【作者】 赵玲；

【导师】 刘荣厚；

【作者基本信息】 沈阳农业大学 ， 农业生物环境与能源工程， 2011， 博士

【摘要】 我国农作物秸秆资源丰富,有效利用秸秆等生物质资源,将其转化为高附加值的沼气及有机副产物是秸秆能源化利用中的一项重要途径。采用厌氧干发酵技术进行秸秆沼气转化由于提高了容积产气能力已经成为秸秆沼气技术的研究热点。然而,农作物秸秆的特点是木质纤维素含量较高,而厌氧微生物的木质纤维素降解消化能力较弱,从而限制了秸秆生物气化的大规模应用。因此,针对原料特点开展秸秆厌氧发酵的预处理技术、干发酵产气工艺及机制、残留物利用等研究对推进秸秆生物气化的产业化进程具有重要意义。本论文以玉米秸秆为研究对象,生物预处理技术为切入点,开展厌氧干发酵产气特性及残留物沼渣基质利用的研究。主要研究内容如下：(1)采用稀释平板法在愈创木酚固体选择培养基、苯胺兰筛选培养基和α-萘酚筛选培养基上,利用定性筛选和定量木质磺酸钙降解试验相结合的方法从林区朽木中分离筛选出漆酶活力较高的木质素降解菌S4,对该菌株的生物学特性和液体发酵产木质素降解酶的条件进行了研究。结果显示菌株S4主要产生漆酶和木质素过氧化物酶,培养最佳碳氮源为葡萄糖和酒石酸铵,培养条件为28℃、pH值为5.0、接种量5%,影响Lac产量的主要因素为酒石酸铵、Tween80和葡萄糖,最优培养基组合为葡萄糖15g/L,酒石酸铵0.2g/L, MnSO40. 01g/L, Tween80 1g/L,优化后Lac产量为279.5U/L,是优化前的1.21倍；影响LiP产量的主要因素为葡萄糖和酒石酸铵,最优培养基组合为葡萄糖15g/L,酒石酸铵0.4g/L,Tween80 0.5g/L,MnS040.015g/L,优化后LiP产量为221.3U/L,是优化前的1.16倍。(2)综合酶产生能力和兼容性试验结果,利用产酶互补的原则将Phanerochaete chrysosporium, Trametes versicolor及与环境中筛选出的能协同降解木质素的菌株S4进行混合培养降解玉米秸秆,通过对温度、含水率、接种量和培养时间等降解条件进行研究,结果表明混合菌降解玉米秸秆较优的培养条件为28℃、含水量65%、接种量5%,此时7d-15d木质素降解速率较快,20d固态发酵的木质素降解率为35.75%。将混合菌用于玉米秸秆厌氧干发酵前的预处理,结合木质素的降解和厌氧干发酵启动及30d累积产沼气情况,确定预处理时间为11d,预处理后木质素、纤维素和半纤维素的降解率分别为28.74%、17.74%和28.35%,有效缩短后续发酵的启动时间。利用扫描式电子显微镜和红外光谱分析仪对降解前后的玉米秸秆对比分析,发现玉米秸秆表面形态结构发生明显的变化,红外光谱中的芳香环和醚键吸收减少,说明混合菌对木质素有较强降解作用。(3)在比较不同生物预处理产气效果的基础上,研究了玉米秸秆经混合菌生物预处理后,在温度、粒径、总固体浓度、接种物添加量、C/N不同的条件下,其厌氧干发酵沼气生产过程稳定性及沼气产量的变化。结果表明混合菌预处理玉米秸秆厌氧干发酵效果最好,累积产气量高于堆腐处理组21.2%,青贮后玉米秸秆用于发酵需定期监测并调节基质pH值,否则易引起消化障碍；高温发酵有优势,但能耗大,中温35℃较适合玉米秸秆厌氧干发酵；减小玉米秸秆粒径对厌氧消化具有促进作用,一定范围内粒径越小达到产气高峰的时间越短,但粒径过小影响底物疏松状态,试验条件下粒径为1-3mm的玉米秸秆干发酵效果及产气稳定性最好。总固体浓度对玉米秸秆的厌氧发酵也有显著影响,在保证较高的处理效率的情况下,选择较低的总固体浓度有利于后期产甲烷阶段的进行。在单因素试验基础上,选择混合菌预处理、中温(35℃),粒径1-3mm的条件下,利用响应面法进行进一步的工艺参数优化,确定出最优的工艺条件为原料TS浓度为21.4%,接种物添加量为25.5%,初始C/N为30.2：1,累积产气量相比优化前提高23.15%。最优工艺条件下,100d发酵周期的玉米秸秆干发酵试验,发酵原料TS产气率为0.476L/g,沼气中甲烷体积分数平均为50.13%。优化后工艺条件有效促进了玉米秸秆厌氧干发酵产沼气的进行,为玉米秸秆厌氧干发酵系统的合理设计与运行提供理论依据。(4)通过对玉米秸秆厌氧干发酵残留物的营养成分及污染性指标测试分析,结果表明：以玉米秸秆为原料的发酵残留物中含有丰富的营养性物质,其中沼渣中各种营养成分明显高于沼液,沼渣中全氮、全磷和全钾含量均较高,相对于畜禽粪便发酵后的沼渣相比处于较高水平。对照农用污泥污染物控制标准(GB4284-84),本试验条件下的沼渣中各重金属元素含量均在标准范围内。表明沼渣不仅含有丰富的营养元素,并且可作为一种高效无公害有机基质的原料长期使用。(5)不同配比的沼渣基质容重、含水量、pH值、EC值等指标均在适宜范围内,其中沼渣：蛭石为1：2-3的处理促进根系发育,干物质积累多,光系统(PSⅡ)有较大的光合电子受体库(Sm),可接受较多的电子,有利于在电子传递链中的传递,使光能的最大转化效率(Fv/Fm)及PSⅡ的潜在活性(Fv/Fo)提高。添加一定比例的沼渣后,改变了基质的微生物区系组成,明显提高草莓根际基质中放线菌和细菌的比例,其中沼渣：蛭石为1：2处理中细菌/真菌为对照的3.09倍,放线菌/真菌比例达76.23倍,基质中活性物质的提高改善了草莓根际环境,表现为草莓根系活力比对照提高了19.17%,根系总呼吸速率较对照显著提高47.67%,根系生化呼吸代谢途径向产能较高的TCA呼吸途径转化,为植株良好发育提供能量。(6)干旱逆境下,沼渣：蛭石为1：2基质的草莓植株净光合速率较对照显著提高44.39%,植株的水分利用效率提高,草莓叶片PSⅡ电子传递体未受到明显伤害；低温条件下沼渣：蛭石为1：3处理叶片Pn处于较高水平,但PSⅡ受体侧电子从QA-向QB-的传递受到一定的抑制；强光照条件下沼渣：蛭石为1：2处理光化学性能指数(PIABC)明显高于其他处理,光合电子传递效率高。研究表明沼渣：蛭石为1：2处理的草莓植株抗逆性强,是一种较为理想的栽培基质。更多还原

【Abstract】 It’s universally acknowleged that straw energy-oriented use can be achieved through the transformation of biomass energy crops such as straw into high value-added methane and organic fertilizer,especially in China with her abundant crops straw resources.Currently the dry anaerobic fermentation technology has become a research focus because it can improve the production volume.Howerever,because of the characteristic of high cellulose and the weak ability to digestion for anaerobic microbial,the application and production of straw biogas has been seriously limited.Therefore it’s extremely significant to have a further reseach on pretreatment technology, efficient biogas production of dry fermentation and the depth utilization of the residue.My thesis revolve around corn stalk and bio-pretreatment to expound on the features and the factors of dry anaerobic fermentation.Based on the previous experiment,the conclusion has been reached that the process of mixed bacteria bio-pretreatment corn stalk and dry anaerobic fermentation biogas production.Through the analysis of the composition of fermentation residue, investigating feasibility of using residue as the matrix to cultivat strawberry. The main research contents are as follows:(1) Dilution-plate method is used on the guaiacol solid selective medium、aniline blue screening medium and a-naphthol screening medium,made use of the method that combine the qualitative screening with quantitative calcium lignin sulfonate degradation experiment to separate the lignin-degrading bacteria S4 which laccase activity is higher from rotten wood in the forest, the biological characteristics of this strains and the conditions of liquid fermentation produce lignin degradation enzyme were studied. Through the conditions for enzyme production of carbon source、nitrogen source、mentalion、surfactant、temperature、PH、inoculation amount and incubation time to research, the results show that the best carbon and nitrogen source of strains S4 is glucose and ammonium tartrate, temperature is 28℃、PH is 5.0、inoculation amount is 5% that is the best culture conditions, orthogonal design ascertain three main factors for influence Lac production is ammonium tartrate、Tween80 and glucose, the optimal combination of culture medium is that the glucose is 15g/L, ammonium tartrate is 0.2g/L, MnSO40.01g/L,Tween is 801/L; the main factors to affect Lip production is glucose and ammonium tartrate, the optimal combination of culture medium is that the glucose is 15g/L, ammonium tartrate is 0.4g/L, Tween is 800.5/L,MnSO40.015g/L.(2) Comprehensive the ability of produce enzyme and the results of compatibility experiment,using the principle of enzyme production complementary to make Phanerochaete chrysosporium,Trametes versicolor and strains S4 which could cooperate with degrade lignin screening from environment to mixed cultivation degradation corn stalks, through the degradation conditions of temperature、moisture、inoculation amount and incubation time to studied, the results showed that the culture conditions of mixed bacteria degradation corn stalks is 28℃、water content is 65%、inoculation amount is 5%,this moment, the degradation rate of lignin is faster after 7-15d disposed, the lignin degradation rate of solid state fermentation is 35.75% after disposed 20 days.The mixed bacteria will be used for the Bio-pretreatment before corn stalks dry anaerobic fermentation, combined the situation with the degradation of lignin、start-up dry anaerobic fermentation and produced biogas accumulated in 30d, determined that the time of mixed bacteria Bio-pretreatment corn stalks is 11d, after pretreatment the degradation rate of lignin is 28.74%,the degradation rate of cellulose is 17.74%,the degradation rate of hemicellulose is 28.35%. Using scanning electron microscope and infrared spectrometric analyzer to comparative analysis corn stalks degradation from beginning to end.From scanning electron microscopy we found that the surface morphology structure of corn stalks have change obviously, the aromatic rings and the ether bonds in the FTIR absorbed reduce, which speak volumes for that the mixed bacteria has strong degradation function to lignin.(3) On the basis of comparing gas production effect of different Bio-pretreatment, researched that the corn stalks after Bio-pretreatment with the mixed bacteria, in the different condition of temperature, particle diameter, solid concentration, the inoculum additives, C/N, the changes of stability of dry anaerobic fermentation production biogas and biogas yield, and adapt the Response Surface Methods to make sure that the best condition of dry anaerobic fermentation is the mixed bacteria Bio-pretreatment, temperature (35℃), particle size 1～3 mm, density of TS is 21.4%, the inoculum additives is 25.5%, the initial C/N is 30.2:1.In the optimal technological conditions, make the corn stalks dry fermentation experiment in a 1L reactor in a 100d fermentation period, the cumulative gas production is 31.78L,TS which is the fermentation materials that the gas yield is 0.476 L/g and the volume fraction of methane in biogas is 50.13% on average. It explain that after optimized the process conditions can effectively promote produce methane in the way of stalks dry anaerobic fermentation, and provide the theory basis for the reasonable design and operation of the corn stalks dry anaerobic fermentation system.(4) Though the experiment analysis of nutrient content and pollution index of the residue of corn stalks dry anaerobic fermentation. The results shows that the fermentation residue which the raw material is stalks rich of nutritional substances, among it all kinds of the nutrient content of residue is higher than biogas slurry, in residue the content of total-N、total-P and total-K are all higher, relative to the residue of the poultry feces after fermented in a higher level. Compare contamination control standards in agricultural sludge(GB4284-84),each heavy metal element content of residue in this laboratory condition are all in the standard range. It indicate that residue not only rich of nutrient element, and also can be used long-term used as a kind of raw material of efficient pollution-free organic matrix.(5) The study of making use of anaerobically digested slurry. The different ratio of the anaerobically digested slurry, the index like volume density、moisture content、pH value、EC value are all in a suitable scope, among it will promote the root development when residue:vermiculite is 1:2-3 and dry matter accumulated more, photosystem (PSⅡ) have larger photosynthetic electron acceptor library(Sm)which could accept more electronic, contributed to transfer in electron-transport chain and made the maximum conversion efficiency of light energy (Fv/Fm) and potential activity of PSⅡ(Fv/Fo) improve. The microflora composition of substance is changed after added proportional biogas residue and the presence of actinomycetes and bacterium in the strawberry rhizosphere matrix is improved obviously, in it if residue:vermiculite is 1:2 that bacteria/fungi is 3.09 times of control subjects and the proportion of actinomyces/fungi is as high as 76.23 times, the strawberry rhizosphere environment is improved when active agent of substance improve, it expressed that strawberry root vigor is improved 19.17% compared by control subjects and total root respiratory rate is improved significantly 47.67% compared by control subjects, the root system biochemical breathing metabolic pathway is converted into TCA breath pathway which is full of higher output and improve energy to plants for fine growth.(6) Under drought adverse environmental stresses, when residue:vermiculite=1:2 strawberry plants net photosynthetic rate of substance is improved significantly 44.39% compared by control subjects, increase the efficiency of water of plants usage, strawberry leaves PSⅡtransfer electrons are suffering considerable damage; under low temperature conditions when residue:vermiculite is 1:3 and made the Pn of leaves in high level,but the receptor side electronic of PSⅡtransfers from QA- to QB- has been a degree of restraint; under the condition of strong light when residue:vermiculite is 1:2 PIABS is higher than other processes obviously, the efficiency is high of photosynthetic electronic transfer. The research shows that when residue:vermiculite is 1:2 the stress resistance is strong of strawberry plants and it is an ideal culture substrate.更多还原