The structure of lignin has not yet been completely elucidated. Milled wood lignin (MWL) and cellulolytic enzyme lignin (CEL) have been used as suitable lignin samples to characterize natural lignin in wood. However, MWL and CEL are isolated from wood meal after rather extensive ball milling, and it has been demonstrated that the structure of lignin could be changed during ball milling. In this dissertation, the first topic was to investigate the relationship between the structure of hemicelluloses and that of lignin by two methods. One is the relationship between syringyl ratio (syringyl / (syringyl+guiaicyl)) and neutral sugar in 65 woods, another one is the analysis of fractions from beech and red pine which were extracted by LiCl/DMSO dissolution system. The second topic was to prepare the CEL by a new pretreatment method. The structure of lignin could be changed during ball milling. Thus, CEL was tried to obtain after mild EDA pretreatment instead of ball milling. As third topic, 65 woods were analyzed by IR spectroscopy to examine if syringyl ratios can be precisely expressed by area ratios of characteristic peaks of aromatic ring in IR spectrum.

Relationships between hemicellulose composition and lignin structure

The compositions and the absolute amounts of neutral sugars were determined for 48 hardwood species (including 17 hardwoods of genera Acacia, 14 hardwoods of genera Eucalyptus and 17 hardwoods of different other genera) and 17 softwood species by alditol-acetate method, and their relationships to the syringyl ratios of lignin, which was determined by nitrobenzene oxidation, were investigated. In 48 hardwood species, with the increase in syringyl ratio of lignin, xylose/glucose ratio and rhamnose/glucose of 48 hardwoods seemed to show upward tendency and mannose/xylose ratios of 48 hardwoods showed downward tendency, while, absolute amounts of glucose of 48 hardwoods remained almost constants. In 17 softwood species, with the increase in lignin contents, mannose /glucose ratio decreased, as well as absolute amounts of glucose, remained almost constants. Therefore, in both hardwoods and softwood, it was hemicellulose but not cellulose that changed in the conjugation with lignin structure and amount. Accordingly, the diversity of hemicellulose was closely related to the structure of lignin.

Relationships between hemicellulose composition and lignin structure of LCC fractions

New fractionation method of wood cell wall components was applied to selected wood species (beech and red pine). Stepwise extraction (Fig. 1, Scheme I) and individual extraction (Fig. 2, Scheme II) were used to obtain fractions.

Finely ground wood meal was first extracted by aqueous dioxane and DMSO to obtain conventional Bjorkman lignin (MWL) and Bjorkman LCC. The residue was then subjected to successive extraction by DMSO with increasing content of LiCl (0.5 to 3.0%) in order to obtain new LCC fractions which have not been extracted by a conventional method.

By the analysis of conventional MWL and LCC fractions together with new LCC fractions by stepwise extraction, the following results were obtained. 1) In case of beech, syringyl ratio of soluble fractions was always lower than that of insoluble fractions. 2) For both beech and red pine, lignin structure of SS1 fraction (MWL) was quite different from other soluble fractions SS2 to SS9. 3) Fractions with higher lignin content tended to exhibit higher xylan and lower glucan content in neutral sugar composition in beech. In case of red pine, no clear tendency was observed. 4) Fraction SS6 of beech and red pine seemed to be very important. Because from SS6 to SS9, lignin content, xylan content are lower, and glucan content is higher.

In case of individual extraction, the results indicated that red pine was more difficult to be dissolved than beech. Lignin of red pine seems to be more difficult to be dissolved than lignin of beech. For both beech and red pine, solubilization of milled wood by the LiCl/DMSO solvent system was basically controlled by the solubilization of cellulose. However, lignin of red pine seems to be playing more important role to prevent cell wall dissolution than lignin of beech.

Isolation of cellulolytic enzyme lignin (CEL) after pretreatment by lignocelluloses dissolution method

Compared to MWL, CEL was considered to be more representative of the total lignin in wood than MWL due to the higher yield. However, both MWL and CEL need to go through rather extensive ball milling pretreatment. In this chapter, EDA pretreatment before enzymatic hydrolysis was used instead of ball milling as shown in Fig. 3. The results show that after pretreatment and enzymatic hydrolysis, more than 80% of lignin was recovered in the residual fraction (WNLi8E), and no obvious change of the structural characteristics of lignin was observed. The carbohydrate content of WNLi8E is 29.5%. Although this value is higher than that of MWLi6E (usual CEL obtained by the use of ball milling pretreatment), it is quite lower than that of WLi8E (CEL obtained without any pretreatment). It suggested that EDA pretreatment is an effective method for the lignin separation by enzymatic hydrolysis.

Analysis of lignin aromatic structure based on the IR spectrum

17 softwoods and 48 hardwoods were analyzed by IR spectroscopy to examine if lignin syringyl ratio (syringyl/(syringyl+guaiacyl)) obtained by nitrobenzene oxidation analysis can be precisely expressed by area ratios of characteristic peaks of aromatic ring in IR spectrum. Area ratio of two peaks is referred to as that of two wavenumber domains, represented by "wavenumber 1/ wavenumber 2". Examined peak area ratios were 1595/1509, 1509/1460, 1275/1220, 1130/1032 and 835/(855+815). Among these ratios, log(1595/1509) and log(1275/1220) showed significant linear relationship with the syringyl ratios with a correlation coefficient of 0.98 for all 65 woods. These two ratios could also be used to distinguish all the hardwoods from the softwoods.

Preparation, characterization and Photodegradation of epoxy chloropropane modified rice straw

Whole wood dissolution system (dissolution into 6% LiCl/DMSO after slight ball milling) was applied to dissolve modified rice straw obtained by etherification reaction by the use of epichlorohydrin. The purpose of this modification was to convert straw into a thermoplastic material. From IR analysis, neutral sugar analysis and weight percent gain (WPG), it was suggested that the reaction of cellulose hydroxyl group with epichlorohydrin proceeded well as expected. When the modified rice straw was subjected to whole wood dissolution system, 4 hr milling was required to be completely dissolved instead of 2 hr milling which is usually required for the dissolution of wood. Considering that grasses are usually much easier to be dissolved than wood, the necessity for the longer milling time seemed to be due to the matrix formation in cellulose by the reaction with epichlorohydrin. Moreover, Modified rice straw has good photo degradability as well as natural rice straw.

Fig. 1 Scheme I for the isolation procedure of various fractions by the stepwise extraction from milled wood meals

Fig. 2 Scheme II for isolation procedure of various fractions by the individual extraction from milled wood (2 hr)

Fig. 3 The method to obtain enzymatic hydrolysis lignin

セルロース、リグニン、ヘミセルロースの三者は、細胞壁中で物理的・化学的に密接に関連しあって存在し、物理的にも化学的にも強固で安定な木質細胞壁を形成している。リグニンおよびヘミセルロースについては、裸子植物および被子植物では化学構造が異なっていることが古くより知られている。一方、東京大学木材化学研究室における一連の研究は、広範な多様性を示す被子植物リグニンの化学構造上の特徴は、シリンギル比(芳香核に占めるシリンギル核の割合)を指標として統一的に示すことができることを明らかにした。

細胞壁におけるリグニンと多糖類の密接な関係を考えるならば、リグニン構造の多様性と、多糖類、特にヘミセルロースの化学構造の多様性は対応していると考えるのが自然である。本研究は、ヘミセルロースの化学構造上の多様性がリグニンのシリンギル比との対応において記述できるかどうかを検討した。

まず、広範な樹種(広葉樹48種、針葉樹14種)について、多糖類の構造解析と各種のリグニン構造解析を行った。細胞壁中のグルカンの含有量はほぼ40~50%の範囲に見出されるがシリンギル比には影響されなかった。一方、キシラン含有量はシリンギル比の増大とともに上昇する傾向が見られた。シリンギル比が高くなるとともにリグニン量は減少するが、その際にリグニン量の減少を補って上昇するのはセルロースでは無くヘミセルロースであることが示唆された。へミセスロース内の変動を見ると、シリンギル比の増大とともにキシラン/マンナンの比が上昇する傾向が見られた。

この結果は、シリンギル比の高いリグニンは、キシラン/マンナン比の高いヘミセルロースとより密接な関係にあることを示唆するが、この点を検証するために、一つの樹種(ブナ)について細胞壁成分を分画し各フラクションに含まれるリグニンとヘミセルロースの構造の関係を調べた。細胞壁成分の分画には、含水ジオキサン抽出についで、塩化リチウム(LiCl)濃度を段階的に高めたジメチルスルホキシド(DMSO)を用いて順次細胞壁を抽出する方法を用いた。各段階における可溶部と不溶部のリグニン構造を比較すると、シリンギル比の高いリグニンの方が溶解しにくいことがわかった。多糖類では、最初の二段階、すなわち、含水ジオキサン抽出区分とLiClを含まないDMSOで抽出されるリグニン含有量が極めて高い区分を除けば、それ以降の各抽出段階では、不溶部の方が可溶部よりも高いキシラン/マンナン比を示した。これらの結果は、高いシリンギル比を持つリグニンと高いキシラン/マンナン比を持つヘミセルロースがより強い相互作用を有することによって抽出に対して抵抗性を示していることを意味する。

以上で見られた、高シリンギル比と高キシラン/マンナン比の相関は、リグニンとヘミセルロースという互いに独立したポリマー間の相互作用として発現していると考えられた。一方、リグニンとヘミセルロースの間には、独立したポリマー間の相互作用のみではなく、化学結合を含む非常に強固な結合あるいは会合も存在していると考えられている。そのような結合あるいは会合に関与するヘミセルロースの性状を知る上では、単離リグニンフラクションに含まれる多糖の解析が有効である。単離リグニン試料として広く用いられる摩砕リグニン (MSL) はリグニン収率が低い等の理由により、細胞壁中のリグニン試料を代表するものとは言えない。そこで、新しく、摩砕を必要とせず高収率で単離リグニンを得ることのできる方法を開発し、MWLなどの低収率で得られる単離リグニンと比較したところ、キシラン/マンナン比が、単離リグニンの性状の違いを非常によく反映する事が分かった。

このようにリグニンのシリンギル比は、リグニン構造や含有量と相関するのみならず、ヘミセルロースの構造とも相関しており、細胞壁全体の化学的特徴を特徴付ける重要な指標であることが示された。もしシリンギル比が赤外分光スペクトルによって簡便にそして正確に表現できるとすれば、その意義は高い。針葉樹17種、広葉樹48種について、リグニンに関連する赤外スペクトル上のさまざまな波数を検討した結果、シリンギル比は、二つのピーク(1595cm-1と1509cm-1)の面積比の指数、すなわち、log (1595/1509)の一次関数として、0.98と言う高い相関係数で表されることがわかった。アカシア属17樹種、あるいは、ユーカリ属14樹種というシリンギル比の分布範囲が狭いグループ内に限っても、0.90以上の相関係数が保たれた。また、これにより広葉樹と針葉樹は完全に分離された。

このように本研究では、リグニン化学構造の指標としてシリンギル比をとることによって、リグニン化学構造とヘミセルロースの化学構造の間に一定の相関があることを示すことができた。従って、審査委員一同は、本論文が博士(農学)の学位論文として価値あるものと認めた。