水旱轮作稻田土壤厌氧氨氧化及其影响因素的研究

发布时间：2018-04-13 01:20

本文选题：厌氧氨氧化 + 水旱轮作　；参考：《浙江大学》2017年硕士论文

【摘要】：目前,厌氧氨氧化(Anammox)过程作为公认的脱氮途径,广泛存在缺氧的自然环境中,但它在稻田土壤氮素循环的作用和地位仍缺乏深入了解。本项研究在借鉴国内外在陆地生态系统中Anammox活性等方面的研究成果的基础上,以水旱轮作稻田土壤Anammox过程为主线,联合室内培养和长期定位肥料试验基础,运用15N同位素示踪技术、荧光定量PCR、DNA-SIP、克隆文库和高通量测序等先进技术,探究水旱轮作体系土壤Anammox对氮气产生的贡献、Anammox微生物丰度和群落结构及其影响因素等。主要研究结果如下:1)结果表明:无论是小麦季还是水稻季均检测到了 Anammox活性,而且种植季节之间存在显著差异,其中水稻季Anammox速率为0.9nmolN2·g-1-h-1,小麦季为0.46 nmol N2·g-1·h-1,不同施肥处理之间差异不显著。从全年来看,水旱轮作过程中土壤中通过Anammox反应流失的氮含量占总氮气生成量的3.15%-9.62%。进一步对Anammox细菌丰度进行分析,发现水稻和小麦季均测检到了高丰度的Anammox细菌,长期不同施肥处理条件下Anammox细菌的丰度表现为:猪粪堆肥配施化学肥料(PMCF)秸秆还田配施化学肥料(SRCF)和化学肥料(CF)不施肥(CK)。Anammox肼合成酶β亚基(hzs-β)基因序列分析表明,在水稻种植季节,Anammox 细菌与 Ca.Brocadia,Ca.Scalindua 和 Caa.Jettenia 属同源性较高。相比之下,所有的Anammox肼氧化酶(hzo)基因仅与Ca.Brocadia属表现出较高的同源性,表明hzs基因比hzo基因有更高的敏感性。PMCF处理中的序列与Ca.Jettenia和Ca.Brocadia有较高的同源性,并显示出最高的多样性。2)通过实验室培养实验研究了不同有机碳源(无外源碳施用(CK)、施用尿素(UR)、施用秸秆(SR)、施用猪粪(PM)、施用淀粉(ST)和施用葡萄糖(GL))对Anammox过程和活性的影响。结果显示,添加不同有机碳源进行培养之后,Anammox过程的氮气产生速率和对氮气的贡献率在1.04-3.19 nmol N2·g-1·h-1和4.22%-22.25%。虽然所有处理中均能检测到Anammox活性,但各处理产生氮气量不同,CK处理Anammox速率最高,其他添加有机碳源之后的Anammox速率较低,差异显著,说明有机碳源的添加对Anammox活性具有显著的抑制作用,同时有机碳对反硝化活性具有显著的促进作用。碳源添加对Anammox速率的影响如下:GLST,URSR。定量PCR结果表明,不同有机碳源均对Anammox微生物丰度具有一定的抑制作用,但PM处理对Anammox丰度的影响最小。选取活性最高的PM和CK处理的13C-Anammox肼合成酶β亚基(hzs-β)功能基因序列进行DNA-SIP分析,发现活性厌氧氨氧化细菌与Ca.Brocadia属具有较高的同源性。3)利用长期定位试验土壤,通过室内培养试验,研究了不同水分状态及溶解氧浓度(70%FC、干湿交替(淹水7天,落干7天)、淹水Ⅰ:溶解氧浓度为5.8 mg·L-1、淹水Ⅱ:溶解氧浓度为2.6 mg·L-1)对土壤Anammox过程的影响。结果表明,Anammox速率范围在0.56-1.47nmolN2·g-1·h-1之间。70%田间持水量(70%FC)处理中的Anammox速率最低(0.61 nmol N2.g 1·h-1),淹水Ⅱ处理最高(].14 nmol N2·g-1·h-1);说明溶解氧含量较高时会降低Anammox速率。水分处理会明显增加Anammox细菌数量,不论是干湿交替还是淹水处理,Anammox细菌丰度较高,不同施肥处理中,CF和SRCF处理中Anammox细菌hzs-β基因拷贝数较高。Anammox细菌群落结构在不同水分处理后差异并不明显,绝大部分Anammox细菌与Ca.Brocadia属具有较高的同源性,Ca.Jettenia属数量较少。
[Abstract]:At present, the anaerobic ammonia oxidation (Anammox) process for nitrogen removal routes recognized, widespread anoxic environment, but its role and status in the paddy soil nitrogen cycle is still poorly understood. In this study, on the basis of research results of Anammox activity at home and abroad in the terrestrial ecosystem area, drought and flood the rotation of paddy soil Anammox process as the main line, combined with indoor culture and long-term base fertilizer experiment using 15N isotope tracer technique, fluorescence quantitative PCR, DNA-SIP, clone libraries and high-throughput sequencing and other advanced technology, explore the rotation system of soil Anammox to produce nitrogen contribution, Anammox microbial abundance and community structure and its influencing factors. The main results are as follows: 1) the results show that both the wheat or rice season were detected in the Anammox activity, but there are significant differences between the planting season, The rice season Anammox rate was 0.9nmolN2 g-1-h-1, 0.46 nmol N2 wheat season g-1 H-1, there was no significant difference between different treatments. From the view of the rotation process, soil total nitrogen content by 3.15%-9.62%. nitrogen Anammox reaction loss further analysis of Anammox found that the abundance of bacteria. Rice and wheat season were the detection to the high abundance of Anammox bacteria, long-term abundance performance under different fertilizer treatments of Anammox bacteria: pig manure combined with chemical fertilizer (PMCF) straw fertilizer chemical fertilizer (SRCF) and chemical fertilizer (CF) fertilizer (CK) hydrazine synthase beta subunit (.Anammox hzs- beta) gene sequence analysis showed that the planting season in rice, Anammox bacteria and Ca.Brocadia and Caa.Jettenia belong to Ca.Scalindua, the homology is high. In contrast, Anammox (HZO) of all the hydrazine oxidase gene and Ca.Broc The genus adia showed high homology, suggesting that the HZS gene has higher sensitivity to.PMCF treatment in Ca.Jettenia and Ca.Brocadia and has higher sequence homology than HZO gene, and showed the highest diversity of.2) the experimental study on the cultivation of different organic carbon sources through the laboratory (without exogenous application of carbon (CK), application urea (UR), straw (SR), pig manure (PM), the application of starch (ST) and application of glucose (GL)) influence on the Anammox process and activity. The results showed that after training with different organic carbon sources, nitrogen Anammox process for nitrogen production rate and the contribution rate of 1.04-3.19 in nmol N2 - g-1 - H-1 and 4.22%-22.25%. although all treatments were detected in Anammox activity, but the generation of nitrogen content is different, CK Anammox rate is the highest, the other after the addition of organic carbon source Anammox rate is low, indicating significant difference, organic carbon source Add has a significant inhibitory effect on the activity of Anammox, while organic carbon has significant effect on the denitrification activity. Adding carbon source effect on the rate of Anammox is as follows: GLST, URSR. quantitative PCR results showed that different organic carbon source has certain inhibitory effect on Anammox microbial abundance, but the effect of PM treatment on Anammox the abundance of selected minimum. The highest activity PM and CK 13C-Anammox hydrazine synthase beta subunit (hzs-) gene sequence by DNA-SIP analysis, found that the activity of anammox bacteria and the genus Ca.Brocadia has high homology of.3) using soil long-term experiment, through laboratory incubation experiment of different moisture state and dissolved oxygen concentration (70%FC, alternate (7 days of flooding, dry 7 days), flooding I: dissolved oxygen concentration of 5.8 mg L-1, flooding II: dissolved oxygen concentration of 2.6 mg L-1) on soil Anammox process Effect. The results show that the Anammox rate in the range between 0.56-1.47nmolN2 - g-1 - H-1.70% of field capacity (70%FC) in the treatment of Anammox was the lowest (0.61 nmol N2.g 1 - h-1), flooding treatment was the highest (].14 nmol II N2 - g-1 - h-1); it showed that the higher the content of dissolved oxygen will reduce the rate of Anammox water. Anammox treatment significantly increased the number of bacteria, whether or waterlogged dry wet alternation Anammox bacterial abundance was higher in different treatments, CF and SRCF in Anammox gene of bacterial hzs- beta copy bacterial community structure in a higher number of.Anammox in different water treatments after the difference is not obvious, the majority of Anammox bacteria and the genus Ca.Brocadia have high homology Ca.Jettenia, is a smaller number.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S153

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