【作者】 张鑫；

【导师】 高聚林； 青格尔；

【作者基本信息】 内蒙古农业大学 ， 作物栽培学与耕作学， 2022， 博士

【摘要】 为促进寒旱地区还田玉米秸秆的快速腐解,本研究以内蒙古不同生态区55个样品为菌源材料,经限制性继代培养与驯化,筛选适应于内蒙古不同生态区的秸秆低温降解复合菌,探究其生物学特性、菌种组成特性、适应性及稳定性,同时利用宏基因组测序技术进一步阐释其秸秆降解机理;通过在内蒙古不同生态区的田间秸秆降解试验探明秸秆降解菌的作用效果。主要研究结果如下:1.玉米秸秆低温高效降解复合菌系的筛选:以来自内蒙古不同生态区的腐殖质、多年秸秆还田土壤、牛羊粪等菌源样品作为筛选对象,通过富集培养以及低温限制性继代培养技术筛选得到9个复合菌系,以秸秆降解率和酶活性为筛选指标,通过初筛以及复筛,得到具有稳定高效降解玉米秸秆潜力的5个复合菌系,编号为M1、M2、M14、M36和M44。菌系M1中主要由Pseudomonas、Trichococcus、Enterococcus、Devosia组成;菌系M2中主要含有Brevundimonas、Enterobacter、Enterococcus;菌系M14主要由Pseudomonas、Enterococcus、Brevundimonas、Azospirillum组成;菌系M36中主要包括Trichococcus、Enterococcus、Pseudomonas、Nocardioides、Sphingomonas菌属;菌系M44中优势菌为Pseudomona、Brevundimonas、Acinetobacter、Azospirillumr。2.玉米秸秆低温高效降解复合菌系的生物学特性:在15℃条件下,各复合菌系在培养前期OD₆₀₀值迅速进入对数生长期,且具有良好的p H值自我调节能力。随玉米秸秆的分解,各复合菌系滤纸酶、内切葡聚糖酶、木聚糖酶、漆酶和木素过氧化物酶在培养第5 d或10 d时达到峰值,秸秆及其组成成分的降解速率呈前期快、后期慢的变化特征。在温度10～15℃、p H 6～8、装液量40～50 m L条件下,各复合菌系秸秆降解特性保持稳定,连续继代培养5代,复合菌秸秆降解特性及菌种组成均无显著变化,其中菌系M44表现出稳定高效的降解秸秆能力,具有良好的应用价值。3.玉米秸秆低温高效降解复合菌M44的秸秆降解机理:通过对复合菌M44秸秆降解过程中的酶活性、秸秆表观形态及结构进行分析可知,降解前期（1～14 d）,滤纸酶、内切葡聚糖酶和木聚糖酶活性增加幅度较高,烷氧碳、双氧烷基碳显著下降,主要降解纤维素、半纤维素等易分解物质;降解后期（14～28 d）,漆酶和木素过氧化物酶活性增加,芳基碳、羧基碳显著上升,以降解木质素、蜡质、单宁等难分解物质为主。基于宏基因组分析可知,纤维素、半纤维素主要由Pseudomonas、Delftia、Leclercia、Raoultella、Sphingobium、Microbacterium等产生的内切β-1,4-葡聚糖酶、外切β-葡聚糖酶、β-葡萄糖苷酶等纤维素降解酶和甘露聚糖酶、β-1,4-甘露糖苷酶、内切-β-1,4-木聚糖酶等半纤维素降解酶进行降解,木质素主要由Pseudomonas、Enterobacter、Delftia、Chryseobacterium、Sphingobium、Stenotrophomonas等分泌的漆酶、锰过氧化物酶、木质素过氧化物酶、纤维二糖脱氢酶等活性酶进行降解,秸秆木质纤维素的降解是多种微生物分泌多种酶共同作用的结果。4.玉米秸秆低温高效降解复合菌M44的应用效果:秸秆还田配施低温秸秆降解菌可显著提高腐解0～210 d的秸秆腐解速率,促进秸秆木质纤维素的分解,提高秸秆养分释放速率。在腐解结束时,施用秸秆降解菌剂处理下各试验点玉米秸秆降解率、纤维素、半纤维素、木质素降解率分别为58.13%～62.53%、39.38%～47.71%、39.91%～49.96%、30.97%～38.79%,较对照分别增加3.82～8.29、4.24～8.54、4.45～6.97、4.93～10.84个百分点。同时施用秸秆低温降解菌可以提高各试验点0～45 cm土层土壤酶活性,在15～30 cm土层土壤过氧化氢酶、脲酶、碱性磷酸酶、蔗糖酶、纤维素酶的活性较对照分别提高5.84%～9.37%、1.80%～8.47%、3.61%～10.70%、3.67%～11.48%和6.43%～17.31%。更多还原

【Abstract】 To promote the rapid decomposition of corn straw returned to fields in frigid regions,the present study,the microbial consortia suitable for frigid region with straw degradation at low temperature were screened by restrictive transgenerational cultivation and cold domestication technology from the 55 samples that different ecological regions.Then the biological characteristics,microbial composition,corn straw degradation activity,adaptability and stability of microbial consortia were investigated,so as to provide microbial decomposer in frigid region of northern China.Furthermore,the decomposition mechanism of M44 was evaluated by metagenomic sequencing technology.Meanwhile,straw returning degradation experiments in different ecological regions of Inner Mongolia were carried out to verify the application effect of straw degradation microbial consortium.The main results were as follows:1.Microbial consortia with high efficiency degradation straw at low temperature were screened out.9 groups of microbial consortia were isolated from the collected humus,corn straw returned soils,cow and sheep dung from different ecological regions of Inner Mongolia by enrichment cultivation and low-temperature restriction subculture technology.Taking the degradation ratio and enzyme activities as indicators,the microbial consortium M1,M2,M14,M36 and M44 with the potential to degrade corn straw stably and efficiently were obtained through primary screening and re-screening.In which,the M1 mainly included Pseudomonas,Trichococcus,Enterococcus,Devosia,the M2 was consist of Brevundimonas,Enterobacter and Enterococcus,the M14 mainly contained Pseudomonas,Enterococcus,Brevundimonas and Azospirillum,the M36 mainly included Trichococcus,Enterococcus,Pseudomonas,Nocardioides,Sphingomonas,and dominant genera of the M44 were Pseudomona,Brevundimonas,Acinetobacter and Azospirillumr.2.Efficiency degradation characteristics of corn straw at low temperature was identified.The microbial consortia went into logarithmic growth phase in the early stage of culture,and could p H self-adjustmenta at 15 ℃ conditions.With the decomposition of straw,filter paper enzyme,endoglucanase,xylanase,laccase and lignin peroxidase of microbial consortium reached the highest in the culture 5th or 10 th day,and the degradation rate of corn straw and lignocellulose showed the characteristics of fasert in the early stage and slowly in the later stage.Moreover,microbial consortia remained relaticely stable in straw degradation characteristics and microbial composition under temperature 10～15 ℃,p H 6～8,liquid loading 30～50 m L conditions and differen subgenerations.Among them,the microbial consortium M44 showed excellent stability and adaptability to volatile environment,had good application potential.3.Degradation mechanism of microbial consortium M44 with high efficiency degradation straw at low temperature was evaluated.In the early stage(1～14 d),filter paper enzyme,endoglymediase and xylanase activity greatly increase,and the Alkyl-C and di-OAlkyl-C decreased significantly,mainly decomposed cellulose,hemicellulose and other easily decomposable substances.And in the later stage(14～28 d),the activities of laccase and lignoperoxidase increased,and Aryl-C and Carbonyl-C increased significantly,mainly degrading lignin,wax,tannin and other refractory substances.Based on metagenomic analysis,cellulose and hemicellulose were mainly decomposed by associated degradation enzymes that endocut β-1,4-glucanase,exocut β-glucanase and β-glucosidase Glycanase,β-1,4-mannosidase,endonuclease-β-1,4-xylanase secreted by Pseudomonas,Delftia,Leclercia,Raoultella,Sphingobium,Microbacterium,and lignin was mainly decomposed by lignin active enzymes which laccase,manganese peroxidase,lignin peroxidase,cellulose dehydrogenase and others secreted by Pseudomonas,Enterobacter,Delftia,Chryseobacterium,Sphingobium,Stenotrophomonas.It can be seen that the degradation of lignocellulose is the result of the joint action of various enzymes secreted by various microorganisms.4.Application effects of microbial consortium M44 with high efficiency degradation straw at low temperature were expounded.Straw returning combined with microbial decomposetr M44 could significantly increase the straw and lignocellulose decomposition rate,nutrient release ratio in 0～210 d.At the end of decomposition,the degradation rates of corn straw,cellulose,hemicellulose and lignin were processed by RM treatment at each test site were 58.13%～62.53%,39.38%～47.71%,39.91～49.96%,30.97%～38.79%,respectively,and 3.82～8.29,4.24～8.54,4.45～6.97 and 4.93～10.84 percentage points higher than the control.In addition,the soil enzyme activities in 0～45 cm soil layer of each test site could be improved by applying degrading agent of each test site,and the activities of soil catalase,urease,alkaline phosphatase,sucrase and cellulase in 15～30 cm soil layer increased by5.84%～9.37%,1.80%～8.47%,3.61%～10.70%,3.67%～11.48% and 6.43%～17.31%respectively than the control.更多还原