摘要
木质纤维素原料复杂的结构抗性,极大地限制了纤维质乙醇的转化效率。虽然环境友好、低能耗的白腐菌生物预处理技术可降低原料酶解抗性屏障,增强乙醇转化效率,极具应用前景。但是,预处理效率仍有待提高,白腐菌预处理如何促进酶解反应的机制也亟待深入解析。因此,本论文以新型预处理菌株乳白耙菌(Irpex lacteus)为研究对象,通过研究预处理过程中的关键影响因素,建立了简单高效的生物预处理玉米秸秆转化乙醇新技术,并明晰关键因子Mn2+对生物预处理的影响机理;在此基础上,进一步利用分子结构表征技术,从木质素结构、木质纤维素结构及其与纤维素酶相互作用等方面研究了关键因子Mn2+添加前后产生的结构及反应差异,揭示了生物高效预处理技术如何通过降低木质纤维素结构抗性促进酶解反应的作用机制。
系统研究了环境及添加因子对乳白耙菌预处理的影响,发现Mn2+是促进乳白耙菌生物预处理效率的关键因子,由此建立了简单高效的白腐菌生物预处理秸秆转化乙醇新技术:即在玉米秸秆中添加0.01mM/g MnSO4进行生物预处理28天后,葡萄糖产量达到308.98mg/g秸秆,乙醇产量达到144.03mg/g秸秆,为当前同类研究的最高水平。对不同预处理条件下的玉米秸秆组分变化、酶反应性和乙醇转化的相关性研究表明,预处理后木质素组分的变化与秸秆酶解增效密切相关。
乳白耙菌产酶特性和及其降解木质素结构类似物的研究结果表明,锰过氧化物酶(Manganese Peroxidase, MnP)在生物高效降解和改性木质素过程中起关键作用。Mn2+一方面提高乳白耙菌胞外MnP的催化活性,另一方面增强处理过程中乳白耙菌产生自由基的能力,超氧阴离子自由基较单独生物培养体系提高3.25倍,从而促进对木质素的选择性生物改性。
利用红外光谱、核磁共振、热裂解气质联用等表征手段,对添加Mn2+和未添加Mn2+预处理条件下生物改性木质素结构进行差异性分析。结果表明,β-0-4醚键、羟基、甲氧基、苯环等木质素关键结构的生物改性与乳白耙菌预处理酶解增效密切相关。Mn2+促进了预处理过程中木质素关键结构的生物改性,较未添加Mn2+预处理,β-O-4醚键的含量降低50%左右,羟基、甲氧基含量进一步降低,紫丁香基与愈创木基含量降低30%以上,从而导致木质素大分子显著解聚,木质素苯环侧链的修饰增强,进而促进木质素苯环的断裂,使木质素网状结构解体。
进一步研究木质素生物改性、秸秆基质理化性质和基质与酶相互作用规律之间的关联。结果表明,乳白耙菌预处理使玉米秸秆比表面积增加49.51%,亲水性增加,从而降低纤维素酶解的空间位阻。而在添加Mn2+的高效生物预处理过程中,乳白耙菌通过木质素关键结构生物改性的增强,进一步破坏木质素大分子的网状结构;较未添加Mn2+体系,高效生物改性后的玉米秸秆在15-30nmm、60-100nm范围内孔径分布增多,亲水性进一步增加,进而导致木质纤维素—纤维素酶的吸附率增加了15.38%。添加Mn2+的高效生物预处理使改性后的玉米秸秆近乎完全解除酶解的抗性屏障,实现生物改性秸秆的高效酶解和乙醇转化。
本论文从白腐菌预处理玉米秸秆酶解增效现象出发,以关键性因子为媒介,通过差异性分析,明晰玉米秸秆结构生物改性与酶解增效的关键靶点。不仅建立简单高效的生物预处理体系,更为提升酶解糖化效率提供结构上的关键改性位点。阐明木质纤维素酶解抗性的关键性因素,明确木质素关键结构改性与酶解增效之间的关系,为构建人工改性木质素增强底物转化效率提供理论基础。
The complex structural recalcitrance of lignocelluloses is the key factor to inhibit the efficiency of bioethanol production. White rot fungal pretreatment is an environmental friendly and low energy comsuption technology, which can reduce the resistance of lignocelluluose to enzymatic hydrolysis and enchance the efficiency of bioethanol conversion. Fungal pretreatment has a great potential in the production of biofuels from lignocelluloses. However, the mechanism of biological pretreatment to improve the hydrolysis of lignocelluloses is not clear and the efficiency of fungal pretreatment is still to be improved. In this study, a simple and efficient bio-pretreatment was established by investigating the key factor to biological pretreatment using the novel white rot fungus Irpex lacteus, and the mechanism of the key factor Mn2+to the biological pretreatment was clarified; on the basis, by analysising the differences of structures of lignin and lignocelluloses and the interactions between cellulase and substrates under different pretreatment conditions, it was clarified that the mechanism of the reduction of structural resistance to improve the efficiency of enzymatic hydrolysis by biological pretreatment.
According to the study of effects of environmental factors and additives to biological pretreatment using/. lacteus, it showed that Mn2+was the key factor to promote the efficiency of fungal pretreatment. An novel and efficient technology to produce bioethanol from lignocelluloses was established:the efficiency of biological pretreatment has been achieved the highest level in the current studies, the yield of glucose and ethanol from the pretreated corn stover were308.98mg/g and144.03mg/g respectively with additive of0.01mM/g MnSO4in substrates. Moreover, the correlation of component composition of corn stover, reaction of cellulases and the production of ethanol was also investigated, results showed that the efficiency of enzymatic hydrolysis was closely related to the the content of lignin in pretreated corn stover.
The determination of extracellular enzymes from I. lacteus during pretreatment and the degradation of lignin model compounds by I. lacteus showed that manganese peroxidase played an important part in the process of biodegradation and biomodification of lignin. The supplement of Mn2+improved the activity of MnP from I. lacteus. On the other hand, the ability of I. lacteus to generate free radicals was also enhanced during biological pretreatment. The production of superoxide anion radical was enhanced by3.5-fold compared with conventional biological pretreatment, thereby promoting the selective modification of lignin during fungal pretreatment.
Structural characteristics of pretreated lignin with Mn2+present and absent during biological pretreatment were evaluated by the technologies of infrared spectroscopy, nuclear magnetic resonance spectroscopy and pyrolysis-gas chromatography/mass spectroscopy, etc. Results showed that the improvement of enzymatic hydrolysis after fungal pretreatment was closely related to deconstruction of key structures of/β-O-4ether bond, hydroxyl group, methoxy group and phenyl ring in lignin. Mn2+promoted the process of lignin bio-modification during pretreatment using I. lacteus. In comparsion with the structural alteration of pretreated lignin with Mn2+absent in biomass, the content of β-O-4ether bond decreased by50%, hydroxyl and methoxy group were further reduced and more than30%of guaiacyl lignin and syringyl lignin was deconstructed. Structural modification of lignin resulted in a significant depolymerization of macromolecule lignin and enhancement of modification to side chain of phenyl ring in lignin, which was contributed to the deconstruction of phenyl ring in lignin and decompostion of the network structure of lignin.
The relationships of structural modification of lignin, physical and chemical characteristics of corn stover and the interaction between cellulase and substrates were further investigated. Results showed that the suface area of corn stover was increased by49.51%and hydrophilicity of corn stover was also increased after pretreated by I. lacteus, thereby reducing the steric hindrance of corn stover to enhance hydrolysis of cellulose. The network structure of macromolecular lignin was further destructed caused by the enhancement of modification to the key structure of lignin in the efficient biological pretreatment with Mn2+present in substrates. In comparasion to the pretreated corn stover with Mn2+absent in substrates, there was an increase of the pore size distribution within the range of15-30nm and60-100nm and the hydrophilicity of corn stover was further increased, causing the improvement of adsorption properties of corn stover to cellulase, the adsorption of cellulase increased by15.38%. Supplement of Mn2+in corn stover has greatly improved the efficiency of biological pretreatment. The recalcitrance of corn stover to hydrolysis was almost completely removed, which greatly promoted the efficiency of enzymatic hydrolysis and ethanol production from biopretreated corn stover.
This research was established on the basis of improvement of enzymatic hydrolysis with the supplement of key additive during biological pretreatment, which clarifying the relationship between enhancement of enzymatic hydrolysis and structural modification of corn stover. A simple and efficient biological pretreatment was established, moreover, the key target of structural modification of corn stover to improve efficiency of hydrolysis was also investigated. In this work, the key factor of the resistance of lignocelluloses is clarified and the relationship between enzymatic hydrolysis and structural modification of lignin was elucidated, which provides a theoretical basis for the artificial modification of lignin to enhance the efficiency of bioethanol production from lignocelluloses.
引文
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