【作者】 王伟；

【导师】 戴玉成；

【作者基本信息】 北京林业大学 ， 森林保护学， 2014， 博士

【摘要】 木质纤维素主要由纤维素、半纤维素和木质素三大部分组成,其可再生且来源广泛,是一种制备生物燃料的理想原料。然而由于植物细胞壁结构的复杂性,致使生物质预处理木质纤维素过程仍存在许多关键障碍。本论文针对白腐菌预处理效率的提升,预处理后化学组分对酶水解的影响及预处理后微观结构的变化如何促进酶水解三个方面展开深入的研究。通过愈创木酚平板变色法和定量测定降解前后木片中木质纤维素含量的变化,本实验从23株白腐菌中筛选出3株高效降解黑杨木质素的菌株：白赭多年卧孔菌Perenniporia ochroleuca D9597,粗毛纤孔菌Funalia trogii C6978和东方栓孔菌Trametes orientalis C6320。基于主成分分析发现18株白腐菌存在3种降解类型：选择性降解木质素；选择性降解综纤维素,但对木质纤维素的降解能力极其微弱；强烈地选择性降解综纤维素。筛选出的3株菌分解黑杨木粉20天后,紫外扫描分析黑杨木粉的乙醇提取液表明其中的木质素被显著分解,木质素的芳香环结构遭到了破坏。三株白腐菌Lenzites betulinus, Trametes orientalis和Trametes velutina及其相应的双菌组合预处理毛白杨4周到12周后,单菌T. orientalis在固态培养12周后,可降解高达58.1%的木质素。经过酶水解后,处理后的样品可释放41.3%的还原糖。总体上,三株白腐菌的单菌培养体系在降解木质纤维素和酶水解木质生物质时要优于它们的双菌培养体系,这是由于双菌培养时有拮抗作用的存在,而单菌培养时降解更多的半纤维素和木质素,从而增强了纤维素对水解酶的可渗透性。不同的营养元素添加到白腐菌T.velutinus固态发酵毛白杨中,添加微量元素组引起最多的木质素降解,且半纤维素和纤维素的降解量最少。TE组经过8周预处理后只剩下12.6%的酸不溶木质素。预处理后8周后的样品经过96小时酶水解后,TE组获得了44%的还原糖得率。此外,经过酶水解同步发酵48小时后,预处理8周后的BM+TE组得到了最多的22%的乙醇得率,是未处理对照组的5倍。从4种褐腐菌和7种白腐菌中筛选出高纤维素酶活菌株(Fomitopsis palustrisC7615),并利用响应面优化其产酶条件。在添加4.46g/L的尿素和27.83μL/L的吐温80培养8天后,获得的实际纤维素酶活是130.45FPU/mL。预测值同实际测定值能够很好的拟合,验证了响应面模型的稳定性和优化因子的可靠性。利用优化后所产纤维素粗酶液对热水处理后的毛白杨原料进行了72小时的酶水解。还原糖得率显示,随着热水预处理温度的提升,还原糖得率越高。180℃的热水预处理后的杨木经过酶水解后,释放出最多的25.15%的还原糖,是未处理原料的1.72倍。白腐菌协同热水预处理毛白杨后在去除半纤维素和保存纤维素方面表现良好。L.betulina C5617协同200℃的热水预处理降解了最多的半纤维素高达92.33%。这表明最初的真菌处理使木质纤维生物质的紧密结构变得疏松,从而促进了热水预处理时半纤维素的降解。当毛白杨经过L. betulina C5617与热水在200℃协同预处理后,获得最高的葡萄糖得率60.26%。协同预处理的协同效应在适中条件下更显著,原因可能是半纤维素的损失促进了杨木的酶水解。在上述研究基础上,当生物预处理后的样品和原料经过添加FeCl3的热水预处理后,T.orientalis和F. palustris协助的FeCl3预处理降解了大量的半纤维素,在180℃时半纤维素损失分别达到98.8%和99.7%。T. orientalis和F. palustris协助的FeCl3预处理在180℃条件下分别得到了84.5%和95.4%的还原糖。一种原因是协同处理引起内在表面积和孔隙度的增加,从而降低了酶在木质素表面的可逆性吸附和增加了酶对纤维素的可渗透性。另一个原因是真菌同FeCl3的协同预处理引起了木质素结构的改变,如亲水性酚羟基含量的改变同样引起可逆性酶吸附的减少。白腐菌固态发酵后的残渣进一步溶解在离子液体中以减弱生物质对酶解转化的抗性。真菌处理后的杨木分别经过100℃和120℃的离子液体预处理后,100℃条件下的协同处理同120℃条件下的单独离子液体预处理和协同预处理释放相似含量的葡萄糖,这表明100℃条件下的协同处理比其它两种预处理方式更优异。真菌预处理的发酵周期延长至8周,12周和16周,而后在100℃条件下再离子液体预处理。当第一步的生物预处理的培养周期超过8周后,协同预处理的水解效率并没有实质性的改善,这表明8周的培养周期的真菌预处理协同后续的离子液体预处理已经足够。白腐菌固态发酵后的残渣进一步选择性地去除木质素和半纤维素来研究其中的木质素和半纤维素含量对后续酶水解的影响。真菌处理后的样品经亚氯酸钠再处理后,木质素含量分布为43.66%-77%且半纤维素降解很少。稀酸处理后真菌样品中,半纤维素从79.97%分布至95.09%且仅有小部分木质素被降解。表明木质素和半纤维的交叉反应被降到了最低。酶水解时,不管在何种纤维素酶负荷下,去除木质素后的样品比去除半纤维素能释放更多的葡萄糖和木糖,这表明真菌处理后的残渣中木质素而非半纤维素对酶水解起着最主要的抑制作用。基于过去文献中所阐述的酸性和碱性预处理的机制和本研究中的发现,可推论出真菌预处理同碱性处理的方式相结合比与酸性处理的方式相结合能发挥更大的协同效应。利用高倍率电镜和化学表征技术对白腐菌固态发酵后的残渣进行了多尺度的可视化表征和分析,以探测真菌预处理促进酶水解效率的背后机制。经过16周的固态发酵后,纤维素微弱减少但是半纤维素和总木质素分别逐渐从18.7%到11%和27.7%到12.5%。纤维素和半纤维素转化率随着培养时间的延长而逐渐增加,从预处理4周时的39%和14.5%分别上升到16周预处理时的50.6%和26.1%。本研究中结果可归纳出酶水解能力增强的部分原因是T.orientalis降解了木质素和半纤维素,而不是纤维素结晶度的变化。当预处理后残渣的酶水解数据与各种预处理后的图像比较后,可清晰地表明白腐真菌改善酶水解效率除了通过半纤维素和木质素的部分降解,也通过侵蚀细胞壁形成新的表层界面,使细胞壁上的微纤维得到更多的暴露。表面侵蚀可以作为解释预处理过程中细胞壁结构变化改善酶水解效率能力的重要原因之一更多还原

【Abstract】 Lignocellulosic biomass is composed of three major components:cellulose, hemicelluloses, and lignin. This abundant renewable resource can serve as ideal feedstock for the biofuels production. However, many crucial obstacles should be overcome due to the complicated structure of the cell wall during the pretreatment process. To overcome these obstacles, thorough investigations on improving the performance of biological pretreatment with white-rot fungi, effects of residues from white-rot fungi pretreatment on enzymatic hydrolysis, and how do ultrostructure changes from white-rot fungal decay improve enzymatic hydrolysis, were carried out in this thesis.Based on agar plate tests,18isolates from23white rot fungi were selected to a subsequent wood block decay test. According to the ratio of Klasson lignin losses to holocellulose losses, three isolates, Perenniporia ochroleucaD9597, Funalia trogiiC697S and Trametes orientalisC6320, showed selective delignification on Populus nigra. Based on primary component analysis, three different types of degradation were found during the fermentation of P. nigraby18white rot fungi:type A represents selective delignification; types B and C can selectively decompose holocelluloses, but type C has a stronger capacity than type B. Investigation was carried out on the UV absorbance spectra of ethanol extracts of wood particles treated by P. ochroleucaD9597, F. trogiiC6978and T. orientalisC6320for20days, and lignin in the treated wood was found to be decomposed markedly.Threewhite rot fungi (Lenzites betulinus, Trametes orientalis, and Trametes velutina) as well as their respective paired cultures were used to pretreat Populus tomentosa for enhanced lignocellulosic degradation and enzymatic hydrolysis. Hemicellulose and cellulose were slightly degraded, while a maximum lignin degradation of58.1%was caused by T. velutinaduring the12-week cultivation. After the pretreated samples were subjected to enzymatic hydrolysis for96h, the reducing sugar released by T. orientalis at week12was as high as41.3%, which was in line with the lignin loss at2.2times the control sample. Overall, the monocultures of white-rot fungi exhibited better degradation and saccharification of woody biomass than their co-culture. This can be attributed to the partial removal of lignin and hemicellulose, with an associated increase of cellulose accessibility to enzymes. In addition, different nutrients were added into the solid fermentation of woody biomass, Populus tomentosa, to improve pretreatment by a white rot fungus, Trametes velutina. Fungal pretreatment supplemented with trace elements resulted in large amount of lignin loss but low degradation of carbohydrate. Only12.6%of Klason lignin was left in the residues pretreated by T. velutina for8weeks supplemented with1%trace elements (TE group). When fungal-pretreated residues were subjected to enzymatic hydrolysis for96h, a maximum reducing sugar yield of44%was obtained from the TE group at the8th week,2.3times higher than that of untreated samples. In addition, the highest ethanol yield of22%was observed by the fermentation of8-week pretreated residues from the basic medium plus trace element group, which was five times more than that of untreated samples.Fomitopsis palustris, screened from11wood rotting fungi, was optimized with a sequential optimizationstrategy to produce the largest amount of cellulase, and the efficiency of the enzyme was evaluated. Based on the Plackett-Burman and Box-Behnken designs, the most significant variables, time, urea, and Tween80were varied for optimizing cellulase production. An optimized result for FPase activity with130.45FPU/mL was achieved for an8-day culture containing4.46g/L of urea and27.83μL/L of Tween80, which experimentally matched well with the predicted value from the model. The obtained crude cellulase was subsequently employed in the saccharification of the poplar wood, Populus tomentosa, which was pretreated with liquid hot water (LHW) at different temperatures. A maximum release of25.15%of reducing sugars was observed after a72-h enzymatic hydrolysis of the180℃-LHW-pretreated poplar wood, which is1.72times higher than that from untreated wood (14.66%), indicating that F. palustrishas a potential to produce cellulase for woody biomass hydrolysis.A novel stepwise pretreatment of combination of fungal treatment with liquid hot water (LHW) treatment was conducted to enhance the enzymatic hydrolysis of Populus tomentosa. The results showed that lignin and cellulose increased with the elevating temperature, while significant amount of hemicellulose was degraded during the LHW pretreatment. A highest hemicellulose removal of92.33%was observed by combination of Lenzites betulina C5617with LHW treatment at200℃, which was almost2times higher than that of sole LHW treatment at the same level. Saccharification of poplar co-treated with L. betulina C5617and LHW at200℃resulted in a2.66-fold increase of glucose yield than that of sole LHW treatment, and an increase (2.25-fold) of glucose yield was obtained by the combination of Trametes ochracea C6888with LHW. The combination pretreatment performed well at accelerating the enzymatic hydrolysis of poplar wood. Fungal treatment followed by FeCl3treatment was also used to improve saccharification of wood ofP. tomentosa. Combined treatments accumulated lignin and slightly degraded cellulose, whereas almost all hemicelluloses were removed. The white rot fungus, Trametes orientalis, and the brown rot fungus, Fomitopsis palustris, both accompanied by FeCl3post-treatment resulted in98.8and99.7%of hemicelluloses loss at180℃, respectively. In addition, the solid residue from the T. orientalis-assisted and F. palustrisassisted FeC13treatment at180℃released84.5and95.4%of reducing sugars, respectively. Combined treatments disrupted the intact cell structure and increased accessible surface area of cellulose therefore enhancing the enzymatic digestibility, as evidenced by XRD and SEM analysis data. Wood residues from fungal cultivation with white-rot fungus were further dissolved in ionic liquid (IL) to mitigate the biomass recalcitrance for enhanced bioconversion. Firstly,4-week fungus-pretreated residues were subjected to IL pretreatment at100℃and120℃, respectively. Synergistic pretreatment at100℃can achieve a similar enzymatic digestibility to sole IL pretreatment and synergistic pretreatment at120℃. Therefore, prolonged fungal fermentation followed by IL dissolution at100℃was further investigated. There was no substantial improvement on saccharification of co-treated samples when bio-pretreatment exceeded8weeks. As high as96%of cellulose conversion was achieved by co-treatment with4-week bio-pretreatment and IL pretreatment at100℃, which was3fold,1.3fold and1.2fold higher than that of untreated samples, sole IL pretreatment at100℃and120℃, respectively. This fungi-assisted IL pretreatment would gain enhanced bioconversion at lower severity with minimal costs.Selective delignification and hemicellulose removal were performed on white rot fungus-pretreated residuesto investigate the effects of lignin and hemicellulose removal on enzymatic hydrolysis.43.66-77%of lignin with small part of hemicellulose were degraded by chlorite treatment, while79.97-95.09%of hemicellulose with little lignin were degraded by dilute acid treatment, indicating that cross effect between lignin and hemicellulose was minimized. In subsequent enzymatic digestion, regardless of the cellulase loading, residues from series-grade delignification released more glucose and xylose than that from hemicellulose removal, suggesting that lignin rather than hemicellulose in fungi-pretreated residues played a dominant role in hindering enzymatic hydrolysis. Based on the fundamental mechanisms of acidic/alkaline pretreatments in literature, it is proposed that fungal pretreatment prefers to integrate with alkaline pretreatment rather than acidic pretreatment to maximize the synergy. This indication would be helpful to optimize and renovate the integrated pretreatment.Multi-scale visualization and characterization of poplar wood cell walls were carried out to elucidate the mechanism behind fungal pretreatment with white-rot fungus, Trametes orientalis C6320. During16-week cultivation, cellulose decreased slightly but hemicellulose and total lignin gradually reduced from18.7%to11%and27.7%to12.5%, respectively. Cellulose conversion increased gradually from39%in4-week pretreatment to50.6%in16-week pretreatment, being consistent with the degradation of hemicellulose and lignin. XRD analysis showed thatthe fungal pretreatment had a negligible effect on the cellulose crystalline, indicating that crystallinity is not responsible for the improved enzymatic digestion. Revealed byultrastructural analysis,especiallyby TEM,it can be concluded that the white-rot fungus pretreatmentachieved the improved enzymatic digestibility bycreating extensive new surface area via etching away cell wall matrix and leaving microfibrils exposed on cell wall structures, in addition to partial hemicellulose and lignin removal.更多还原