【作者】 王欣宇；

【导师】 付玉杰；

【作者基本信息】 东北林业大学 ， 植物学， 2020， 硕士

【摘要】 木质纤维素类生物质是自然界中最丰富的可再生资源,也是唯一存在的能够同时为人类提供液体、气体以及固体燃料的可再生资源,并且可以利用木质纤维素作为原料生产多种产品,有重要的社会经济效益。在自然界中,纤维素的降解主要通过微生物完成,微生物可以产生纤维素酶,其中真菌是目前发现的对纤维素降解最有潜力的微生物。因此本论文以筛选具有降解纤维素功能的红豆杉内生真菌为主要研究内容。本研究由健康的东北红豆杉根、茎、叶中分离纯化得到了 164株内生真菌,通过纤维素刚果红透明圈实验初筛得到六株有降解纤维素能力的内生真菌,又通过测定纤维素酶活力对这六株菌株进行了复筛,筛选出一株产纤维素酶活性最高的菌株,并结合形态学和分子生物学特征对其进行了鉴定。对筛选出的产纤维素酶活性最高的菌株进行产酶条件优化,之后在最佳产酶条件下考察其金花葵秸秆废弃物中纤维素的降解效果,为其在降解农林废弃物的应用提供科学依据。研究结果如下:1、筛选出一株产纤维素酶活力最高的东北红豆杉内生真菌,并对其进行分类鉴定:利用纤维素刚果红透明圈实验和纤维素酶活力测定实验,筛选到一株高产纤维素酶的东北红豆杉内生真菌R4。结合形态学和分子生物学,鉴定出该菌株属于青霉菌Penicillium oxalicum,并将菌株R4作为后续实验菌株。2、优化了培养基氮源、pH、培养温度、培养时间等条件,确定了内生真菌R4产纤维素酶的最佳发酵培养条件。最佳工艺参数为:使用PDB培养基,以1%金花葵废弃秸秆粉末为碳源,以硫酸铵为氮源,发酵初始pH为5.0,在34℃,160 rpm条件下培养77 h。在此工艺条件下对内生真菌R4进行培养,其纤维素酶产率最高,滤纸酶活力、内切葡聚糖酶活力、β-葡萄糖苷酶活力分别可以达到1.45 U/mL、5.27 U/mL、6.34 U/mL。3、考察了 R-4菌株对金花葵秸秆废弃物中纤维素降解效果。在液固比20:1条件下,考察和对比了 R4对纤维素降解的效果,研究结果表明:R4菌株对纤维素的降解非常有效,其降解率可达57.11%,此时还原糖含量达到26.95%,红外光谱结果表明R4菌株处理过的金花葵秸秆粉末中有部分纤维素的内部官能团受到破坏,表明R4具有特异性降解纤维素的能力。综上,本论文首次筛选获得一株特异性降解纤维素的东北红豆杉内生真菌,并用其处理天然木质纤维素原料金花葵秸秆粉末中的纤维素,其纤维素的降解率较高且对半纤维素和木质素几乎无影响。该方法具有耗时短,反应条件温和,环保,成本低,易于工业化等优点,因此,红豆杉内生真菌R4是一株有望于工业应用的菌株,为农林废弃物的有效利用提供了新思路。更多还原

【Abstract】 Lignocellulosic biomass is the most abundant renewable resource in nature and the only renewable resource that can provide liquid,gas and solid fuel at the same time,and lignocellulose can also be used as raw material to produce a variety of products,has important economic benefits.In nature,cellulose degradation is mainly accomplished by microorganisms,which can produce cellulase,among which fungi are the most potential microorganisms for cellulose degradation.This study separate 164 strains from health root,stem and leaf of Taxus cuspidata S.et Z,and six strains which can degrade cellulose were obtained through preliminary screening by using cellulose Congo red culture medium,and through the determination of cellulose enzyme on the six strains after screen,select one of cellulose enzyme activity of the highest production strain,and combining the characteristics of morphology and molecular biology,has carried on the appraisal.The selected strain with the highest cellulase activity was optimized for enzyme production conditions,the degradation effect of cellulose on straw waste from Hibiseu manihot L.was investigated under the optimum conditions of enzyme production,it provides scientific basis for its application in degradation of agricultural and forestry waste.The results were as follows:1.A strain of endophytic fungus of Taxus cuspidata S.et Z cuspidate with the highest cellulase activity was screened and identified.R4,a strain of endophytic fungus of Taxus cuspidata S.et Z.cuspidate with high yield of cellulase was screened by means of the experiment of cellulose cone red transparent circle and the experiment of cellulase activity determination.In combination with morphology and molecular biology,R4 was identified as a strain of Penicillium oxalicum,and it was used as a follow-up experimental strain.2.The conditions of nitrogen source,pH,culture temperature and culture time were optimized,and the optimum fermentation and culture conditions for cellulase production by R4 were determined.The optimal process parameters are:Using PDB medium,1%waste straw powder of Hibiseu manihot L.was used as carbon source and ammonium sulfate as nitrogen source.The initial pH of fermentation was 5.0.The culture was conducted at 34℃ and 160 rpm for 77 h.The cellulase yield of endophytic fungus R4 was the highest,with FPase of 1.45 U/mL,CMCase of 5.27 U/mL,β-GLase of 6.34 U/mL.3.The effect of R4 on the degradation of cellulose in the straw waste of Hibiseu manihot L.was investigated.Under the condition of the liquid-solid ratio is 20:1(v/w),investigated and compared the effect of R4 degradation of cellulose.The results show that R4 for the degradation of cellulose is very effective,the degradation rate can reach 57.11%,the reducing sugar content of 26.95%,the FTIR results shows that partial internal functional groups of cellulose are damaged also suggests R4 has the ability of specific degradation of cellulose.In summary,this paper firstly screened and obtained an endophytic fungus of Taxus cuspidata S.et Z.that specifically degrades cellulose,and used it to treat cellulose in the straw powder of Hibiseu manihot L.,the raw material of natural lignocellulose.The degradation rate of cellulose was high and almost had no effect on hemicellulose and lignin.This method has the advantages of short time,mild reaction conditions,environmental protection,low cost and easy industrialization,etc.Therefore,the endophytic fungus R4 of Taxus cuspidata S.et Z.is a strain that is expected to be used in industry,providing a new idea for agricultural and forestry waste treatment.更多还原