【作者】 李玲玲；

【导师】 章永松；

【作者基本信息】 浙江大学 ， 植物营养学， 2010， 博士

【摘要】 本论文针对农业有机废弃物（秸秆和畜禽粪便）处理难、大棚土壤退化严重、生物有机肥货架期短和实际生产中有机肥用量不足等问题,采用实验室和生产相结合的方法,研究了有机废弃物CO2施肥发酵过程对堆肥中抗生素降解、堆肥温度、pH、C、N、微生物和病原菌的影响,并研究了土壤中添加堆肥对抗生素的吸附作用；研究了蜡状芽孢杆菌、枯草芽孢杆菌和EM菌对黄瓜和番茄枯萎病菌的抑制作用；在堆肥结束时,将有益微生物接种到发酵残渣中,施于大棚连作土壤,以番茄（Lycopersicum esculentum Mill）作为供试作物,探讨了残渣对连作土壤的改良和培肥作用及对番茄生长的促进作用,以进一步完善堆肥对大棚退化土壤的修复和培肥技术体系。主要研究结果如下：1.土壤和堆肥中四环素类抗生素（TCs）的提取和HPLC检测方法及其吸附和降解研究用1 mol·L-1 NaCl/0.5 mol·L-1草酸/乙醇（25/25/50,v/v/v）混合溶液可以有效提取土壤和堆肥样品中的土霉素（OTC）、四环素（TC）和金霉素（CTC）等3种四环素类抗生素,其回收率在76.0%-92.5%之间。利用高效液相色谱法（HPLC）对上述3种抗生素的测定条件和方法探索结果表明,采用Agilent Eclipse XDB-C8 （4.6×150 mm,5μm）色谱柱,流动相为0.01 mmo1·L-1草酸/乙腈/甲醇（79/10.5/10.5,v/v/v）,流速1.0 mL·min-1,进样量5μL,紫外检测波长268 nm,以外标法定量,在20 min内就可将它们完全洗脱并达到基线分离；在0-10 mmg·L-1范围内,抗生素浓度与峰面积呈显著的线性关系,相关系数R²均>0.999。吸附试验表明,壤土（Haplaquents）和红土（Kandiudox）和土壤/堆肥混合物对四环素、土霉素和金霉素的吸附均符合Freundlich模型,吸附量随抗生素浓度的增加而增加；3种抗生素在基质中的吸附能力依次为：CTC>TC>OTC；堆肥对抗生素的吸附能力显著高于土壤和土壤/堆肥混合物；红土的吸附能力高于壤土。抗生素在土壤中的吸附受土壤pH、有机碳含量和CEC含量影响。土壤中加入堆肥可以增加对抗生素的吸附,加入的堆肥越多,对抗生素的吸附越多。当加入到土壤中的堆肥使得土壤中有机碳的含量达到5%时,有机质对其吸附的影响占主导地位。四环素、土霉素和金霉素在不同电解质溶液中的吸附趋势为：0.01 mol·L-1 KCl≥0.01 mol·L-1 CaCl2。降解试验表明,抗生素在降解过程中出现可逆的异构化反应,其降解速率除受土壤性质影响外,还与四环素本身的结构和理化性质有关。2.有机废弃物CO2施肥发酵过程堆温、pH、C、N、微生物、病原菌和抗生素的变化连续记录结果表明,采用秸秆和畜禽粪便发酵进行大棚CO2施肥,其堆体升温非常迅速,在第5d时即达到最高值64.5°C。整个发酵过程堆温在50°C以上持续了12 d,55°C以上持续了7d；堆肥第35 d开始,粪大肠菌群值MPN在10^-1-10^-2五之间,均符合国家《粪便无害化卫生标准（GB7959-87）》规定的标准。发酵过程中,pH先迅速上升,在第14 d时达到最高,由初始值7.09上升到8.06；之后逐渐下降,在结束时又略有升高。NH4＋-N浓度也呈先上升后迅速下降态势,最终稳定在0.45 g·kg-1；NO3^--N则由开始几乎为0升高到0.67g·kg-1；有机C含量和C/N比分别降至233 g·kg-1和18/1；堆肥结束时,基质的总C和总N分别损失了69.8%和38.0%。堆肥过程中,细菌数量呈现高-低-高趋势,放线菌和真菌则呈现先升高后降低并最终达到稳定的趋势。达到了粪便无害化卫生标准的要求。发芽指数试验表明发芽率达到98%,另外,堆肥可以迅速降低TCs的浓度。堆肥第7d时,OTC、TC和CTC分别减少了77.6%,77.5%和86.8%。CTC在第14 d,土OTC、TC在第42 d则可被全部降解。试验结果表明该发酵方法实现了有机废弃物的无害化、减量化和资源化。吸附试验结果显示土壤中加入堆肥可以显著提高对四环素类抗生素的吸附能力,降低其环境风险。3.蜡状芽孢杆菌、枯草芽孢杆菌和EM菌对枯萎病菌的抑制作用对峙试验结果表明蜡状芽孢杆菌、枯草芽孢杆菌和EM菌对黄瓜及番茄枯萎病菌具有明显的抑制效果。其中,蜡状芽孢杆菌对4种致病菌的抑制效果最好,抑制率为20%-50%。枯草芽孢杆菌和EM菌对致病菌的抑制率为6.7%-34.8%。孢子萌发试验结果表明,当拮抗菌和EM茵的稀释倍数≤20时,基本可以抑制枯萎病菌孢子的萌发,而浓度为10⁸ cfu.mL-1的拮抗菌和EM茵则可以完全抑制枯萎病菌孢子的萌发。4.发酵残渣接种有益微生物对大棚连作土壤的修复和培肥作用试验选用棚龄为5年的水稻土和棚龄为10年的潮土为供试土壤,番茄为供试作物。结果表明,发酵残渣配施蜡状芽孢杆菌、枯草芽孢杆菌和/或EM菌均可以不同程度地促使退化土壤pH值增加,EC值降低,尤其是同时接种3种微生物的处理效果最明显,这对有效缓解土壤酸化和次生盐渍化起到一定的效果。两种土壤的N、P、K等速效养分和土壤有机质含量显著增加,这可以促进农作物对养分的吸收利用,有利于土壤培肥。同时,改善了土壤微生物区系,促进酶活性提高,对解决连作障碍问题起到一定的作用。盆栽条件下,在添加配施微生物菌剂的残渣之后,番茄植株的株高和鲜重的增加,表明残渣和微生物菌剂可以有效地促进番茄植株的生长和发育。更多还原

【Abstract】 Composting of organic wastes (crop straw and animal manure) is a process of fermentation and microbial decomposition under the conditions of artificial regulation of water, C/N ratio, pH, and ventilation to produce organic fertilizer. It is the fundamental way to achieve the reduction, recycle and safe treatment of the organic wastes. Compost with maturity and stability characteristics can not only improve the soil physical and chemical properties, but also increase crop yields and the quality of agricultural products. This research investigated the effects of fermentation process of organic wastes for CO2 enrichment in greenhouse on the antibiotics degradation and the changes of temperature, pH, C, N, microbial population and pathogenic bacteria in the fermentation medium. After modifying the extraction method of tetracyclines (TCs) in soil and compost, as well as the detection method of TCs using HPLC, the degradation of TCs during composting and the sorption of TCs in soils with addition of compost were studied. The reaserches on the inhibition and mechanism of antagonistic bacteria on cucumber and tomato were also carried out by a series of experiments such as sitzkrieg testing of antagonistic bacteria to pathogenic bacteria, the effect of fermentation residues inoculated with antagonistic bacteria on improving soil fertility and promoting crop growth and so on. The main results obtained from experiments are as follows:1. Modification on the extraction method of TCs in soil and compost and determination method using HPLCThe Oxytetracycline (OTC), Tetracycline (TC) and Chlortetracycline (CTC) in soil and compost could be extracted by a mixture solution of 1 mol·L-1 NaCl/0.5 mol·L-1 oxalic acid/ethanol (25/25/50, v/v/v), with the recoveries from 76.0% to 92.5%. The HPLC method was used to determine the three TCs at the same time. The results indicated that the three TCs could be completely separated in 20 min by a column of Agilent Eclipse XDB-C8 (4.6×150 mm,5μm). The mobile phase was 0.01 mol·L-1 oxalic acid-ACN-MeOH (79/10.5/10.5, v/v/v) at a flow rate of 1.0 mL·min-1. The injection volume of sample solution was 5 pL and the UV detector wavelength was at 268 nm. An external standard method was employed for the quantification. The linear correlation coefficients for three TCs under the concentration of 0-10 mg·L-1 were all larger than 0.999.The sorption experiment showed that the adsorption of OTC, TC and CTC to Haplaquents, Kandiudox, and/or mixtures of compost and soils fit Freundlich model well. The adsorption quantity increased with the increasing concentration of TCs. The adsorptivity of TCs in matrices was in sequence:CTC>TC>OTC. The adsorptivity of TCs in compost was significantly in soils and mixtures of compost and soils. The adsorptivity of Kandiudox was higher than that of Haplaquents. The adsorption of TCs in soils was affected by pH, content of organic carbon and content of CEC. Compost added into soils could enhance the sorption of TCs. The more compost was added, the higher sorption of TCs happened. When the OC content in the soil amended with compost reached 5%, the influence of OC and CEC on sorption became dominant. The trend of sorption of TCs in different electrolyte solutions was 0.01 mol·L-1 KCl≥0.01 mol·L-1 CaCl2. Degradation test showed that the reversible isomerization reaction occurred to TCs. The degradation rate was affected not only by characteristic of soil, but also by the structure and characteristic of tetracycline.2. The changes of temperature, pH, C, N, microbial population, pathogenic bacteria and antibiotics in the medium during CO2 enrichment by fermentation of organic wastesIt was showed by the results of temperature continuous record that the temperature of fermentation medium increased very quickly. It reached the highest temperature of 64.5℃at the 5th day of fermentation. Finally, the temperature higher than 50℃was lasting for 12 d, hiher than 55℃for 7 d. The quantities of fecal coliform group were between 10-1-10-2 after 35 days’composting, which completely met the demand of hygienic standard for innocent treatment of feces (GB7959-87).At the beginning of compost, decomposition occurred to the organic waste due to the microbial activity. NH4+ and NH3 were produced, which resulted in the pH rising rapidly from 7.09 to 8.06 of the maximum at the 14th day. After that, pH decreased gradually, and it increased slightly again to 7.53 at the end of composting. The concentration of NH4+-N also increased first and then decreased rapidly, stable at 0.45 g·kg-1 at last. The concentration of NO3--N reached to 0.67 g·kg-1 finally and the content of organic carbon was 233 g·kg-1. The C/N ratio declined to 18. At the end of composting, the losses of total C and N were 69.8% and 38.0%, respectively.During composting, the trends of changes in bacteria, actinomycetes and fungi were slightly different. The number of bacteria presented in a trend of increase, then decrease and increase again, while the trends for actinomycetes and fungi were increase first, then decrease, and at last reached a plateau. Experiment of germination indices (GI) showed that the GI of fermentation residues reached 98%. In addition, the tetracyclines could be reduced rapidly by composting. CTC and both of OTC and TC could be completely degradated by the composting for 14 d and 42 d, respectively. These results indicated that composting for CO2 enrichment could achieved the aims of reduction, recycle and safe treatment for organic wastes. The results of sorption test demonstrated that soil by adding compost could significantly enhance the sorption ability of tetracyclines and reduce their environmental risks.3. Inhibitory effect of Bacillus cereus, Bacillus subtilis Cohn and effective microorganisms (EM) against Fusarium oxysporum f.sp.The sitzkrieg experiment results showed that the Fusarium oxysporum f.sp. was inhibited obviously by Bacillus cereus, Bacillus subtilis Cohn and EM. Bacillus cereus had the greatest inhibition effect against the four Fusarium oxysporum f.sp., with the inhibition ratio between 20%-50%. The ratio of Bacillus subtilis Cohn and EM against Fusarium oxysporum f.sp. was 6.7%-34.8%. The results of spore germination tests showed that the spore germination of Fusarium oxysporum could be effectively inhibited by the solution of antagonistic bacteria or EM with dilution rate less than 20 times, and completely suppressed by the antagonistic bacteria or EM with the concentration of 108 cfu·mL-1.4. Effects of the fermentation residues inoculated with antagonistic bacteria on the remediation and improvement of continuous cropping soil in greenhouseThe results obtained from the experiments of tomato carried out on a grayey clayed paddy soil and fluvo-aquic soil with continuous cropping for 5 and 10 years, respectively, showed that the application of fermentation residues inoculated with Bacillus cereus, Bacillus subtilis Cohn and/or EM could significantly increase the pH and decrease the EC of the degradation soils, especially in the treatment inoculated simultaneously with three kinds of microbes, which would effectively alleviate soil acidification and secondary salinization. The content of available N, P, K and organic matter were also increased significantly, which could facilitate crops on nutrient absorption and utilization and be beneficial to soil fertility. Furthermore, the application of fermentation residues could also improved and promoted soil microflora and enzyme activity, which played a role in resolving the continuous cropping obstacle in greenhouse. Pot experiment showed that the plant height, fresh weight and dry weigh of tomato were increased, indicating a good effect of the fermentation residues inoculated with beneficial microorganism on promoting tomato growth and development.更多还原