长期施肥对土壤氨氧化微生物的影响

长期施肥可改变土壤碳氮等养分供应,进而影响微生物数量与群落组成。本研究基于棕壤长期定位实验站,分析不同施肥方式下(不施肥,CK;低量无机氮肥,N₂;高量无机氮肥,N₄;有机无机氮肥配施,M₂N₂)土壤氨氧化古菌(AOA)和细菌(AOB)的变化,为土壤氮素转化的微生物学机制和培肥土壤提供依据。结果表明:不同施肥方式下,土壤AOA与AOB的数量比值为2.28～61.95。与CK相比,施肥后土壤AOA数量降低了1.6%～13.6%。N₄处理AOB数量随土壤深度增加呈先降低后升高的趋势,其他处理则相反。土壤AOB群落Shannon多样性指数、均匀度指数和Simpson指数均高于AOA。M₂N₂处理0～20 cm土层土壤AOB多样性增加,但AOA多样性降低。土壤AOB主要因土壤深度发生聚类,AOB和AOA均未因施肥方式发生聚类。综上,长期施肥改变了土壤AOA和AOB的构成状况,AOA对环境变化较为敏感,AOB较为丰富和稳定。     

酸性土壤氨氧化微生物及其影响因素研究进展

自然条件变化和人类活动不仅加剧了土壤酸化,扩大了酸性土壤面积,而且严重影响了土壤氮循环。氨氧化过程作为硝化作用的限速步骤,是全球氮循环的核心环节,受到国内外研究者的广泛关注。探究酸性土壤氨氧化作用及其功能微生物对完善氮循环机制和促进土壤养分循环具有重要意义。本文主要综述了土壤中氨氧化代谢途径,对比了氨氧化细菌(ammoniaoxidizing bacteria, AOB)、氨氧化古菌(ammonia-oxidizing archaea, AOA)和全程硝化菌(complete ammoniaoxidizers,Comammox)对酸性土壤氨氧化作用的相对贡献,分析了微生物内源功能差异及pH、底物浓度等外部环境因素对氨氧化微生物丰度、活性和群落结构的影响,最后对氨氧化微生物研究进行了展望,以期为酸性土壤氨氧化作用研究和微生物修复技术应用与实践提供科学参考。     

Growth of ammonia-oxidizing archaea in soil microcosms is inhibited by acetylene

Autotrophic ammonia-oxidizing bacteria were considered to be responsible for the majority of ammonia oxidation in soil until the recent discovery of the autotrophic ammonia-oxidizing archaea. To assess the relative contributions of bacterial and archaeal ammonia oxidizers to soil ammonia oxidation, their growth was analysed during active nitrification in soil microcosms incubated for 30 days at 30 °C, and the effect of an inhibitor of ammonia oxidation (acetylene) on their growth and soil nitrification kinetics was determined. Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial ammonia oxidizer 16S rRNA genes did not detect any change in their community composition during incubation, and quantitative PCR (qPCR) analysis of bacterial amoA genes indicated a small decrease in abundance in control and acetylene-containing microcosms. DGGE fingerprints of archaeal amoA and 16S rRNA genes demonstrated changes in the relative abundance of specific crenarchaeal phylotypes during active nitrification. Growth was also indicated by increases in crenarchaeal amoA gene copy number, determined by qPCR. In microcosms containing acetylene, nitrification and growth of the crenarchaeal phylotypes were suppressed, suggesting that these crenarchaea are ammonia oxidizers. Growth of only archaeal but not bacterial ammonia oxidizers occurred in microcosms with active nitrification, indicating that ammonia oxidation was mostly due to archaea in the conditions of the present study.     

宏基因组技术研究泥岩母质发育的三种不同pH紫色土硝化微生物群落演变规律

[目的]揭示典型农田旱地紫色土硝化微生物的群落组成及其对pH的响应规律.[方法]针对同一母质发育但pH差异显著的3种紫色土,利用宏基因组技术深度测序研究土壤中硝化微生物丰度和群落,包括氨氧化古菌(ammonia-oxidizing archaea,AOA),氨氧化细菌(ammonia-oxidizing bacteri...     

攀枝花不同海拔高度烤烟农田红壤中氨氧化细菌与氨氧化古菌的群落结构分析

为探究攀枝花干热河谷区农田土壤氨氧化古菌（Ammonia oxidizing archaea,AOA）与氨氧化细菌（Ammonia oxidizing bacteria,AOB）群落对海拔高度的响应特征,深入认识该区域的氮素循环过程。以攀枝花米易县不同海拔（1600 m、1800 m和2000 m）农田红壤为研究对象,运用化学分析和末端限制性片段长度多态性（Terminal restriction fragment length polymorphism,T-RFLP）分别测定土壤理化性质、AOA和AOB群落组成及多样性,研究不同海拔农田土壤中AOA和AOB群落变异及其驱动因子。研究结果显示,不同海拔农田土壤pH均小于7,土壤有机碳（SOC）、全氮（TN）、速效钾（AK）和铵态氮（NH₄⁺-N）含量随海拔升高而降低,碱解氮（AN）、有效磷（AP）和硝态氮（NO₃^--N）含量随海拔升高先增加后降低;随海拔升高,AOA群落多样性指数增加,而AOB群落多样性指数先增加后降低;AOA以亚硝基球菌属（Nitrososphaera）为优势菌群,AOB以亚硝化螺菌属（Nitrosospira）为优势菌群;土壤有机碳（SOC）、速效钾（AK）和硝态氮（NO₃^--N）是影响该区域农田土壤AOA和AOB群落发育的主要因子。总体而言,攀枝花干热河谷区不同海拔农田土壤AOA和AOB群落结构变化明显,土壤硝态氮、速效钾和有机碳是影响AOA和AOB群落结构变异的主要因子;研究结果可为揭示干热河谷区农田红壤氮循环相关微生物的海拔分布格局提供理论依据。     

Lower Abundance of Ammonia-Oxidizing Archaea Than Ammonia-Oxidizing Bacteria Detected in the Subsurface Sediments of the Northern South China Sea

Diversity and abundance of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in samples of the northern South China Sea subsurface sediment were assessed by analyzing the amoA gene sequences retrieved from the samples. The microbial diversity was assessed using rarefaction and phylogenetic analyses. The deep-sea subsurface sediments harbored diverse and distinct AOA and AOB communities, but the abundance of AOA was lower than that of AOB, consistent with many other studies about bacteria and archaea in subsurface sediments. Diversity of AOA shown in the OTUs and Shannon index was correlated with the concentration of nitrite in the Pearson analysis, but no obvious relationships between the diversity or abundance of AOB and the physicochemical parameters could be identified in the present study, indicating the concentration of ammonium may not be an important factor to determine the diversity and abundance of ammonia-oxidizing prokaryotes in the subsurface sediments. Additionally, Nitrosomonas-like AOB was found to be dominant in subsurface sediments of the northern South China Sea showing a different adaption strategy comparing with some Nitrosospira-like AOB lineages. Concentration of nitrite was correlated with diversity of AOA, but no correlations between diversity and abundance of AOB and the physicochemical parameters were established in the study. Supplementary materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental files.     

施肥方式对干旱半干旱地区土壤氨氧化微生物数量和群落的影响

氨氧化是硝化作用的限速步骤,也是评估土壤氮循环和提高氮肥利用效率的重要指标。以内蒙古农牧业科学院旱作实验站长期定位实验为基础,通过实时荧光定量PCR和末端限制性片段长度多态性分析,研究了5种施肥方式（单施氮肥、单施有机肥、氮磷钾配施、有机无机配施和不施肥）对土壤氨氧化古菌（AOA）和氨氧化细菌（AOB）群落丰度、结构和活性的影响。结果表明：单施氮肥、氮磷钾肥配施以及有机无机肥配施均能显著提高AOB的丰度以及土壤硝化潜势。Nitrosospiria cluster 3a.1是不施肥土壤中主要的AOB种群,而施用氮肥后优势种群转变为Nitrosospiria cluster 3a.2。Nitrosospiria cluster 3b的比例在施用有机肥处理土壤中显著升高。在干旱半干旱地区,土壤pH和含水量是解释AOB群落结构变化的关键环境因子。AOA的丰度在单独施用氮肥处理中显著升高,但不同施肥方式对AOA的群落结构没有显著影响。     

Occurrence of ammonia-oxidizing Archaea in activated sludges of a laboratory scale reactor and two wastewater treatment plants

Aims:  Characterization of the ammonia-oxidizing archaea (AOA) community in activated sludge from a nitrogen removal bioreactor and wastewater treatment plants (WWTPs).
Methods and Results:  Three primer sets specific for ammonia mono-oxygenase α -subunit ( amoA ) were used to construct clone libraries for activated sludge sample from a nitrogen removal bioreactor. One primer set resulted in strong nonspecific PCR products. The other two clone libraries retrieved both shared and unique AOA amoA sequences. One primer set was chosen to study the AOA communities of activated sludge samples from Shatin and Stanley WWTPs. In total, 18 AOA amoA sequences were recovered and compared to the previous reported sequences. A phylogenetic analysis indicated that sequences found in this study fell into three clusters.
Conclusions:  Different primers resulted in varied AOA communities from the same sample. The AOA found from Hong Kong WWTPs were closely similar to those from sediment and soil, but distinct from those from activated sludge in other places. A comparison of clone libraries between Shatin WWTP and bioreactor indicated the AOA community significantly shifted only after 30-day enrichment.
Significance and Impact of the Study:  This study confirmed the occurrence of AOA in a laboratory scale nitrogen removal bioreactor and Hong Kong WWTPs treating saline or freshwater wastewater. AOA communities found in this study were significantly different from those found in other places. To retrieve diverse AOA communities from environmental samples, a combination of different primers for the amoA gene is needed.     

亚热带森林转换对土壤氮转化关键功能微生物群落的影响

近年来,由于林地开发和商品林建设等原因,我国亚热带地区大量天然林和次生林经皆伐改造为林分结构简单、树种单一的人工林。氮(N)素是维持森林植被生长和系统初级生产力的重要因子,土壤微生物驱动了森林土壤N转化的关键过程。然而,目前亚热带森林转换对土壤N转化微生物群落的影响仍不清晰。以湖南芦头森林生态系统国家定位观测研究站内典型次生林(CS)及由其转换而成的油茶(YC)、黄桃(HT)、杨梅(YM)和杉木(SM)四种人工林为研究对象,采用实时荧光定量PCR和高通量测序等方法,研究了各林分土壤性质、固N菌和氨氧化微生物功能基因丰度、群落特征及相互关系,旨在探讨亚热带森林转换后土壤N转化关键过程(固N和氨氧化作用)的功能微生物群落变化及驱动因素。结果表明:森林转换显著改变了土壤碳(C)、N含量,降低了土壤nifH基因丰度、固N菌和氨氧化细菌的群落α多样性,但提高了氨氧化微生物amoA基因丰度和氨氧化古菌的群落α多样性;并且,森林转换通过改变各功能微生物优势菌群(如变形菌、蓝细菌、泉古菌和奇古菌等)的相对丰度,显著影响了土壤固N菌和氨氧化微生物的群落组成;冗余分析和结构方程模型表明,土壤有机碳、全氮 、铵态氮含量和pH是驱动土壤固N菌和氨氧化微生物群落变化的关键因素。森林转换后,合理的施肥方式有利于人工林土壤固N菌和氨氧化微生物的群落恢复。研究结果为转换后单一人工林土壤养分恢复、生产力的提高和可持续经营提供了科学依据。     

秸秆还田对玉米根际氨氧化微生物群落及红壤硝化潜势的影响

为探讨酸性红壤根际氨氧化微生物群落以及硝化作用对不同秸秆还田处理的响应,基于中国科学院鹰潭红壤生态实验站设置的秸秆还田长期试验平台(9年),采用荧光定量PCR和高通量测序技术,研究不同秸秆还田处理(不施肥(CK);氮磷钾肥(NPK);氮磷钾肥+秸秆(NPKS);氮磷钾肥+秸秆猪粪配施(NPKSM);氮磷钾肥+秸秆生物炭(NPKB))下玉米根际土壤氨氧化古菌(ammonia-oxidizing archaea, AOA)和细菌(ammonia-oxidizing bacteria, AOB)丰度和群落结构的变化,揭示了秸秆还田对根际氨氧化微生物群落结构和硝化潜势(potential nitrification activity, PNA)的影响机制。结果发现：相比CK和NPK处理,秸秆还田显著提高了土壤养分含量和硝化潜势,其中有机碳(SOC)、全氮(TN)、全磷(TP)、速效磷(AP)、速效钾(AK)、硝态氮(NO~-₃-N)和铵态氮(NH~+₄-N)含量显著增加,NPKSM处理对土壤肥力提升效果最佳。AOA的硝化潜势显著高于AOB,表明AOA...     

轮作模式对冬小麦土壤氨氧化微生物群落多样性和组成的影响

农田温室气体减排已成为农业绿色发展的重要内容,驱动温室气体氧化亚氮(N₂O)产生的氨氧化微生物受到了研究者们的广泛关注。为探究轮作模式对土壤氨氧化微生物群落的影响,基于田间定位试验,研究了夏红小豆-冬小麦、夏绿豆-冬小麦、夏花生-冬小麦、夏大豆-冬小麦和夏玉米-冬小麦5种轮作模式中冬小麦根际和非根际土壤氨氧化古菌(AOA)和氨氧化细菌(AOB)的群落组成和多样性变化特征。结果表明:与夏玉米-冬小麦模式相比,豆禾轮作模式增加了根际土中有机碳和硝态氮含量,以及非根际土中全氮和铵态氮含量。豆禾轮作模式降低了非根际土壤中AOA群落的ACE指数和Chao1指数,并显著降低根际土中AOB群落的ACE指数和Chao1指数(P<0.05)。豆禾轮作显著增加AOA群落中泉古菌门(Crenarchaeota)和AOB群落中变形菌门(Proteobacteria)某些类群的相对丰度(P<0.05)。根际土中豆禾轮作模式与麦玉模式的AOA群落结构发生明显分离,而非根际土中豆禾轮作模式与麦玉模式的AOB群落发生分离(P<0.05)。研究结果表明:豆禾轮作种植改变了AOA和AOB的群落结构和多样性,土壤pH值和速效氮含量是驱动AOA和AOB群落结构变化的重要因子,且根际与非根际土壤中氨氧化微生物存在生态位分离。     

Responses of soil N2O emissions to experimental warming regulated by soil moisture in an alpine steppe

土壤氧化亚氮(N₂O)排放是大气N₂O不可忽视的来源。然而, 目前学术界在气候变暖对土壤N₂O排放影响方面的认识仍存在较大争议, 且调控土壤N₂O排放的微生物机制尚不明确。为此, 该研究以青藏高原高寒草原生态系统为研究对象, 使用透明开顶箱(OTCs)模拟气候变暖, 并基于静态箱法测定了2014和2015年生长季(5-10月)的土壤N₂O通量, 同时利用定量PCR技术测定了表层(0-10 cm)土壤中氨氧化古菌(AOA)和氨氧化细菌(AOB)的基因丰度。结果显示: 增温处理导致2014和2015年生长季表层(0-10 cm)土壤温度分别升高了1.7 ℃和1.6 ℃, 土壤体积含水量下降了2.5%和3.3%, 其他的土壤理化性质没有发生显著变化。土壤N₂O通量呈现年际差异, 2014和2015年生长季的平均值分别为3.23和1.47 μg·m ^-2·h ^-1, 然而, 增温处理并没有显著改变土壤N₂O通量。2014年生长季主导硝化作用的AOA和AOB的基因丰度分别为5.0 × 10 ⁷和4.7 × 10 ⁵拷贝·g ^-1, 2015年为15.2 × 10 ⁷和10.0 × 10 ⁵拷贝·g ^-1。尽管基因丰度存在显著的年际差异, 但在两年中与对照相比并未发生显著变化。在生长季尺度上, 增温导致的土壤N₂O变化量与其引起的土壤水分变化量之间显著正相关, 而与土壤温度的变化量之间没有显著相关关系。以上结果表明, 增温导致的土壤干旱会抑制土壤N₂O通量对增温的响应, 意味着未来评估气候变暖情景下土壤N₂O排放量时需考虑增温引发的土壤干旱等间接效应。     

Ammonia-oxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils

Increasing evidence demonstrated the involvement of ammonia-oxidizing archaea (AOA) in the global nitrogen cycle, but the relative contributions of AOA and ammonia-oxidizing bacteria (AOB) to ammonia oxidation are still in debate. Previous studies suggest that AOA would be more adapted to ammonia-limited oligotrophic conditions, which seems to be favored by protonation of ammonia, turning into ammonium in low-pH environments. Here, we investigated the autotrophic nitrification activity of AOA and AOB in five strongly acidic soils (pH<4.50) during microcosm incubation for 30 days. Significantly positive correlations between nitrate concentration and amoA gene abundance of AOA, but not of AOB, were observed during the active nitrification. ¹³CO₂-DNA-stable isotope probing results showed significant assimilation of ¹³C-labeled carbon source into the amoA gene of AOA, but not of AOB, in one of the selected soil samples. High levels of thaumarchaeal amoA gene abundance were observed during the active nitrification, coupled with increasing intensity of two denaturing gradient gel electrophoresis bands for specific thaumarchaeal community. Addition of the nitrification inhibitor dicyandiamide (DCD) completely inhibited the nitrification activity and CO₂ fixation by AOA, accompanied by decreasing thaumarchaeal amoA gene abundance. Bacterial amoA gene abundance decreased in all microcosms irrespective of DCD addition, and mostly showed no correlation with nitrate concentrations. Phylogenetic analysis of thaumarchaeal amoA gene and 16S rRNA gene revealed active ¹³CO₂-labeled AOA belonged to groups 1.1a-associated and 1.1b. Taken together, these results provided strong evidence that AOA have a more important role than AOB in autotrophic ammonia oxidation in strongly acidic soils.     

不同放牧对滇西北高原泥炭沼泽土壤氨氧化微生物群落的影响

氨氧化由氨氧化细菌(AOB)和氨氧化古菌(AOA)共同执行,是土壤硝化过程的第一步和限速步骤。放牧过程中,动物啃食、排泄和践踏等行为将影响土壤氨氧化微生物群落,但目前关于不同类型放牧对湿地氨氧化微生物群落结构及其多样性的影响尚不清楚。利用Illumina Mise高通量测序技术,对比研究牦牛放牧和藏香猪放养两种放牧类型对泥炭沼泽土壤氨氧化微生物群落结构及其多样性的影响。结果表明,牦牛放牧显著增加土壤容重,显著降低土壤pH、TN、TOC、NH⁺₄-N和NO^-₃-N含量;藏香猪放养显著增加土壤NO^-₃-N含量和硝化潜势(PNR)。牦牛放牧显著降低土壤AOA的丰富度和AOB的α多样性,藏香猪放养降低土壤AOA的α多样性和AOB的丰富度。放牧显著降低泉古菌门(Crenarchaeota)的相对丰度。AOA的α多样性与土壤NO^-₃-N含量和PNR呈显著负相关。AOB的α多样性与pH、TOC、TN和NH⁺     

河口生态系统氨氧化菌生态学研究进展

由amoA基因编码的氨单加氧酶(AMO)所调控的氨氧化作用,是硝化作用的限速步骤和中心环节,而含有amoA基因的氨氧化细菌(AOB)和氨氧化古菌(AOA)多样性与环境因子关系密切,对缓解河口生态系统因人类活动造成的富营养化等环境问题具有特别重要的意义。水、陆和海交汇形成高度变异的具环境因子梯度的河口生态系统,是研究AOA和AOB生态学的天然实验室。河口AOA与AOB的群落组成、丰富度特征和生物有效性,与河口主要环境因子盐度、富营养化程度、植被、温度、碳、氮、硫、铁等,尤其是对盐度和富营养化有着较为强烈的响应。AOA和AOB多样性变化规律及其与河口特有的环境因子之间的相关性,应当是今后我国河口氨氧化菌研究的方向和重点。包括:(1)建立有效的氨氧化菌活性评价方法;(2)研究AOA的同化作用方式;(3)依据氨氧化菌分类和组成对河口环境变化的适应进化机制,建议可作为指示河口环境质量变化的生物标记;(4)将传统的分离培养方法与现代分子生物学研究方法相结合,筛选我国河口高效的氨氧化菌,并将其应用于生产。     

长期施加氮肥及氧化钙调节对酸性土壤硝化作用及氨氧化微生物的影响

以长期施加氮肥及添加氧化钙调节的酸性土壤为研究对象,运用定量PCR和DGGE技术,探讨了土壤氨氧化微生物及硝化作用对不同施肥处理及氧化钙调节的响应。长期施化学氮肥导致酸性土壤p H(KCl)值(3.35—3.47)和硝化潜势(0.02—0.14μg NO-2-N g-1土壤h-1)进一步降低,而添加Ca O后土壤酸化得以缓解(p H值4.10—4.46),土壤硝化潜势(0.22—0.34μg NO-2-N g-1土h-1)显著增加。同时,添加Ca O处理对氨氧化古菌(AOA)的群落结构无明显影响,但明显提高了各施肥处理土壤中氨氧化细菌(AOB)的群落多样性,加Ca O处理的土壤中,AOA的数量降低而AOB的数量增加。这些结果表明虽然酸性土壤中AOA在数量和活性上占主导优势,AOB在功能上冗余,但当添加Ca O后,AOA和AOB对环境变化迅速作出响应,并根据其不同的生态位需求重新分配优势地位,二者交替作用共同驱动酸性土壤硝化作用。     

南美白对虾养殖底泥中氨氧化细菌与氨氧化古菌多态性分析

【目的】本研究皆在了解虾养殖底泥中氨氧化细菌与氨氧化古菌群落多态性。【方法】以功能基因为基础,构建氨氧化细菌(AOB)与氨氧化古菌(AOA)的氨单加氧酶α亚基基因(amoA)克隆文库。利用限制性片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)技术将克隆文库阳性克隆子进行归类分析分成若干个可操作分类单元(Operational Taxa Units,OTUs)。【结果】通过序列多态性分析,表明AOB amoA基因克隆文库中所有序列都属于变形杆菌门β亚纲(β-Proteobacteria)中的亚硝化单细胞菌属(Nitrosomonas)及Nitrosomonas-like,未发现亚硝化螺旋菌属(Nitrosospira)。AOA amoA基因克隆文库中只有一个OTU序列属于未分类的古菌(Unclassified-Archaea),其余序列都属于泉古菌门(Crenarchaeote)。AOA群落结构单一且存在一个绝对优势类群OTU3,其克隆子数目占克隆文库的57.45%。AOB和AOA amoA基因克隆文库分别包括13个OTUs和9个OTUs,其文库覆盖率分别为73.47%和90.43%。AOB amoA基因克隆文库的Shannon-Wiener指数、Evenness指数、Simpson指数、Richness指数均高于AOA。【结论】虾养殖塘底泥中存在氨氧化古菌的amoA基因,且多态性低于氨氧化细菌,表明氨氧化古菌在虾养殖塘底泥的氮循环中可能具有重要的作用。     

小兴安岭酸性泥炭土硝化作用的类型及其驱动因子

以小兴安岭酸性森林泥炭土为研究对象,通过加入10 mL·L^-1乙炔及不同浓度的外源硫酸铵(0、1.2、6.0 mmol N·kg^-1)进行硝化培养试验,探究酸性泥炭土中硝化作用类型及主要驱动因子.结果表明:无论有无外加氮源,酸性泥炭土均存在较强的矿化作用(0.9～1.4 mg N·kg^-1·d^-1),经过2周的培养均发生了硝化作用(0.4～0.6 mg N·kg^-1·d^-1),且不同浓度硫酸铵处理之间无显著差异;而乙炔处理虽有较强的矿化作用(0.8 mg N·kg^-1·d^-1),但未发生明显的硝化作用(0 mg N·kg^-1·d^-1),说明该酸性泥炭土以自养硝化为主,外源无机氮源浓度对硝化作用无显著影响,硝化底物NH₃的主要来源不是外源硫酸铵,更可能来源于土壤中有机氮的矿化.培养0～14 d,无论有无外加氮源,酸性泥炭土氨氧化细菌(AOB)和古菌(AOA)丰度均显著增加,但不同浓度硫酸铵处理间无显著差异,表明外源无机氮浓度对氨氧化微生物的生长无显著促进作用.与不加乙炔的对照相比,乙炔处理AOB和AOA丰度随时间均无显著变化,推测AOA与AOB在该酸性泥炭土的硝化过程中都可能起一定的作用.