【作者】 刘丽；

【导师】 方萍；

【作者基本信息】 浙江大学 ， 植物营养学， 2015， 博士

【摘要】 生物有机肥(Bio-organic fertilizer,BOF)是一类兼具微生物肥料和有机肥效应的新型肥料,是可持续农业的重要生产资料。然而,目前我国生物有机肥产业尚处于起步阶段,基础研究薄弱,市场上BOF产品质量良莠不齐问题十分突出。BOF品质的高低决定于其所含的功能菌数量及活性。因此,研究探讨BOF制备、加工和贮存工艺对功能菌数量的影响机制对于提高其品质至关重要。为此,本文以枯草芽孢杆菌(Bacillus subtilis)F2为功能菌,在对液体培养基进行优化以提高F2活菌密度的基础上,采用平板计数并结合分子生物学手段,研究了二次发酵和吸附制备法、加工工艺及包装方式等对BOF中功能菌数量和微生物群落结构的影响,并通过草莓盆栽试验探讨了BOF的促生作用,取得主要结果如下:1.菌株F2的摇瓶培养对比试验表明,浓缩味精废液(CMGW)的80倍水稀释液(即CMGW培养基)优于肉汤(NB)培养基;为进一步提高菌液的F2活菌密度,通过正交试验对CMGW培养基各类养分进行优化,结果表明在其基础上添加牛肉膏 1.0g/L、蛋白胨4.0g/L、MnSO4·2H20 0.5g/L、H3BO3 0.02 g/L、FeSO4·7H2O 0.1 g//L、MgSO4·7H2O 0.5g/L所培养的F2菌悬液活菌密度分别为CMGW和NB培养基的2.9倍和6.3倍。2.正交设计的二次发酵试验表明,1%接种量和添加CMGW可提高二次发酵后BOF中功能菌F2的芽孢数;添加CMGW和桑枝屑,并调节含水率至60%可促进物料升温并缓解BOF的pH偏高。3.通过CMGW添加与否的二次发酵对比试验发现CMGW作为营养助剂能显著促进F2在BOF中的增殖并提高其优势地位。变性梯度凝胶电泳和illumine-MiSeq测序分析显示,二次发酵前后BOF的微生物群落结构变化明显,添加CMGW可提高BOF的种群丰富度并使其微生物群落结构更稳定。4.包装方式对比试验结果显示,吸附法制备的BOF采用塑料自封袋包装经7m(月)贮存,其F2活菌数显著高于真空包装,且F2活菌丰度的变异系数更小。5.烘干工艺试验结果表明,适当提高烘干温度并缩短烘干时间有利于提高吸附法制备的BOF贮存7m后的F2活菌数、芽孢数及其在贮存中的稳定性。6.对吸附法制备的BOF进行化肥添加试验,结果表明,添加尿素至BOF含氮量为5%时F2数量显著提高,但含氮量≥7.5%时F2数量显著降低;添加尿素会加剧BOF在贮存中的N素损失,并导致BOF的pH偏高、发芽指数下降。7.草莓盆栽试验结果表明,施用以F2为功能菌的BOF能显著提高草莓的生物量和N、P、K含量及积累量;平板计数和T-rflp检测证明F2在草莓根际定殖;BOF处理的根际微生物群落结构与有机肥(OF)处理差异明显,其丰富度指数、均匀度指数和多样性指数均显著低于OF。更多还原

【Abstract】 Bio-organic fertilizer(BOF)is an emerging fertilizer with both effects of microbial fertilizer and organic fertilizer,serving as an important producing mean in sustainable agriculture.However,the domestic BOF industry is still in the initial stage,and the weekness and insuffcient of basal research always results in spotty and instable quality consequently.The quality of BOF and its effect as the microbial fertilizer depends on the quantity and activity of its functional bacteria.Hence,it is very critical for the improvement of BOF quality to study the influence mechanism of BOF preparation,processing technology and storage on the population of functional bacteria.Based on the optimization of liquid fermentation medium,Bacillus subtilis F2,as the functional bacteria of BOF,was traced in this research with plate counting and molecular biology methods,while the microbial community structure was also studied with the molecular biology methods,in order to explore the mechanism of the variation of functional bacteria population and microbial community structure of BOF which prepared by secondary composting or adsorption followed by different processing and packaging technologies.Furthermore,a strawberry pot experiment was conducted to investigate the growth-promoting effect of BOF.Here are the main achievements.1.The contrast experiment of shaking flask culture suggested that the CMGW medium,the 80 fold-dilution of concentrated monosodium glutamate waste(CMGW),can substitute for nutrient broth(NB)medium to culture strain F2.To further improve the F2 density in medium,the CMGW medium was optimized by the orthogonal experiment as follows:CMGW 12.5 g/L,beef extracts 1.0 g/L,peptone 4.0 g/L,MnSO4·2H2O 0.5 g/L,H3BO3 0.02 g/L,FeSO4·7H2O 0.1 g/L and MgSO4·7H2O 0.5 g/L.The viable cells density of F2 suspension in the optimized CMGW medium was 2.9 and 6.3 times as high as the CMGW medium and NB medium,respectively.2.The orthogonal secondary composting experiment for preparing BOF indicates that the 1%inoculation and the application of CMGW increased F2 spores population in BOF,and the addition of CMGW and mulberry branch sawdust and the adjustment of moisture content to 60%significantly increased the material temperature and alleviated the excessively high pH.3.The contrast experiment of CMGW application or not in secondary composting for preparing BOF inferred that CMGW as nutrient additive significantly promoted the F2 multiplication and enhanced its dominance in BOF.Results of denaturing gradient gel electrophoresis(DGGE)and illumine-MiSeq sequencing revealed that the microbial community structure of BOF varied obviously after secondary composting,and the addition of CMGW contributed to the change by improving species richness and enhancing the stability of microbial community structure of BOF.4.Two packaging technologies were implemented on BOF prepared by absorption in storage experiment.The results showed that the population of F2 viable cells was significantly larger in ordinary plastic package than that in vacuum package,and the variable coefficient of F2 viable cells abundance was smaller in ordinary package.5.Consequences of drying technology experiment of BOF prepared by absorption implied that raising heating temperature and decreasing heating time properly could increase the quantity of F2 viable cells,F2 spores and their stability during storage.6.Experiments of adding chemical fertilizer to BOF prepared by absorption indicated that the addition of urea significantly raised the population of F2 when the nitrogen content of BOF was 5%,and decreased it when the nitrogen content of BOF was no less than 7.5%.Besides,the addition of urea would enhance the loss of nitrogen content,result in excessively high pH and reduce the germination index of BOF during storage.7.The strawberry pot experiment showed that,BOF prepared with F2 as functional bacteria improved the plant biomass and the N,P,K nutrient content and accumulation;both the plate counting and terminal restriction fragment length polymorphism(T-rflp)analysis results indicated F2 colonization in strawberry rhizosphere;and the rhizosphere microbial community structure of BOF treatment was different with organic fertilizer(OF)treatment,while its Margalef,Pielou,Simpson and Shannon-wiener index were all lower than OF.更多还原