated and NaOHtreated rice straw was examined in detail. with TC-1 (GL Science Inc., Tokyo, Japan). The operating conditions of the instrument were as follows Materials and Methods Sodium hydroxide treatment of rice straw Rice straw was purchased from Japan Horse Feed Co. Ltd. (Miho-mura, Ibaraki) and was hand cut (3 cm length) with trimming shears. The trimmed straw was evenly hand sprayed with an aqueous solution containing NaOH to give it a 35% moisture content, and to provide a 4% NaOH concentration of the rice straw DM. The straw was then stored in a vinyl bag for 3 weeks at room temperature. And, the untreated straw was sprayed with distilled water to give it a 35% moisture content and stored in a vinyl bag for 3 weeks at room temperature. After these treatments, the materials were artificially dried in an to pass through a lmm screen. The milled material was treated three times with boiling 80% aqueous ethanol for 1.5h, and then treated with boiling chloroform for 1h. The treated residues were dried in a following analysis. General chemical analysis The contents of neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), crude cellulose, crude hemicellulose, and lignin of the materials were determined following the methods described in the previous report9). Analysis of monosaccharides The materials were hydrolyzed in 2N trifluoroacetic fractionated from the hydrolysate using a Dowex 50-X8 (H form) according to the procedures described by Minato et al.13). The neutral sugars were determined as their alditol acetates derivatives according to the methods reported by Albersheim et al.1) and by Goto et al.7). The derivatives of neutral sugars were analyzed by gas-liquid chromatography (GLC) using a Shimadzu GC-16 A gas chromatograph on a flexible fused silica capillary Anim. Sci. J. 72 (1): 19-25, carrier gas. Uronic acid was measured using the colorimetric method described by Scott15). Alkaline nitrobenzene oxidation and analysis of the products by GLC Alkaline nitrobenzene oxidation of the materials and the analysis of the products by GLC were performed as described in the previous report9). Determination of esterified and etherified phenolic acids The determination of esterified and etherified phenolic acids of the materials was performed using the method described in the previous report9). The phenolic acids were trimethylsilated with N, O-bis (trimethylsilyl) acetamide and were analyzed by GLC. In situ disappearance of constituents of rice straw In situ disappearance of the constituents of rice straw was determined according to the protocol described by Van Keuren and Heineman16). Five grams of the materials were weighed into nylon bags that were closed and tied with nylon string. The bags were placed in the rumen of ruminally fistulated sheep, which were fed hay and concentrate (4:1) twice daily, and incubated for 48h in the rumen. After removal from the rumen, the bags were washed mechanically with tap water until the water that was squeezed from the bags was clear. And then, the 48h prior to the determination of DM, NDF, ADF, crude cellulose, crude hemicellulose, lignin, phenolics and monosaccharides. Results and Discussion Comparison of cell wall constituents of untreated and NaOH-treated rice straw The chemical compositions of the untreated and NaOH-treated rice straw are shown in Table 1. The contents of the cell wall substances of untreated and NaOH-treated rice straw were 86.2 and 76.6%, respectively. The result demonstrates that the NaOHtreatment of rice straw increased the amount of water-

3 In situ Digestion of Rice Straw Table 1. Cell wall composition of untreated and NaOH-treated rice straw 1) 80% Ethanol extracted residue. 2) Mean of six determinations. 3) Figure in parentheses shows the percentage of substance released from rice straw by NaOH-treatment. and ethanol-soluble materials in the cell wall. After NaOH-treatment, the hemicellulose content of rice straw remarkably decreased to 31.8% as compared with that of the untreated rice straw. Hemicellulose in the cell walls contained galactose, xylose, arabinose, glucose, and uronic acid. Therefore, it is assumed on the basis of the informations described by Carpita and Gibeaut3) that the hemicellulose of this rice straw was comprised of glucuronoarabinoxylans, xyloglucans, arabinogalactans and polygalacturonic acid. The content of hemicellulosic monosaccharides in the NaOH-treated rice straw was lower than that of the untreated rice straw. And the most abundant monosaccharides released from rice straw by treatment with 4% NaOH were xylose and galactose, followed by arabinose and glucose. The uronic acid content of the rice straw did not change by treatment with NaOH. Lignin is a complex biopolymer, which is built up from guaiacyl-, syringyl- and p-hydroxyphenylpropane units. Lignin undergoes an oxidativecleavage reaction to produce phenolic aldehydes when heated in an alkaline aqueous solution containing nitrobenzene. The alkaline nitrobenzene oxidation is Anim. Sci. J. 72 (1): 19-25,

4 HARADA, SAITO, NAKAMURA and MINATO established as an important method for qualitative and quantitative determination of the building units of lignin. As shown in Table 1, the amount of the nitrobenzene oxidation products of the untreated rice straw was more abundant than that of the NaOH-treated rice straw. And, the most abundant phenolics in lignins released from rice straw by treatment with NaOH were vanillin, followed by p-coumaric acid, ferulic acid and p-hydroxybenzaldehyde. The results show that lignins rich in guaiacyl nuclei are more easily released treatment from the cell walls of rice straw by with NaOH. The amounts of esterified and etherified phenolic acids in the untreated and NaOH-treated rice straw was presented in Table 3. In the untreated rice straw, the total amount of esterified and etherified p-coumaric acids was much more abundant than that of the ferulic or syringic acids. Most of the p- coumaric acids were in ester-linked form. About 46% of the esterified p-coumaric acid were released from rice straw by the NaOH-treatment. On the other hand, the etherified forms of p-coumaric, ferulic and syringic acids were hardly released from rice straw through treatment with NaOH. In situ disappearance of cell wall constituents of untreated and NaOH-treated rice straw In situ disappearance of the constituents of untreated and NaOH-treated rice straw were measured and the obtained results by the incubation of 48h are presented in Table 2 and 3. In situ cell wall digestibilities of the untreated and NaOH-treated rice straw were 46.9 and 75.4%, respectively. In situ DM digestibility of rice straw was remarkably improved by the NaOH-treatment. Especially, the digestibility of cellulose among the constituents of rice straw cell wall was markedly improved by treatment with NaOH. In addition, as shown in Table 1, 60.9% of cell wall hemicellulose was released from rice straw by the NaOH treatment. These facts suggest that the partial destruction of a network structure built up from hemicellulosic polysaccharides and lignin in rice straw cell walls by NaOH-treatment, and the release of abundant hemicellulose from a cell wall by NaOHtreatment together, make it easy for the access of Table 2. In situ disappearance of cell wall constituents of untreated and NaOH-treated rice straw 1) 80% Ethanol extracted residue. 2) Mean of three dete rminations. cellulolytic microorganisms and their cellulases to cellulose and accelerates the in situ cellulose digestion of rice straw. In situ digestibilities of hemicellulosic monosaccharides of the untreated and NaOH-treated rice straw were 46.1 and 74.3%, respectively. In situ digestibility of galactose in hemicellulosic monosaccharides was superior to that of other mono - saccharides. Conversely, in situ digestibility of xylose of hemicellulosic monosaccharides was inferior to that of other monosaccharides. However, in situ Anim. Sci. J. 72 (1): 19-25,

5 digestion of xylose of rice straw treated with NaOH was remarkably improved, as compared with that of the untreated rice straw. The contents of the hemicellulose determined according to the method developed by Van Soest17) were smaller than the values estimated by the summation of the monosaccharides which comprised cell wall hemicellulose (Tables 1 and 2). This discrepancy shows the possibility that a part of the cell wall hemicellulose was solubilized by neutral detergent treatment and was lost from rice straw. In situ disappearance of hemicellulosic monosaccharides of rice straw was measured with the lapse of incubation time in the rumen. The rate of disappearance of hemicellulosic monosaccharides in HaOH treated rice straw was higher than in the untreated rice straw. A rapid disappearance of galactose, glucose and arabinose of hemicellulose were observed during the 12h incubation period, and a slow linear disappearances of these monosaccharides were observed during the incubation time of 12 to 48h. The rate of disappearance of uronic acid showed a similar tendency as these three monosaccharides. A disappearance of xylose was much slower than other monosaccharides and uronic acid, but a steady disappearance continued during the incubation of 48h (data not shown). In situ disappearance of lignin from untreated and NaOH-treated rice straw during the incubation of 48 h was 25.9 and 34.7%, respectively. In addition, the amount of lignin that disappeared from the untreated and the NaOH-treated rice straw during the incubation of 48h was 51.7 and 51.8mg per g of straw, respectively. These results agree approximately with those reported by Dekker et al.5) and Muntifering et al.14) that the rate of in situ disappearance of lignin In situ Digestion of Rice Straw digestion. For the untreated rice straw, in situ dis- appearance of ester-linked p-coumaric and ferulic When the amounts of phenolics disappeared from the rice straw in in situ digestion were determined by the alkaline nitrobenzene oxidation method, the amount of phenolics disappeared from the untreated rice straw was larger than from the NaOH-treated rice straw. While, when the amounts of lignin were determined by the acetyl bromide method, the amount of lignin that disappeared from the untreated and the Anim. Sci. J. 72 (1): 19-25, NaOH-treated rice straw during the 48h incubation were almost same. These facts suggest that the lignin which is resistant to the alkaline nitrobenzene oxidation treatment was released more abundantly from the NaOH-treated rice straw than from the untreated rice straw during in situ digestion. In addition, almost the same amount of guaiacyl- and syringyl-lignins was released from the untreated rice straw during in situ digestion. On the other hand, more abundant syringyl-lignin than guaiacyl-lignin was released from NaOH-treated rice straw during in situ digestion. Because Gaillard and Richards6) pointed out that the lignin of grass decomposed into soluble ligninpolysaccharide complexes in the bovine rumen, it is assumed that the phenolics disappeared from the rice straw during in situ digestion were released as soluble lignin-polysaccharide complexes in the rumen. Ferulic acids are involved in the formation of bridged linkages between polysaccharides and lignins. Particularly, ferulic acids are covalently linked to polysaccharides through ester linkages and to lignins through ether linkages. In addition, dehydrodiferulic acid in diester linkages between polysaccharides is etherified to lignin. On the other hand, p-coumaric acids are directly esterified to polysaccharides, and are directly esterified or esterified to lignins. But, p- coumaric acids are not involved in the formation of bridged linkages between polysaccharides and lignins10) In situ disappearance of esterified and etherified phenolic acids from the untreated and NaOH-treated rice straw during the 48h incubation was measured and the obtained results are shown in Table 3. The small amount of phenolic acid was released from the untreated and NaOH-treated rice straw during in situ acids was larger than that of the ether-linked acids. This fact suggests that ester-bonds of p-coumaric and ferulic acids are more susceptible for breakdown by microorganisms than ether-bonds of these acids. This result does not contradict the data that the amount of ester-linked phenolics cloven from sorghum rind by Neocallimastix patriciarum was larger than that of the ether-linked phenolics12). While, for the

6 HARADA, SAITO, NAKAMURA and MINATO Table 3. In situ disappearance of esterified and etherified phenolic acids in cell wall of untreated and NaOH-treated rice straw 1) Mean of six determinations. 2) Mean of three determinations. NaOH-treated rice straw, in situ disappearance of ether-linked p-coumaric and ferulic acids were larger than that of the ester-linked acids. This result is partly due to the fact that a large amount of p- coumaric acid esterified to polysaccharides or lignins was released from the rice straw by NaOH-treatment and the amount of ester-bondedp-coumaric acid easily accessible by microorganisms decreased. In situ disappearance of syringic acid was much smaller than other phenolic acids. The phenolics produced by alkaline nitrobenzene oxidation of NaOH-treated rice straw were smaller by about 30% in quantity than the untreated rice straw. About 46% of the esterified p-coumaric acid were released from rice straw by the NaOH- treatment. In addition, about 60% of the hemicellulose from rice straw was solubilized and lost by treatment with NaOH. While, in situ DM digestibility, especially cellulose digestibility, of rice straw was remarkably improved by the NaOH-treatment (Table 1). These results suggest that a network structure built up from hemicellulosic polysaccharides and lignins, which embeds cellulose microfibrils, and phenolic acids linked to them inhibits in situ DM digestion of the rice straw. Acknowledgments The authors wish to thank Dr. Masakazu Goto, Faculty of Bioresources, Mie University, Japan, for his valuable suggestions in this study. This research was financially supported by grant-in-aid for scientific research (C) from the Ministry of Education of Japan. References 1) Albersheim P, Nevins DJ, English PD, Karr A. A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. Carbohydrate Research, 5: ) Canale CJ, Abrams SM, Varga GA, Muller LD. Alkali-treated orchardgrass and alfalfa: composition and in situ digestion of dry matter and cell wall components. Journal of Dairy Science, 73: ) Carpita NC, Gibeaut DM. Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. The Plant Journal, 3: ) Chesson A. Effects of sodium hydroxide on cereal straws in relation to the enhanced degradation of structural polysaccharides by rumen Anim. Sci. J. 72 (1): 19-25,

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