内蒙古锡林郭勒草原土壤中氨氧化微生物对氨和pH的响应
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摘要
大气氮沉降的加剧使土壤中的氮素积累日益严重。由微生物催化的硝化作用是全球氮循环的一个重要环节,与土壤养分的供应和硝酸盐污染等问题有着紧密的联系,但是关于内蒙地区氮素积累对硝化过程的影响及机制还缺乏深入的研究。氨氧化微生物是硝化过程的主要驱动者,在硝化过程中起着关键作用。氨氧化微生物主要有氨氧化细菌(Ammonia-oxidizing bacteria, AOB)和氨氧化古菌(Ammonia-oxidizing archaea,AOA)两大类,目前关于它们的不同生理特点还缺乏足够的认识,同时研究AOB和AOA的组成和活性及其影响因素对于深入了解并进一步调控土壤硝化过程具有重要意义。
氮素积累在导致土壤中氨浓度升高的同时往往伴随着土壤因硝化作用增加而导致土壤酸化,氨浓度和pH值的变化都是影响氨氧化微生物组成和活性的重要因素,也很可能成为氮素积累影响氨氧化过程的主要机制。本论文利用中国科学院内蒙古草原生态系统定位试验站的不同氮素添加和不同pH实验样地,以编码催化氨氧化过程的氨单加氧酶的功能基因amoA基因为分子标记物,通过分子生物学手段(Real-timePCR、克隆测序、T-RFLP等),对长期添加不同浓度氮素(0、1.75、5.25、10.5、17.5、28 g N m-2 yr-1)和不同pH值下土壤中氨氧化细菌(AOB)和氨氧化古菌(AOA)的数量和群落结构进行了分析,同时结合土壤氨氧化潜势的测定和室内培养实验,对影响氨氧化微生物组成和氨氧化活性的主要因素进行分析,以探讨内蒙古草原土壤中氮素积累对氨氧化过程的影响及其机制,结果如下：
在长期添加氮素处理中,随着氮素添加浓度的升高,土壤pH值逐渐下降(从6.63下降至4.93),氨氧化潜势(PNR)也呈下降趋势且与pH呈极显著正相关性(p<0.01)。AOB的数量随添加氮素浓度升高呈上升趋势,与土壤中氨浓度呈极显著正相关性,与土壤pH和PNR呈极显著负相关性；而AOA的数量在不同加氮水平处理间没有显著差异,除了在最高氮素添加水平(28gNm-2yr-1)时数量有明显下降,与土壤氨浓度呈极显著负相关,与pH和PNR均极显著正相关。克隆测序和聚类分析结果表明,各处理中AOB均归属于亚硝化螺菌属(Nitrosospira),氮素添加导致AOB群落结构发生了改变,在不添加氮素、添加中浓度和高浓度氮素下,AOB的主要组成类群分别为Cluster3a1、Cluster3a2和Cluster 2。而T-RFLP图谱分析结果显示AOA的群落结构没有发生明显改变。
在添加硫酸处理的不同pH样地中,随着pH的下降,PNR明显下降,且与pH呈极显著正相关(p<0.01),与添加不同浓度氮素实验的结果相似。但随着pH的下降AOB数量也呈下降趋势,而AOA数量明显上升,只在最低pH值(4.6)处理下显著下降。T-RFLP图谱分析表明,不同pH处理土壤中AOA的群落组成变化不大。综合氮素添加和不同pH处理的分析结果,可以看出,在这些处理中,土壤pH值是决定PNR的主要因素,而土壤中氨浓度的变化则对AOB和AOA的数量起着重要的影响。
为了进一步验证氨浓度和pH值对氨氧化微生物组成和活性的影响,分别在中性和酸性条件下对不施肥土样(NO)和最高施肥浓度土样(N28)进行加氨培养(40天)。结果表明,优势类群为Cluster 3a1的NO处理土样在中性条件下加氨培养后,所得AOB序列全部归属于Cluster 3a2;而在酸性条件下培养后约56%归属于Cluster 2。优势种群为Cluster 2的N28处理在中性条件下培养后约56%归属于Cluster 3a2;酸性条件下培养后87%的序列归属于Cluster 2。由此推测Cluster 3a2喜中性高氨环境,而Cluster2喜酸性环境,这些结果也解释了氮素添加平台中AOB群落结构变化的原因。
Nitrification is a central process in nitrogen cycle in soil and is important for soil nutrition and nitrate pollution. Ammonia-oxidation is the key step of nitrification which is driven by ammonia-oxidizing microorganisms, mainly including ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). However, very little is known about their distinct physiological characteristics. So the research for the influence of environmental factors on the abundance and community composition of AOA and AOB are of great significance for understanding the soil nitrification process in Inner Mongolia.
In this study, soil samples were collected from the Inner Mongolia Grassland Ecosystem Research Station (IMGERS) of Chinese Academy of Sciences. The abundance and community structure of soil ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in soils with long-term different N addition rates (0, 1.75,5.25,10.5,17.5, and 28 g N m-2 yr-1) and vitriol-loading rates for pH adjustment were investigated using quantitative real-time PCR, cloning and sequencing by targeting on amoA gene. In addition, soil potential ammonia oxidation rate was analyzed and short-term lab incubation experiment was completed. We analyzed the central factors which affected the soil ammonia-oxidizing microorganisms and ammonia activity, and discussed the influence and mechanism of ammonia oxidation resulted from N accumulation in the soil of Inner Mongolia Grassland. The results are as follow.
With the increase of nitrogen added, soil pH declined significantly from 6.6 without N addition to 4.9 with the highest N addition rate, and potential ammonia oxidation rate also declined which was positively correlated with soil pH (p<0.01). Contrary to the variation of pH and potential ammonia oxidation rate, the copy number of bacterial amoA gene increased with nitrogen addition rates and it was positively (p<0.01) correlated with ammonia concentrations in soil while it was negatively correlated with PNR and pH. The archaeal amoA gene copy number did not change a lot with the increase of N addition, but decreased significantly under high N concentration. Sequencing of clone libraries revealed that all bacterial amoA sequences belong to genus of Nitrosospira. In the treatment without N addition, middle-level N addition, and high N addition, AOB was dominated by Cluster 3al,Cluster 3a2, Cluster 2, respectively., The profile of T-RFLP showed that no significant variation on community structure of AOA was found among all the treatments.
With the increase of vitriol added, soil pH and PNR declined significantly and positively correlation was found between them. The results were similarly to N addition experiments. Differently, with the decreased of soil pH, the copy number of bacterial amoA gene declined, while the amoA gene copies of archaea increase significantly, though the copies declined under the lowest pH. The profile of T-RFLP showed that the composition of AOA change little among all the treatments. From the results of N addition experiment and vitriol addition experiment, we conclude that soil PNR may be determined by pH, and the change on abundance of AOB and AOA may be resulted from the influence of soil ammonia concentration.
To further validate the effect of ammonia concentration and pH which acted on the compositions and activity of soil ammonia-oxidizing microorganisms, we made a cultivation of two soil sample(NO treatment without N addition, N28 treatment with addition of 28g N/m2) under acid and neutral condition respectively. Before cultivating, AOB has been dominated by Cluster 3a1 for N0 and Cluster 2 for N28. Through adding N, for N0, all the sequences we got grouped into Cluster 3a2 under neutral medium while about 56% sequences grouped into Cluster 2 under acid medium; for N28, near 56% for Cluster 3a2 under neutral medium while near 87% for Cluster 2 under acid medium. This result just give the evidence of the result before that composition of AOB can be changed with N adding. From this we can conclude that the AOB which can be grouped into Cluster 3a2 may adjust to the neutral environment of high N concentration and AOB which can be grouped into Cluster 2 may adjust to the acid environment.
氮素积累在导致土壤中氨浓度升高的同时往往伴随着土壤因硝化作用增加而导致土壤酸化,氨浓度和pH值的变化都是影响氨氧化微生物组成和活性的重要因素,也很可能成为氮素积累影响氨氧化过程的主要机制。本论文利用中国科学院内蒙古草原生态系统定位试验站的不同氮素添加和不同pH实验样地,以编码催化氨氧化过程的氨单加氧酶的功能基因amoA基因为分子标记物,通过分子生物学手段(Real-timePCR、克隆测序、T-RFLP等),对长期添加不同浓度氮素(0、1.75、5.25、10.5、17.5、28 g N m-2 yr-1)和不同pH值下土壤中氨氧化细菌(AOB)和氨氧化古菌(AOA)的数量和群落结构进行了分析,同时结合土壤氨氧化潜势的测定和室内培养实验,对影响氨氧化微生物组成和氨氧化活性的主要因素进行分析,以探讨内蒙古草原土壤中氮素积累对氨氧化过程的影响及其机制,结果如下：
在长期添加氮素处理中,随着氮素添加浓度的升高,土壤pH值逐渐下降(从6.63下降至4.93),氨氧化潜势(PNR)也呈下降趋势且与pH呈极显著正相关性(p<0.01)。AOB的数量随添加氮素浓度升高呈上升趋势,与土壤中氨浓度呈极显著正相关性,与土壤pH和PNR呈极显著负相关性；而AOA的数量在不同加氮水平处理间没有显著差异,除了在最高氮素添加水平(28gNm-2yr-1)时数量有明显下降,与土壤氨浓度呈极显著负相关,与pH和PNR均极显著正相关。克隆测序和聚类分析结果表明,各处理中AOB均归属于亚硝化螺菌属(Nitrosospira),氮素添加导致AOB群落结构发生了改变,在不添加氮素、添加中浓度和高浓度氮素下,AOB的主要组成类群分别为Cluster3a1、Cluster3a2和Cluster 2。而T-RFLP图谱分析结果显示AOA的群落结构没有发生明显改变。
在添加硫酸处理的不同pH样地中,随着pH的下降,PNR明显下降,且与pH呈极显著正相关(p<0.01),与添加不同浓度氮素实验的结果相似。但随着pH的下降AOB数量也呈下降趋势,而AOA数量明显上升,只在最低pH值(4.6)处理下显著下降。T-RFLP图谱分析表明,不同pH处理土壤中AOA的群落组成变化不大。综合氮素添加和不同pH处理的分析结果,可以看出,在这些处理中,土壤pH值是决定PNR的主要因素,而土壤中氨浓度的变化则对AOB和AOA的数量起着重要的影响。
为了进一步验证氨浓度和pH值对氨氧化微生物组成和活性的影响,分别在中性和酸性条件下对不施肥土样(NO)和最高施肥浓度土样(N28)进行加氨培养(40天)。结果表明,优势类群为Cluster 3a1的NO处理土样在中性条件下加氨培养后,所得AOB序列全部归属于Cluster 3a2;而在酸性条件下培养后约56%归属于Cluster 2。优势种群为Cluster 2的N28处理在中性条件下培养后约56%归属于Cluster 3a2;酸性条件下培养后87%的序列归属于Cluster 2。由此推测Cluster 3a2喜中性高氨环境,而Cluster2喜酸性环境,这些结果也解释了氮素添加平台中AOB群落结构变化的原因。
Nitrification is a central process in nitrogen cycle in soil and is important for soil nutrition and nitrate pollution. Ammonia-oxidation is the key step of nitrification which is driven by ammonia-oxidizing microorganisms, mainly including ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). However, very little is known about their distinct physiological characteristics. So the research for the influence of environmental factors on the abundance and community composition of AOA and AOB are of great significance for understanding the soil nitrification process in Inner Mongolia.
In this study, soil samples were collected from the Inner Mongolia Grassland Ecosystem Research Station (IMGERS) of Chinese Academy of Sciences. The abundance and community structure of soil ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in soils with long-term different N addition rates (0, 1.75,5.25,10.5,17.5, and 28 g N m-2 yr-1) and vitriol-loading rates for pH adjustment were investigated using quantitative real-time PCR, cloning and sequencing by targeting on amoA gene. In addition, soil potential ammonia oxidation rate was analyzed and short-term lab incubation experiment was completed. We analyzed the central factors which affected the soil ammonia-oxidizing microorganisms and ammonia activity, and discussed the influence and mechanism of ammonia oxidation resulted from N accumulation in the soil of Inner Mongolia Grassland. The results are as follow.
With the increase of nitrogen added, soil pH declined significantly from 6.6 without N addition to 4.9 with the highest N addition rate, and potential ammonia oxidation rate also declined which was positively correlated with soil pH (p<0.01). Contrary to the variation of pH and potential ammonia oxidation rate, the copy number of bacterial amoA gene increased with nitrogen addition rates and it was positively (p<0.01) correlated with ammonia concentrations in soil while it was negatively correlated with PNR and pH. The archaeal amoA gene copy number did not change a lot with the increase of N addition, but decreased significantly under high N concentration. Sequencing of clone libraries revealed that all bacterial amoA sequences belong to genus of Nitrosospira. In the treatment without N addition, middle-level N addition, and high N addition, AOB was dominated by Cluster 3al,Cluster 3a2, Cluster 2, respectively., The profile of T-RFLP showed that no significant variation on community structure of AOA was found among all the treatments.
With the increase of vitriol added, soil pH and PNR declined significantly and positively correlation was found between them. The results were similarly to N addition experiments. Differently, with the decreased of soil pH, the copy number of bacterial amoA gene declined, while the amoA gene copies of archaea increase significantly, though the copies declined under the lowest pH. The profile of T-RFLP showed that the composition of AOA change little among all the treatments. From the results of N addition experiment and vitriol addition experiment, we conclude that soil PNR may be determined by pH, and the change on abundance of AOB and AOA may be resulted from the influence of soil ammonia concentration.
To further validate the effect of ammonia concentration and pH which acted on the compositions and activity of soil ammonia-oxidizing microorganisms, we made a cultivation of two soil sample(NO treatment without N addition, N28 treatment with addition of 28g N/m2) under acid and neutral condition respectively. Before cultivating, AOB has been dominated by Cluster 3a1 for N0 and Cluster 2 for N28. Through adding N, for N0, all the sequences we got grouped into Cluster 3a2 under neutral medium while about 56% sequences grouped into Cluster 2 under acid medium; for N28, near 56% for Cluster 3a2 under neutral medium while near 87% for Cluster 2 under acid medium. This result just give the evidence of the result before that composition of AOB can be changed with N adding. From this we can conclude that the AOB which can be grouped into Cluster 3a2 may adjust to the neutral environment of high N concentration and AOB which can be grouped into Cluster 2 may adjust to the acid environment.
引文
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