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南极苔原土壤细菌群落和酶活性分布特征及其影响因素
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摘要
土壤微生物是土壤圈的重要组成部分。微生物在调节生态系统功能,如碳氮循环过程、有机质分解和温室气体排放中起重要作用,是生物地球化学循环的主要驱动者。环境条件改变会直接或间接地影响微生物群落组成和功能,从而影响生态系统的稳定。在南北极陆地生态系统,极端的气候条件导致极区几乎没有大型植物生长,微生物在土壤生态系统中占绝对优势,其对全球气候变化具有敏感的指示作用。本文应用PCR-DGGE.克隆测序、Bar-coded焦磷酸测序和荧光定量PCR等先进的分子生物学技术和多种统计分析方法,结合土壤酶活性、微生物量碳(Cmin)和土壤呼吸指标等,系统地研究了南极典型生态区土壤细菌群落的分布特征;探讨了企鹅和海豹活动、人类活动以及自然生态因子等对南极土壤细菌群落结构的影响,丰富和拓展了极地微生物学研究领域;为从微生物层次上阐明南极陆地生态系统C、N循环过程和机理,以及预测未来气候变化对南极生态系统的影响及其反馈作用,提供了理论依据。该论文主要研究内容及结果如下:
     (1)东南极企鹅、海豹聚居地细菌群落的分布特征
     对东南极西福尔丘陵地区四个企鹅、海豹聚居地粪土剖面中酶活性和细菌群落分布特征进行了详细研究,发现企鹅和海豹聚居地粪土剖面中Cmin、土壤呼吸和酶活性随深度增加而递减,且企鹅聚居地粪土含量高于海豹聚居地(1-2倍)。土壤细菌丰度与土壤pH值(p=0.024;p=0.048)、含水率(p=0.021)、有机碳(p<0.001)和总氮(p<0.001)含量呈显著正相关,细菌丰度与企鹅粪、海豹粪相对含量随土壤深度的变化高度一致,表明土壤细菌的丰度取决于企鹅粪和海豹粪的沉降量。16S rDNA-DGGE分子指纹图谱和香浓多样性指数结果表明企鹅和海豹聚居地粪土含有复杂的细菌群落结构。DGGE图谱条带丰富度和优势条带数目都随土壤深度增加而递减。聚类分析和非度量多维标度(NMDS)分析表明:四个土壤剖面细菌组成差异明显,且聚为四类。切取优势条带克隆测序表明:优势细菌门类是变形菌(Proteobacteria,36.84%)、放线菌(Actinobacteria,24.65%)、拟杆菌(Bacteroidetes,21.05%)、异常球菌(Deinococcus-Thermus,5.26%)、绿弯菌(Chloroflexi,5.26%)(?)口厚壁菌(Firmicutes,3.51%)。研究结果表明:动物排泄物沉降量引起土壤中有机碳、总氮、pH值和含水率等的不同,可能是造成细菌丰度和多样性在土壤垂直深度上较大差异的主要原因。
     (2)西南极典型生态区土壤细菌群落的分布特征
     对西南极法尔兹半岛地区企鹅聚居地、海豹聚居地、人类活动区、苔藓植被区、湖泊沉积物和背景地区等不同生态区土壤进行了系统地对比分析研究,结果表明:土壤转化酶、磷酸酶、脲酶活性、细菌丰度在企鹅、海豹聚居地粪土中高于其它类型土壤。土壤转化酶、磷酸酶活性与土壤碳氮磷含量呈正相关,细菌丰度与土壤磷酸酶(p=0.006)、脲酶(p<0.001)、含水率(p<0.05)、总碳(p<0.001)、总氮(p=0.005)、C/N(p=0.007)和总磷(p=0.05)呈显著正相关。DGGE分子指纹图谱显示动物聚居地粪土相对于其它类型土壤,含有较密较粗的条带,一些片段较大的优势条带富集在图谱上层。高通量测序表明:主要优势门类菌种为:β/变形菌(Betaproteobacteria)、放线菌(Actinobacterium)、拟杆菌(Bacteroidetes)、γ-变形菌(Gammaproteobacteria)、芽单胞菌(Gemmatimonadetes)、厚壁菌(Firmicutes)、绿弯菌(Chloroflexi)、酸杆菌(Acidobacteria)和α-变形菌(Alphaproteobacteria),占86.7%。企鹅、海豹以及人类活动对法尔兹半岛细菌群落影响显著,不同生态区土壤细菌群落结构差异明显。背景地区有高的细菌系统发育多样性和丰富度指数(OUT=976;PD=85.79),动物活动和人类活动降低细菌多样性(OUT=273; PD=21.34).皮尔森相关性分析表明:土壤TN(p=0.0031; p<0.001), TC (p=0.011;p=0.0024), C/N (p=0.031;p=0.0028)和Zn(p=0.030;p=0.025)含量是影响土壤细菌系统发育多样性和丰富度指数的主要环境因子。土壤碳、氮含量同拟杆菌相对丰度呈显著正相关(p=0.003;p=0.007),同p-变形菌和放线菌相对丰度呈负相关。
     (3)Y2湖沉积剖面细菌群落分布特征及其影响因素
     对西南极阿德雷岛Y2湖受企鹅粪影响的沉积剖面中细菌群落分布特征进行了详细研究。Y2湖沉积剖面pH、含水率、总碳、总氮、Cu、Zn, Sr、Ba、Fe、 Mn、Mg和Al等12种理化指标随深度变化表现出高度一致性,总体上变化可以分为四个区域:0-7cm,表层较高,随深度增加而递减;7-23cm,随深度增加缓慢增加;23-34cm,显著锐减,最低值在32cm;34-48cm,随深度增加而增加。Fe、Mn、Mg和Al四种元素在23-34cm和34-48cm变化趋势相反,且最大值在32cm。在DGGE指纹图谱分为三个区域:1-22cm,整体上条带变动不大;23-36cm,条带密度和优势特征带明显增加;37-48cm,变化范围不大。细菌丰度随深度分布分为四个区间:最表层含有高的细菌丰度,随着深度减小(0-8cm);缓慢地增加(8-23cm);显著增加(23-32cm);缓慢地降低(32-48cm)的变化趋势,最大值出现在32cm。主要的优势门类菌种为:β-变形菌(Betaproteobacteria)、放线菌(Actinobacterium)、γ-变形菌(Gammaproteobacteria)、拟杆菌(Bacteroidetes)、芽单胞菌(Gemmatimonadetes)、酸杆菌(Acidobacteria)、绿弯菌(Firmicutes),占据87.2%。综合理化性质、DGGE图谱、定量PCR和454高通量测序结果表明:历史时期企鹅数量变动和企鹅粪沉积量是影响沉积剖面中细菌群落结构多样性的重要因素。
     (4)南北极土壤剖面磷酸酶活性变化特征及其对磷循环的影响
     对东南极西福尔丘陵、拉斯曼丘陵和北极新奥尔松地区,不同环境类型土壤剖面中碱性磷酸酶活性进行了测定,发现剖面中碱性磷酸酶活力随深度呈一定规律性分布,最大值出现在最表层,随深度增加而递减。碱性磷酸酶活性与土壤中有机碳、总氮含量呈显著正相关,与各形态磷含量呈正相关。碱性磷酸酶活力与土壤中铜、锌含量呈负相关,表明重金属对土壤中磷酸酶活力有抑制和破坏作用。基质结合态磷化氢(MBP)在深度剖面上表现出在土壤表层先增加,然后随深度增加而递减的规律。MBP和土壤碱性磷酸酶活性呈显著正相关(P<0.001),表明在富含磷的粪土和沉积物中,是微生物过程而不是磷含量对MBP产生量起决定作用。总之,在极地土壤中碳、氮等养分含量是影响土壤磷酸酶活性的主要因素。土壤磷酸酶活性可作为指示土壤微生物活性和土壤肥力的一个重要指标。
Soil microorganisms are a major component of the soil biosphere. Microbes regulate ecosystem function, and play a crucial role in the processes of ecosystem carbon and nitrogen cycles, such as the cycling of nutrient and the decomposition of organic matter, and emissions of greenhouse gases. They are the main driver of biogeochemical cycle. Changes in environmental conditions can directly or indirectly affect the microbial community compositions and function, thus affect the stability of ecosystem function. Terrestrial trophic interactions are generally dominated by microorganisms due to harsh environments, which restricts the development of higher plant communities. Furthermore soil microbes are very sensitive to the global climate changes in Arctic and Antarctica. In this paper, The soil bacterial community characteristics of typical environments in Antarctica have been studied using PCR-DGGE, cloning sequencing,454Bar-coded pyrosequencing and quantitative PCR, multi-statistical methods, combining with soil enzyme activity, microbial biomass carbon (Cmin), soil respiration. Effects of penguin and seal activities, human activities and natural ecological factors on soil bacterial community structure were detailedly discussed in the paper. This study will enrich and enlarge the research areas about polar microbiology, and will be helpful to understand the effects of soil microbes on the processes carbon and nitrogen cycles in Antarctic terrestrial ecosystems, and to predict the response and feedback of Antarctic terrestrial ecosystem to future climate changes. Main research contents and results are as follows:
     (1) Distribution characteristics of bacteria community in soil profile of penguins and seal colonies of east Antarctica
     Distribution characteristics of bacteria community were studied in four penguin and seal colony soil profiles collected on Vesfold Hills of east Antarctica. It was found that soil Cmin, soil respiration and soil enzyme activity increased with depth in penguin and seal colonies. Overall they were much higher (one to two times) in the penguin colony than in seal colony. Bacterial abundance was significantly positively correlated with soil pH (p=0.024; p=0.048), soil moisture (p=0.021), organic carbon (p<0.001) and total nitrogen content (p<0.001), indicating that bacterial abundance was affected by the nutrients from marine animal excrement in these animal colonies.16S rDNA-DGGE molecular fingerprint and Shannon-wiener diversity index showed that the soils in penguin and seal colonies contained complex bacteria community structure. DGGE bands richness and dominant bands decreased with depths. The result of Nonmetric multidimensional scaling (NMDS) and cluster showed obvious differences in four soil profiles. The predominant bacteria bacterium categories are: Proteobacteria (36.84%), Actinobacteria (24.65%), Bacteroidetes (21.05%), Deinococcus-Thermus (5.26%), Chloroflexi (5.26%) and Firmicutes (3.51%). The results indicated that the differences of soil organic carbon, total nitrogen, pH and moisture content from marine animal excreta might be the main reasons to bacterial abundance and diversity in the soil profiles.
     (2) Distribution characteristics of bacteria community in soil of typical biome of western Antarctic
     Distribution characteristics of bacteria community were studied in six typical soils collected on Fildes peninsula of western Antarctic. It was found that soil physicochemical properties, soil invertase, phosphatase, urease activity and soil bacterial abundance were much higher in the animal colonies and human activity areas than background soils. Soil invertase and phosphatase were significantly positively correlated with soil total carbon, total nitrogen and total phosphorus content. Bacterial abundance was significantly positively correlated with soil phosphatase (p=0.006), urease (p<0.001), moisture (p<0.05), total carbon (p<0.001), total nitrogen (p=0.58), C/N (p=0.52) and total phosphorus content (p=0.35). Molecular fingerprint shows that the soils in penguin, seal colonies and human areas contained complex bacteria community structure, and some dominant bands enrichment in the upper mapping. The predominant bacteria bacterium categories are:Betaproteobacteria, Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Chloroflexi, Alphaproteobacteria, Gammaproteobacteria, Acidobacteria, Gemmatimonadetes and Firmicutes, accounting for86.7%. Penguins, seals and human activity have a significant impact on bacteria community. Background regions have a high bacteria phylogenetic diversity (PD=85.79) and phylotype richness (OUT=976), whereas animal and human activities reduce the diversity. Pearson correlation analysis showed that soil TN (p=0.0031; p<0.001), TC (p=0.011; p=0.0024), C/N (p=0.031; p=0.0028) and Zn (p=0.030; p=0.025) contents are the major environmental factors affecting soil bacteria phylogenetic diversity and phylotype richness. The relative abundance of Bacteroides were significantly positively correlated with soil carbon and nitrogen (p=0.003; p=0.007), with a negative correlation for the relative abundance Betaproteobacteria and Actinobacteria.
     (3) The Y2lake sedimentary section bacteria changes and the historical relationship of climate change
     Distribution characteristics of bacteria community were studied in sediments collected on Y2lake sediment of Fildes peninsula, western Antarctic. It was found twelve kinds of physicochemical indicators show a high consistency with depth change and were divided into four regions:0to7cm, the surface is high, and decreases with depth;7to23cm, increases with depth slowly;23to34cm, significantly reduced, the lowest value in the32cm;34to48cm, increase with depth. The whole fingerprint can be divided into three regions:1to22cm, without a big change in the overall stripe;23to36cm obviously increasing in bands richness and dominant bands;37to48cm without a large range. Bacterial abundance in depth distribution was also divided into four sections:the first surface containing a high bacteria abundance and decreasing with depth (0to8cm); the second section slowly increasing until23cm; the third section with a significant increase (23to32cm); the last one a slowly lower increasing speed (32to48cm), and thus the maximum appeared in the32cm. The advantage of bacteria bacterium categories are: Betaproteobacteria, Actinobacteria, Gemmatimonadetes, Bacteroidetes, Acidobacteria, Gammaproteobacteria and Chloroflexi, occupy87.2%of all. Combination of physicochemical properties, DGGE fingerprint, quantitative PCR, and454sequencing results, we found the penguin populations and excretion significantly affect the diversity of bacterial community structure in the sedimentary.
     (4) Phosphatase activity characteristics in soil profiles and effects on the phosphorus cycle
     Alkaline phosphatase activity (APA) were determined in the soil profiles collected from Vesfold Hills, Lasemann Hills in east Antarctica, and Ny-Alesund in Arctic. Soil APA showed a regular distribution with depths, the maximum appeared in surface soil layers, and then decreased with depths. APA showed a significant positive correlation with soil organic carbon, total nitrogen contents, and the contents of phosphorus fractions, whereas soil APA showed a negative correlation with Cu and Zn contents, suggesting that heavy metals might limit the soil APA. Phosphine increased first in soil surface layer and then decreased with depth. Alkaline phosphatase was significantly affected phosphine along with the profile distribution. Overall, the activity of soil phosphorus was mostly affected by carbon, nitrogen and the other organic matters in the polar regions, and thus soil phosphatase activity can be used as an important indicator for soil microbial activity and soil fertility.
引文
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