【作者】 李文涛；

【导师】 李文哲；

【作者基本信息】 东北农业大学 ， 农业机械化工程， 2013， 博士

【摘要】 近年来我国大中型沼气工程发展迅速,在新能源发展和农村环境改善等方面发挥了巨大作用,然而厌氧发酵剩余物(沼液)存在量大、集中的特点,其无害化消纳已成为制约沼气工程正常运行的瓶颈问题。沼液中含有多种作物所需的水溶性营养成分,速效营养能力强,养分可利用率高,进行田间施用时不仅能提高作物的产量和品质,还可以起到防病抗逆的作用,并且可以改善土壤结构,是一种理想的绿色有机肥料。但是目前沼液防病抑菌的机理和沼液施肥方式对其有效利用的影响都尚不能完全明确,为此本论文对沼液抑菌的机理进行了深入的研究；同时利用沼液暗灌施肥,研究了其对土壤肥力特性和土壤微生物群落结构的影响,并在此基础上提出了沼液暗灌施肥机的总体设计方案,对关键部件分配器进行了设计。获得的研究成果主要有以下几个方面：(1)沼液抑菌机理试验研究1)沼液和沼液的离心上清液均随着浓度的增加,对5种病原菌(立枯丝核菌、雪腐镰刀菌、尖孢镰刀菌、茄腐镰刀菌和核盘菌)的抑制率也逐渐提高,且表现为浓度100%的处理抑制效果最好,但随时间延长抑制率有所下降。100%沼液对立枯丝核菌、雪腐镰刀菌、尖孢菌、茄腐镰刀菌的抑制作用显著,抑制率分别为70%、40%、68%、70%,对核盘菌的抑制效果较差,抑制率为25%；经过灭菌处理100%沼液的抑制作用较小,最高抑制率分别为30%、15%、35%、25%和10%；100%沼液离心上清液的抑制率较100%沼液略小,分别为48%、35%、47%、55%和18%,而100%沼液离心上清液灭菌处理后的抑制率远小于灭菌前,分别为23%、14%、22%、20%和13%。2)为确定沼液中起抑制作用的因子,将沼液进行了5种处理即原沼液、沼液灭菌、离心上清液、无菌膜滤液和菌体溶液,并进行了抑菌试验。抑制效果表现为：菌体溶液最好,其它依次为原沼液、离心上清液、无菌膜滤液和沼液灭菌,菌体溶液的抑制率分别为74%、45%、68%、64%和24%,且抑制作用时间较原沼液长,说明对病原菌起主要抑制作用的是沼液中的微生物。3)对沼液中微生物进行了筛选和纯化,最终获得到了48株细菌、8株真菌和3株放线菌,对其进行了编号,并分别与作物5种病原菌进行了对峙试验,在真菌与病原菌对峙试验中筛选出了2株具有强抑制作用的菌株nfd-9和nfd-16。电镜扫描结果表明：nfd-9的菌丝主要表现为牢牢的缠绕在供试的5种作物病原菌的菌丝上,有些菌丝则是穿过病原菌的菌丝,从空间上限制了病原菌的生长,同时还通过与病原菌竞争生长必需的营养物质,从而达到抑制作用的目的,说明重寄生、竞争作用是nfd-9菌具有抑制作用的一个重要机制。nfd-16的抑菌机制为不断覆盖、侵入各病原真菌的菌落,侵占各病原真菌的生长空间,形成包围圈,阻止其接触到植物根系和叶片表面,从而达到抑制的目的。细菌对立枯丝核菌、核盘菌的抑制效果最好,抑制率在90%以上,对茄腐镰刀菌、尖孢和雪腐镰刀菌的抑制率在70%～80%之间。4)对具有抑制作用的菌株进行分子生物学鉴定,确定菌株nfd-9和nfd-16分别为米根霉(Rhizopus oryzae)和哈茨木霉(Trichoderma harzianum)。拮抗性细菌主要属于芽孢杆菌(Bacillus),其中对5种病原菌均有抑制作用的细菌有6株,沼液中的细菌中以芽孢杆菌(Bacillus)为主。(2)沼液暗灌施肥对土壤肥力特性和土壤微生物多样性的影响1).沼液可不同程度提高土壤的pH、铵态氮、速效钾、有机质的含量。T3处理能够使土壤的pH由初始的7.3提高到7.6左右,提高了4.11%；T2处理次之,可提高3.91%；T1处理最差,仅提高2.61%。3组清水对照组C1、C2、C3的pH基本没有变化；随着沼液施肥量的增加,铵态氮含量也相应提高,T3处理的土壤铵态氮含量最高,为34.52mg/kg,对照处理铵态氮的含量几乎没有变化；而沼液对速效磷的影响小于沼液对土壤氮的影响；T3处理对土壤速效钾的含量提高幅度最大,含量最高时可达到364.42mg/kg,与种植前相比提高了32.55%,其次为T2处理和T4处理,与种植前相比分别提高了26.98%和36.7%；沼液对提高土壤有机质的作用优于复合肥,T3处理的土壤有机质含量达到23.48g/kg,T2处理的土壤有机质含量达到23.13g/kg,T1处理的土壤有机质含量达到22.84g/kg,影响显著。2)随着沼液施用量的增加,土壤中细菌的数量也相应增加,施肥后第一次进行土壤细菌计数时T3处理的细菌数量最多,由32×105CFU/g增加到59.97×105CFU/g,T2增加到53.13×105CFU/g,T1处理的细菌数量达到49.00×105CFU/g,而C1、C2、C3的细菌数量没有显著性变化；土壤真菌数量的变化规律与土壤细菌具有类似性,但增加程度较低。T3处理土壤真菌数量最大时约为31×104CFU/g,提高约40.91%,而T2处理和T1处理分别提高了36.36%和31.81%。复合肥处理的真菌数量略有下降。放线菌的初始数量平均值为23.67×104CFU/g,施肥后最大可达27×104CFU/g左右,与初始值相比增加了26.09%,对照处理和复合肥处理的放线菌数量变化较小,基本在22～24×104CFU/g范围内,且低于沼液处理的数量。3)对土壤细菌的DGGE图谱分析得出细菌主要由27类菌种构成,多样性指数(Dsh)为4.16。沼液施肥后DGGE图谱的条带清晰、明亮,但条带数有所减少,说明沼液施肥降低了土壤中细菌的种类；土壤真菌DGGE图谱中主要的条带共计13条,较清晰明亮,且多数属于共同条带,特异性条带较少,沼液对土壤真菌的影响较细菌影响小。(3)沼液施肥机的设计提出沼液施肥机总体方案为拖拉机牵引沼液罐车提供前进和转弯动力,施肥机具悬挂在沼液罐车后面进行施肥和其他作业。并针对沼液黏度大、易堵塞等问题,设计了转子防堵型分配器,对其原理和结构进行了分析。更多还原

【Abstract】 In recent years, the rapid development of large and medium biogas projects played an important role in domains such as the new energy development, rural environment improvement, and so on. However, the anaerobic fermentation residues such as biogas slurry have the characteristics of continuous, large amount and centralization, its harmless treatment have become bottleneck for the normal operation of biogas projects. Biogas slurry contains a variety of water-soluble nutrients, which are available and high efficient for crops. When applied in field, the biogas slurry could not only improve crop yield and quality, but also prevent disease and improve soil structure. Thus, biogas slurry is considered as an ideal organic fertilizer. However, the antibacterial mechanism of biogas slurry and the way of effective utilization as fertilization are not yet entirely clear. In this research, the antibacterial mechanism of biogas slurry was investigated; impacts of biogas slurry subsurface irrigation on soil fertility characteristics and microbial community in soil were studied. Based on results of above research, the key components of biogas slurry fertilizer machine are designed. The main research results are as follows:(1) Antibacterial mechanism of biogas slurry1) With the concentration increased from20%to100%by step of20%, inhibition rate of both biogas slurry and supernatant on five kinds of pathogens(Rhizoctonia solani, Fusarium nivale, Fusarium oxysporum, Fusarium solani, Sclerotinia sclerotiorum) gradually improve, and the concentration of100%biogas slurry has the best inhibitory effect, but inhibition rate decline with time.100%biogas slurry has significant inhibition on Rhizoctonia solani, Fusarium nivale, Fusarium oxysporum, Fusarium solani, inhibition rates are70%,40%,68%,70%, respectively. Inhibitory rate of Sclerotinia sclerotiorum is poor, only25%.100%sterilized biogas slurry has little inhibition effect, and the maximum inhibition rates were30%,15%,35%,25%and10%, respectively.100%supernatant has tiny inhibition effect, and the maximum inhibition rates were48%,35%,47%,55%and18%, respectively.while the sterilized100%supernatant is far less than before, with inhibition rate of23%,14%,22%,20%and13%, respectively.2) In order to find the inhibitory factors in the biogas slurry, the slurry was pretreated by five methods, namely, untreated, sterilization, centrifugal supernatant, sterile filtration and bacteria solution. Then the antibacterial test was carried out. The result showed that the antibacterial effect: bacteria solution is the best, and the others are untreated, centrifugal supernatant, sterile filtration and sterilization, respectively. The inhibition rate of bacteria solution were74%,45%, 68%,64%and24%, respectively, and the inhibition time are longer than raw slurry. This suggested that the microbial of bacteria solution played a main role in inhibiting pathogens. In addition, the inhibition rate of sterilization was still the smallest.3) After the isolation, screening and purification for the microbe from biogas slurry, finally,8fungi,48bacteria and3strains of Actinomycetes were obtained (No.1-59). Then, the co-culture experiments were respectively conducted using each microbe with the pathogens. As a result, there were two strains showing stronger inhibitory effect to the pathogens was detected, namely, nfd-9and nfd-16. The scan electron microscope observation showed that the mycelia of pathogens was firmly tangled or pierced by the mycelia of nfd-9, thus, the growth space of r. solani was limited. Additionally, nfd-9could rival the necessary nutrients for growth with pathogens, thus playing the inhibitory effect. These results suggested that the mycoparasitism was an important mechanism for nfd-9as antagonistic microorganism. The antibacterial mechanism of nfd-16was constantly to invade the growth space of pathogens, or covered, or intruded into the pathogenic colony, which could form a ring of encirclement to prevent the pathogens get in touch with the plant root and leaf surface, thereby playing the inhibitory effect. The inhibitory effect of Antagonistic bacteria against R.solani and Sclerotinia sclerotiorum was best, and the inhibition rate of above90%. Moreover, the inhibitory rate for Fusarium solani, Fusarium fungi and Fusarium nivale was at the range of70-80%.4) strains are identified by molecular biological technology, strain nfd-9and nfd-16were Rhizopus oryzae and Trichoderma harzianum. Antagonistic bacteria are mainly belonging to bacillus, illustrating huge bacteria in biogas slurry.(2) Soil fertility characteristics and microbial diversity1) Biogas slurry can improve the pH, content of ammonium nitrogen, nitrate nitrogen, available phosphorus, available potassium and organic matter. The pH of T3treatment can increase from7.3to7.6, improved about4.11%, T2is the next, about3.91%, the worst one is T1, only2.61%. The pH of three groups of water control is unchanged. With the increase of biogas slurry, ammonium nitrogen increased. Soil ammonium nitrogen of T3has the highest content,34.52mg/kg, and the control treatment has almost no change. The effects of biogas slurry on available phosphorus is less than nitrogen, and T3treatment has the biggest available potassium content in soil, about364.42mg/kg, improved32.55%compared with the pre-implantation, T2and T4are the next, about26.98%and36.7%, respectively. Biogas slurry has superior and significant effect on soil organic matter to conventional chemical fertilizers. Soil organic matter content of T3, T2and T1is23.48g/kg,23.13g/kg and22.84g/kg, respectively.2) With the increased of biogas slurry, the number of bacteria also corresponding increase, the number of bacteria of T3after fertilization is the most, counting up by32×105CFU/g to59.97×105CFU/g, T2is to53.13×105CFU/g, and T1reach49.00×105CFU/g. But the control did not change significantly. The number of soil fungi has similar laws with soil bacteria; however, the level of increase is low. The largest number soil fungus of T3is approximately31×104CFU/g, an increase of about40.91%, and T2and T1increased about36.36%and31.81%. Conventional chemical fertilizers reduce slightly the number of fungi. The maximum number of actinomycetes is about27×104CFU/g, increased by26.09%, compared to the average initial number of actinomycetes of23.67×104CFU/g. Actinomycetes of control treatment Conventional chemical fertilizers has almost no change, which are between22×104CFU/g and24×104CFU/g, and less than biogas slurry tretments.3)27categories bacterial strains are derived from the DGGE profiles of bacteria and actinomyces in soil. The diversity index is4.16, slightly higher than in the control treatment, and evenness indexes are basically the same. Biogas slurry significantly reduced species of bacteria in the soil.13major bands are derived from the DGGE profiles of soil fungi, and these bands are clear and bright, belonging to the common bands, lack of peculiar bands. Biogas slurry has little impact on fungi compare with bacteria.(3) Design of biogas fertilizer machineThe total design scheme is that forward and turning power of biogas slurry tanker is provided by tractor, while fertilization and other operations are carried out by fertilizing machine behind the tanker. An antiblocking type distributor is designed to slave the problem of high viscosity and blockage of the biogas slurry, and its principle and structure are discussed.更多还原