【作者】 黄莹；

【导师】 王光火； 姚槐应；

【作者基本信息】 浙江大学 ， 农业资源利用， 2014， 博士

【摘要】 氧化亚氮(N2O)是一种重要的温室气体。它参与各种光化学反应、破坏臭氧层,并且在大气中滞留时间较长,对人类居住环境和地球生物构成很大威胁。农田土壤是全球最主要的N2O排放源,对N2O排放的贡献率高达70%-90%。茶园土壤广泛分布于中国南方丘陵地区,由于大量施用氮肥,土壤严重酸化并伴随着大量N2O排放。石灰、不同形态氮肥、硝化抑制剂的施用等茶园施肥措施会影响土壤氮素循环相关微生物活性从而影响N2O排放。迄今,国内外对于N2O的研究主要针对于农田、稻田系统,而对我国茶园土壤N2O排放的研究尚不多见。本论文选取杭州市西湖区梅家坞茶园土壤为研究对象,利用田间调查与实验室培养的方法,综合运用传统和现代微生物分析技术(包括定量PCR、克隆文库和末端限制性片段长度多态性),结合茶园施肥措施对强酸性茶园土壤的N2O排放来源及其微生物机制等方面进行了较系统的研究,获得以下主要研究结果：1茶园土壤(3年、50年和100年)主要理化性质土壤酸度、有机质、全氮、硝氮、氨氮和有效磷含量随植茶年龄的增加而显著增加,均以100年茶园土壤最高。50年和100年茶园土壤的净硝化速率较大,分别为0.99和0.79mg/kg·d,100年茶园土壤的反硝化潜势为10.5mg/kg·h,显著高于其余土壤；添加不同氮肥与硝化抑制剂的研究表明：不同处理以单施硝酸钾产生的N2O最高,为0.87mg/kg,是单施硫酸铵的7倍。硝酸钾与硝化抑制剂混施对N2O排放的抑制效果不显著,而硫酸铵与抑制剂结合可显著降低硝化势和N2O排放。对不同处理编码N2O还原酶的nosZ基因的定量结果表明,添加硝态氮处理的nosZ基因丰度显著低于其余处理；对茶园土壤中添加不同氮肥种类(硝酸钾、硫酸铵、尿素、硝酸铵、对照)的研究表明,硝酸钾处理N2O排放总量最高,为744.8ng/g,是对照的16倍；尿素、硫酸铵处理的土壤硝化潜势显著高于硝酸钾处理,而N2O排放量却显著低于后者；通过添加石灰将pH为3.71的强酸性茶园土壤调至5.11、6.19、和7.41三个梯度,并结合施用硫酸铵的实验结果表明：添加硫酸铵以后,pH为7.41的土壤硝化势比pH3.71的土壤提高了88%,N2O释放量是pH为3.71土壤排放的7.3倍。添加硫酸铵和乙炔后,pH7.41土壤的硝化势和N2O分别降低了91%和93%,而pH3.71的土壤虽然硝化势降低了79%,但N2O释放量与未添加乙炔处理的N2O释放量差异不显著。以上实验结果表明：中性茶园土壤中硝化作用是产生N2O的主要来源,而强酸性茶园土壤中,反硝化作用是N2O排放的主要来源,并且在低pH条件下,NO3-对nosZ基因丰度具有一定的抑制作用。2通过’5N同位素示踪的研究表明,经过3天和7天的好氧培养,反硝化作用释放的15N-N2O分别占总15N-N20排放的71.1%和78.9%,自养硝化对15N-N20的贡献率分别为23.7%和17.8%。用乙炔抑制法测定反硝化和异养硝化的贡献率分别为80.6%和88.9%,自养硝化作用对总N20排放的贡献率分别为19.4%和11.1%。进一步说明强酸性茶园土壤中N20排放主要来源于反硝化作用。3通过向施石灰土壤中添加不同浓度硝酸钾的研究表明,不同pH(3.71,5.11,6.19和7.41)茶园土壤以pH3.71土壤添加1000mg/kg的硝酸钾时,净N20排放量最大,是其余pH土壤的21.1-65.2倍。利用乙炔抑制不同pH土壤的N20还原过程,pH3.71-6.19这三个酸性土壤被还原的N20占总N20的89%-99%,显著高于pH为7.41的土壤。说明在供试强酸性茶园土壤中,N20大量排放与低pH抑制N20还原过程这一机制无关。通过TRFLP和克隆测序,本文在pH3.71-5.11的克隆文库中找到的耐酸反硝化微生物在前人的研究中同样存在于酸性土壤中。说明这些反硝化微生物在酸性茶园土壤中可能具有很高的活性,强酸性茶园土壤大量N20排放主要与反硝化微生物长期以来对酸性环境的适应机制有关。4通过添加真菌和细菌的生物抑制剂-放线菌酮和链霉素,发现茶园土壤中真菌反硝化作用对N20排放的贡献率为70%。因此茶园土壤中真菌反硝化作用值得深入研究。随后,我们利用孟加拉红培养基在pH为3.8的茶园土壤中分离出了2株耐酸的反硝化真菌,根据其18S rDNA测序分析,鉴定二者分别为青霉菌和肉座菌。其中,青霉菌在液体培养基中的N20排放量高达328mg/kg’g菌株干重,占加入的N03--N的16.4%。我们将活性较强的青霉菌接种到γ射线灭菌的土壤中,培养前3天,添加硝酸钾的N20释放速率显著高于未灭菌土壤。上述结果揭示了茶园土壤中N20排放很可能与真菌的反硝化作用密切相关。更多还原

【Abstract】 N2O is one of the green-house gases. It not only participates in photochemical reaction and destruction of stratospheric ozone, but also has negative effect on biosphere. Soil is the major N2O source and contributes70%of N2O emission to atomosphere. Tea orchard soil is highly acid and widely distributed in the southern hilly area of China. Soil acidification and N2O emission were enhanced due to heavy nitrogen fertilization application in tea orchard. The agricultural managements, e.g. liming, fertilizer application in tea orchard can affect the changes in microbial community composition and the rate of N2O emission. However, the mechanism between soil microbial community and high N2O emission rate in tea orchard remains unclear. In this study, a series of tea orchard soils, adjacent wasteland and forest derived from the same parent material were collected from Meijiawu tea orchard, located in West Lake district of Hangzhou, Zhejiang Province in Southeast of China. A series of traditional and molecular methods (quantitative PCR, clone library and terminal restriction fragment length polymorphism (TRFLP)) were carried out to study the effect of fertilization treatments on N2O emission and its microbiological mechanism.The main results are as follows:1With the succeeding development of tea orchard ecosystems, the primary chemical and microbial properties in tea orchard soils, i.e., soil acidity, organic carbon, total N, inorganic N, available P showed a significantly increasing trend with increasing ages. The higher nitrification potential and denitrification enzyme activity were observed in the50-year-old (0.99mg/kg-d) and100-year-old tea orchard soil (0.79mg/kg-d). The application of nitrogen fertilizers and nitrification inhibitors showed that the highest amount of N2O emission was observed in KNO3treatments (0.87mg/kg) which was7times higher than that in (NHO2SO4alone. Effect of KNO3with DCD or DMPP on N2O emission was not significant whereas (NH4)2SO4with DCD or DMPP could significantly inhibit N2O emission. Quantitative PCR of nosZ gene, a key gene encoded N2O reduction enzyme, suggested that nosZ gene abundance in KNO3-associated treatments was the lowest. The application of different nitrogen fertilizers (KNO3,(NH4)2SO4, Urea, NH4NO3, control) showed that N2O emission in KNO3treatment was744.8ng/g, that was16times higher than control. The nitrification potential in Urea or (NH4)2SO4treatments was significantly higher than that in KNO3treatment, but N2O emission was lower. Soil pH (3.71) was adjusted to5.11,6.19and7.41after liming. With (NH4)2SO4addition, soil nitrification potential in soil with pH7.41was increased by88%and N2O emission was7.3times higher than that in pH3.71treatment. Combined (NH4)2SO4with C2H2, soil nitrification potential and N2O emission decreased by91%and93%, respectively in soils with pH7.41. In soils with pH3.71, though soil nitrification potential was decreased by79%, N2O emission showed no significant difference with (NH4)SO4alone treatment. The above results suggested that nitrification is the dominant source of N2O emission in the neutral soil, but denitrification dominated in N2O emission in highly acidic soil. The inhibition of nosZ by NO3" addition may result in high N2O emission at low pH.2The15N tracer method showed dentirification is the dominant source of15N-N2O during3days or7days aerobic incubation, accounting for71.1%and78.9%of total15N-N2O, respectively. The contribution of autotrophic nitrification to15N-N2O was smaller than denitrification, accounting for23.7%and17.8%, respectively. Using C2H2inhibition method, the contribution of autotrophic nitrification was19.4%(3days) and11.1%(7days), respectively and denitrification accounted for80.6%(3days) and88.9%(7days), respectively.3In the nitrate amendment experiment, the highest N2O emission (2.0mg/kg,21days) was produced at1000mg/kg NO3-in soils at pH3.71, ranging from21.1-65.2times higher than the rest three pH soils.10Pa C2H2was used to inhibit N2O reduction and the result suggested that the reduced N2O accounted for89%-99%of total N2O production in soils with pH3.71-6.19, which is significantly higher than that in soils with pH7.41. We provide strong evidence that the impaired reduction of N2O under acidic conditions is not likely to be responsible for the high N2O emission in this acidic soil. Using TRFLP, cloning and sequencing, the acid-tolerant denitrifiers were found in clone library3.71-5.11, which were also found in the acidic soils in the previous studies. The detected denitrifiers may have a high activity in the acidic soils and the large amounts of N2O emission was mainly related with the adaptation of acid-tolerant denitrifying populations.4Using fungal and bacterial inbitors, cycloheximide and streptomycin, fungal denitrification accounted for70%of N2O emission in tea soil and therefore fungal denitrification in tea soil plays an important role in N2O emission. Then, two acid-tolerant fungi, were isolated in soils with pH3.8using rose-bengal medium. According to the morphological characteristics and the phylogenetic analysis based on18S rDNA sequence, the two strains were identified to be penicillium and hypocrea, respectively. With the inoculation of the two strains into the liquid medium, the N2O emission from penicillium was328mg/kg, accounting for16.4%of added NO3-N, but the hypocrea showed no significant N2O emission with control. The representative penicillium was also inoculated into soils with y-sterilization. The result suggested that on day3, N2O emission rate was significantly higher than that in the nonsterile soils with NO3-N addition. The above results suggested N2O emission in the tested100-year-old tea soil is closely related with fungal denitrification.更多还原