1. Introduction

Currently, attention to the utilization of lignocellulose which account for roughly 20% of the terrestrial feedstock carbon storage as a sustainable feedstock for biorefinery has been growing. Saccharification of cellulose in the lignocellulose by saccharification enzymes, namely cellulase (i.e. CBHs; cellobiohydrolases, EGs; endo-glucanases and β-glucosidase) is a key step of biorefinery of lignocellulose. However, the intrinsic three-dimensional cell-wall structure of lignocellulose composed of cellulose microfibril aggregates linked with a lignin and hemicellulose matrix strongly interferes with the access of cellulase to cellulose (Fig.1) and lowers the efficiency of synergetic interaction of cellualse (Fig. 2).

Therefore the increase of cellulase accessibility to cellulose has been the main technical issue and various pretreatments combined with severe conditions (i.e. high pressure, temperature and dosage of chemicals) are introduced to achieve sufficient saccharification of the cellulose in lignocellulose. However, because economics of biorefinery are strongly affected by severity of pretreatment and dosage of enzyme loading, economic commercial scale process is yet to be proposed. Among a number of studies dealing with these technical and economical issues, the addition of surfactants to saccharification process has been attracting a great deal of attention as a possible solution for above issues.

Most of previous studies in this field have focused on the effect of surfactants on the interaction between cellulase and lignin and reported that surfactants lower the non-biospecific and irreversible adsorption of cellualse onto lignin1), 2). Nevertheless, a number of studies qualitatively assessed the various roles of surfactants on the saccharification, the major role of surfactants controlling the behaviors of cellulase and the saccharification efficiency was not fully understood. Besides, structural effects of surfactants on lignocellulose are rarely discussed so far. Knowing these things is very important to maximize the surfactant's effects on saccharification of cellulose in lignocellulose.

To propose a better way for effective utilization of lignocellulose by surfactants, the author clarified i) the major role of a surfactant on the saccharification, ii) the structural changes of lignocellulose by a surfactant and their effects on cellulase adsorption (equilibrium and rate) and saccharification and proposed iii) the quantitative model which describes effects of a surfactant on saccharification of cellulose in lignocellulose in this work.

2. The major role of Tween 20 on cellulase reaction

2.1 Material and methods

The author prepared lignocellulose of different lignin contents (R: 31, P1: 25, P2: 15, P3:10, P4: 5%) and microcrystalline cellulose (Avicel) as samples. Non-ionic surfactant, Tween 20 was used as a model surfactant. Knowing the adsorption behaviors of Tween 20 on samples is necessary to interpret the reason for the enhancement of cellulose conversion by surfactants. Therefore, batch Tween 20 adsorption was carried out at room temperature for 24hrs. As shown in Fig.2, the author assessed the role of Tween 20 on liquid phase (cellobiose hydrolysis; step IV) and solid surface interaction (cellulose hydrolysis; step I-III), respectively. For liquid phase interaction, batch cellobiose hydrolysis (50℃) with and without Tween 20 was performed. For solid surface interaction, analysis of Tween 20's effects on cellulase reaction is not easy since adsorption and hydrolysis occur simultaneously. Therefore the author examined the effects of Tween 20 on cellulase adsorption and cellulose conversion, respectively. Batch cellulase adsorption experiment (0℃for 6hrs) and enzymatic hydrolysis (50℃ for 72hrs) were carried out.

2.2 Results and Discussion.

As shown in Fig.3, both lignocellulose and pure cellulose showed Langmuir-type adsorption isotherms of Tween 20, which means that Tween 20 is adsorbed to form monomers and that no hemi-micelles or admicelles are formed on the adsorbent surface. Considering that Tween series adsorb on the adsorbent by H-bonding with oxygen ethylene of the hydrophilic head and hydrophobic interaction with hydrophobic tails, it can be said that adsorbed Tween 20 as a monomer on surfaces by H-bonding and hydrophobic interaction and free Tween 20 (monomer and/or micelle) in solution affect the interactions between enzymes and lignocellulose.

Fig. 4 shows the effects of Tween 20 on cellobiose hydrolysis by β-glucosidase (step IV). Under the various enzyme/ substrate ratio, Tween 20 had no effects on liquid phase interaction. Fig. 5 shows the Tween 20's effects on cellulase adsorption on lignocellulose at around 0℃ at which the catalytic activity of cellulase is negligible (without hydrolysis). Tween 20 increased amount adsorbed of cellulase onto both samples P1 (high lignin contents) and P4 (low lignin contents).

Cellulase adsorption (eq.1) and cellulose conversion (eq.2) during hydrolysis were examined using previously reported empirical equations3) as below. During the hydrolysis of cellulose, Tween 20 also increased the cellulose conversion as well as the amount adsorbed of cellulase onto cellulose as shown in Fig. 6a, b.

Analysis using empirical equations showed that Tween 20 increased cellulose conversion rate not by increasing the cellulase activity itself but by increasing the adsorption site (cellulose surface) of cellulase (Fig 6c).

3. Structural effects of Tween 20 on lignocellulose

3.1 Material and methods

To verify the above inference that is Tween 20 increases the accessible cellulose surface, the author treated various samples of different properties (i.e. lignin contents and crystallinity) with different concentration of Tween 20 in a stirred vessel for 24-48hrs and also monitored the macroscopic structural change of specimens. Moreover, x-ray diffraction (XRD) and differential scanning calorimetry (DSC) analysis were carried out for inner structure study.

3.2 Results and discussion

It was found that Tween 20 treatment contributed to the cell wall collapse of most of samples as shown in Fig. 7 except for those with high lignin contents and high crystallinity. Through the XRD analysis, no substantial changes in crystallite size of samples due to cell wall collapse were observed. This means that cellulose elementary fibrils in lignocellulose were rarely affected by Tween 20. DSC analysis showed the changes in pore water contents by Tween 20 treatment. As shown in Fig. 8, Cell wall collapse by Tween 20 contributed to the formation of 10- to 50-nm pores. Moreover, non-freezing bound water, generally considered as pore water which does not freeze due to the intimate hydrogen-bond to a cellulose chain, also increased largely. From the DSC analysis, it can conclude that Tween 20 treatment contributed to water intrusion into cellulose microfibrils in lignocellulose and increase of accessible cellulose surface.

Fig. 9 shows the structural effects of Tween 20 on cellulase adsorption amount and rate.

Cellulse adsorption rate was quantified using the general numerical diffusion model4) for cylindrical particle as below.

Cell wall collapse with Tween 20 treatment not only increased the monolayer saturation amount of adsorbed cellulase about 3-3.6 times (Fig. 9a) but also increased the cellulase adsorption rate (De/r2) about 160 -880 times (Fig. 9b). Considering the dimensions of cellulase (EGI: 10-20 nm, CBHII: 5-10 nm), it is thought that 10- to 50-nm size pores newly formed by Tween 20 treatment are sufficient to enhance accessibility of cellulase into cellulose microfibrils in lignocellulose.

4. Construction of cellulose conversion model with consideration of Tween 20's effect

Through this study the author verified that Tween 20 increased the accessible cellulose surface and it contributed to enhancement of cellulase adsorption onto cellulose. Judging from results obtained, the structural effects of Tween 20 can be shown in Fig. 10. Water intrusion into cellulose microfibrils by Tween 20 exposes cellulose elementary fibrils to cellulase reaction. Hence, the cellulose radius for cellulase reaction is decreased. Cellulose conversion model based on the physical evidences obtained from this work was constructed as below equation.

5. Summary

Although application of surfactants has been attracting a great attention, conventional pretreatment methods are still limited to injection as a supplement of saccharification for pretreated lignocellulose. However, the author showed for the first time that structural effects of Tween 20 contributed to significant enhancement of cellulase adsorption and saccharification. The author believes that a new aspect of Tween 20's roles on saccharification of cellulose in lignocellulose proposed in this work can contribute to expansion of surfactant application field for biorefinery and effective saccharification of cellulose in lignocellulose.

Fig.1 Three-dimensional structure of lignocellulose

Fig.2 Steps for synergetic interaction cellulase(Saccharification)

Fig.3 Tween 20 adsorption isotherms

Fig.4 Effect of Tween 20 on cellobic hydrolysis

Fig.5 Effect of Tween 20 on cellulase adsorption

Fig.6 Effects of Tween 20 on cellulose conversion(a) and cellulase adsorption(b) and Relationship between cellulose conversion rate and parameters related with availability of cellulose to cellulase(c)

Fig.7 Cell wall collapse of lignocellulose by Tween 20 (upper.not treated,down:Tween 20 teeated)

Fig.8 Variation of pore water by Tween 20 treatment (a) Freeze bound water (b)Nonfreeze bound water

Fig.9 Variation of cellulase adsorption (a) Amount (b) Adsorptionrate by Tween 20 treatment

Fig.10 Structural effect of Tween 20

本論文は、「Effects of Lignocellulose Pretreatment Using Surfactants on Enzymatic Saccharification(界面活性剤を用いたリグノセルロースの前処理が酵素糖化に及ぼす影響)」と題し、リグノセルロース系バイオマスの酵素糖化において、非イオン性界面活性剤であるTween20を用いた全く新しい前処理法を提案し、そのメカニズムを一連の実験と数理モデル解析によって定量的に明らかにするとともに、実用化のための具体的プロセスの提示までを行なったものであり、全5章からなる。

第1章は緒論であり、リグノセルロース系バイオマスの酵素糖化において、これまでに提案されている種々の前処理法について、それらの物質収支、エネルギー収支、環境負荷、経済性等を詳細に調査分析し、問題点を整理している。従来の前処理においては、リグノセルロースからのリグニンおよびヘミセルロースの除去、セルロースと糖化酵素の吸着平衡にのみ注目されており、複雑な構造を有するリグノセルロース中のセルロースへのバルク中からの糖化酵素の物質移動についてはほとんど研究がなされていない。そこで、リグノセルロースを高効率に酵素糖化するためには、セルロースに対する酵素のアクセシビリティを改善すること、すなわち円滑な物質移動を実現することがブレークスルーになると着想し、非イオン性界面活性剤Tween20を用いてリグノセルロースの高次フィブリル構造を改変させるという全く新しい前処理法を提案している。そして、本論文の目的を、提案する前処理法において、糖化酵素のセルロースへのアクセシビリティ改善の定量的評価、糖化速度および糖化率を向上させるメカニズムの解明と定量的記述、そして実用プロセスの提案であるとし、本論文の構成を示している。

第2章では、リグニン含有量が異なるリグノセルロース試料を木質バイオマスから調製し、酵素糖化および酵素吸着に対するTween20添加の影響を実験および数理モデルにより検討している。まず、本論文では3種類の酵素の混合物であると言われる糖化酵素セルラーゼを、単成分酵素として扱う簡便化について述べた後に、Tween20およびセルラーゼの種々のリグノセルロース試料への吸着平衡と吸着速度を、それぞれの単成分系および混合系で測定している。そして、Tween20がリグニン表面に吸着することによってセルラーゼの吸着を阻害するブロッキング効果を確認する共に、Tween20の添加の影響はむしろセルラーゼの吸着速度により大きく現れることを実験的に明らかにしている。そして、これまでに提案されている複数の半経験的酵素糖化数理モデルを組合せ、さらに糖化の進行によるセルロースの減少の影響を新たに組込んだ数理モデルを構築し、それを用いた計算結果と実験結果と比較することにより、Tween20の添加による糖化速度の向上の主要因は、リグノセルロース内部のセルロースに対するセルラーゼのアクセシビリティの向上であると結論づけている。

第3章では、第2章で結論付けたことを証明するために、第2章で調製した各種のリグノセルロース試料や市販の結晶性セルロースについて、Tween20添加によるセルロースフィブリルのナノ構造変化とセルラーゼの吸着平衡および吸着速度との関係を体系化している。構造分析としては、セルロースフィブリルの水に浸った状態における比表面積と細孔分布の分析評価、セルロースの還元末端濃度分析などを行なっている。そして、50%程度以上のリグニンを除去したリグノセルロース試料においては、Tween20の添加によりセルロースフィブリルが細分化され、細分化された新しいフィブリル間にセルラーゼが侵入可能な10~50nmの空隙が形成されること、ならびにセルロース還元末端濃度が高くなることを確認している。

第4章は、Tween20添加によるセルロースフィブリルのナノ構造変化を考慮した酵素糖化の数理モデルを新たに構築し、それを用いた計算結果と実験結果との対比からその妥当性を検証した上で、種々の操作条件における糖化速度の予測を行い、当該前処理法の有効性を定量的に論じている。つまり、一定量のリグニンの除去が行われたリグノセルロースにおいては、当該前処理法によって酵素投入量を著しく低減し、かつ糖化時間を短縮することが可能であることを定量的に示している。また、一定量のリグニンをあらかじめ除去する必要性を確認した上で、当該前処理法の実用化のためには経済的な脱リグニン法との組合わせが不可欠であると結論づけている。

第5章は結論であり、本論文の内容を総括した上で、得られた知見に基づいて生物的脱リグニン法と当該前処理法を組合わせた複合前処理プロセスを提案しており、今後の研究課題の整理と将来展望を行っている。

以上要するに本論文は、リグノセルロースの酵素糖化の全く新しい前処理法を提案し、そのメカニズムと有効性を定量的に明らかにしたものであり、リグノセルロース系バイオマスの利活用を飛躍的に高効率化する工学的に高い価値を有し、吸着工学および化学システム工学への貢献は大きい。

よって、本論文は博士(工学)の学位請求論文として合格と認められる。