东湖表层沉积物中氨氧化古菌和氨氧化细菌丰度及多样性研究

研究通过高通量测序和荧光定量PCR等分子生物学分析方法, 以氨单加氧酶基因(amoA)为分子标记, 研究了东湖表层沉积物中AOA和AOB的群落多样性、丰度及其与环境因子的关系。结果表明, 东湖沉积物AOA主要为Nitrosopumilus, 其群落结构与沉积物中总氮含量显著相关, 而AOB主要为Nitrosomonas, 群落结构与沉积物中总有机碳和总磷显著相关。此外, 不同季节AOA丰度均高于AOB, 且沉积物AOA数量与温度呈显著负相关, 但AOB丰度变化不明显。东湖沉积物中AOA可能主导了氨氧化过程。     

季节性冻结初期川西亚高山/高山森林土壤细菌多样性

高山/亚高山显著的季节性冻结过程可能对土壤细菌多样性产生重要影响。为了解季节性冻结初期土壤完全冻结前后川西亚高山/高山森林群落土壤细菌多样性变化特征,于2008年11月5日(土壤冻结前期)—11月25日(土壤完全冻结期)期间,采用PCR-DGGE技术同步研究了原始冷杉(Abies faxoniana)林(PF)、针阔混交林(MF)和次生冷杉林(SF)的土壤细菌群落多样性变化特征。土壤完全冻结后,3个森林群落仍然具有较高的土壤细菌多样性。3个森林的土壤细菌类群总数在土壤冻结前表现为MFSFPF,但在土壤完全冻结后表现为PFMFSF。土壤冻结明显降低了土壤细菌多样性,但提高了土壤细菌群落的优势度。冻结作用对土壤细菌群落的影响随着土壤深度增加而降低,随着海拔升高而降低。这些结果表明季节性冻结过程对亚高山/高山森林土壤细菌多样性有着显著的影响,这对深入认识冬季土壤生态过程具有重要意义。     

川西亚高山/高山森林土壤氧化还原酶活性及其对季节性冻融的响应

川西亚高山/高山森林土壤通常具有明显的季节性冻融特征。为深入了解川西亚高山/高山森林冬季土壤生态过程,于2008年11月-2009年10月,在土壤初冻期、冻结期和融化期及生长季节,研究了不同海拔(3582 m、3292 m和3023 m)岷江冷杉林的土壤氧化还原酶活性及其对土壤冻融的响应。土壤冻结时间和冻融循环次数随海拔的增加而增加。冻融格局显著影响了土壤氧化还原酶活性,但不同土壤酶在不同海拔表现出明显差异。土壤过氧化物酶和脱氢酶活性受初冻期冻融循环和温度降低影响显著下降,而过氧化氢酶活性明显上升。3种土壤氧化还原酶活性在土壤温度相对稳定的冻结期变化不显著,但在融化期随着土壤温度急剧增加经历一个明显的活性高峰后快速降低,且冻结时间最长和冻融循环次数最多的3582 m变化更为显著。此外,海拔和土层的交互作用显著影响了过氧化物物活性,但对脱氢酶和过氧化氢酶活性不显著。脱氢酶活性与土壤温度极显著相关,但过氧化物酶和过氧化氢酶活性与土壤温度的相关性随海拔差异而不同。这些结果表明川西亚高山/高山森林冬季土壤氧化还原酶仍然具有较高的活性,但受到季节性冻融及其变化的显著影响。     

川西亚高山/高山森林土壤线虫多样性

为了解青藏高原东缘亚高山/高山森林土壤线虫多样性,于2015年7月以该地区岷江冷杉原始林、混交林和次生林为研究对象,采用淘洗-过筛-蔗糖离心的方法分离土壤线虫,研究了3个海拔森林土壤线虫群落的组成与结构特征.结果表明: 共捕获线虫37950条,隶属于20科27属,平均为4217 条·100 g^-1干土,原始林以丝尾垫刃属为优势属,混交林和次生林以丝尾垫刃属和拟盘旋属为优势属,且优势属个体数量受林型的影响显著.原始林和次生林的优势营养类群为食真菌线虫,混交林则为食细菌线虫.土壤线虫c-p (colonizer-persister)类群c-p 1、c-p 2、 c-p 3和c-p 4数量分别占总数的6.1%、51.1%、30.0%和12.7%.3个海拔森林土壤线虫的自由生活线虫成熟度指数(MI)、总成熟度指数(∑MI)和植物寄生线虫成熟度指数(PPI)随海拔增加而逐渐降低.土壤线虫通路指数(NCR)在混交林高于0.5,在原始林和次生林低于0.5.林型显著影响了土壤线虫成熟度和NCR指数,但林型、土层及二者的交互作用对多样性指数影响不显著.川西亚高山/高山不同森林土壤线虫的组成、营养结构和能流通道存在明显差异,为深入理解土壤线虫在该区森林土壤生态过程中的作用提供了参考.     

冻融末期川西亚高山/高山森林土壤水解酶活性特征

冻融末期是连接冬季与生长季节的关键时期,期间强烈的温度变化可能深刻影响土壤生态过程.为了解冻融末期川西亚高山/高山森林土壤的生化过程,2009年3月5日-4月25日土壤融化期间,研究了该区典型冷杉原始林、针阔混交林和冷杉次生林土壤转化酶、脲酶和磷酸酶(中性、酸性和碱性磷酸酶)活性特征.结果表明:在土壤完全冻结期,3个森林群落各水解酶的活性仍相对较高.在土壤融化前期,随土壤温度升高,除中性磷酸酶外,其他水解酶活性均出现了一个爆发性增高然后迅速降低的过程.随后,除转化酶外,其他水解酶活性均随土壤温度的升高而持续增高.相对于矿质土壤层,冻融末期土壤有机层的水解酶活性更高,对土壤温度变化的响应更加明显.     

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。     

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。     

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。     

施氮肥对华北平原土壤氨氧化细菌和古菌数量及群落结构的影响

利用荧光定量PCR、末端限制性片段长度多样性(T-RFLP)和基因克隆文库技术,比较了4种施氮水平(不施氮肥,0 kg N/hm~2,CK;施低水平氮肥,75 kg N/hm~2,N1;施中水平氮肥,150 kg N/hm~2,N2;施高水平氮肥,225 kg N/hm~2,N3)下华北平原地区小麦季表层(0—20 cm)土壤总细菌、氨氧化细菌(AOB)和氨氧化古菌(AOA)的丰度和群落结构。结果表明,土壤总细菌、AOB和AOA数量分别在每克干土5.74×10~9—7.50×10~9、8.89×10~6—2.66×10~7和3.83×10~8—7.78×10~8之间。不同施氮量土壤AOA数量均高于AOB数量,AOA/AOB值在81.72—14.38之间。增施氮肥显著显著提高AOB数量(P0.05),对总细菌和AOA数量的影响不显著(P0.05)。与CK相比,处理N1、N2和N3中AOB数量分别提高了0.64、1.50和1.99倍。增施氮肥显著改变了AOB和AOA的群落结构,且不同施氮量处理中AOB群落结构差异更大。系统进化分析显示,施氮肥小麦土壤AOB主要为Nitrosospira属类群,分布在Cluster 3的两个分支中;AOA分布在Cluster S的4个分支中。相关性分析显示,AOB数量与全氮和铵态氮含量呈显著正相关关系,与土壤pH和碳氮比呈显著负相关关系(P0.05);AOA数量与硝态氮含量和土壤pH呈显著正相关关系,与铵态氮含量呈显著负相关关系(P0.05)。研究结果表明:增施氮肥可显著改变华北平原地区碱性土壤AOB数量与群落结构,该地区小麦土壤中AOB比AOA对氮肥响应更敏感。     

氨氧化古菌的生态学研究进展

上百年来细菌一直被认为是地球氨氧化过程的主要驱动者,2005年海洋中分离到迄今唯一的非极端环境泉古菌,发现其氧化氨态氮获得能源生长,是氨氧化古菌。氨氧化古菌和细菌对地球氨氧化过程的相对贡献率,是目前全球氮循环研究最重要的微生物生态学问题之一。已有的证据表明古菌在海洋氨氧化过程中发挥了重要作用,细菌则是土壤氨氧化过程的主要驱动者。本文重点探讨了原位自然环境下氨氧化古菌的生态学研究进展。     

土壤动物对川西高山/亚高山森林凋落物分解的贡献

采用凋落物分解袋法,研究了土壤动物对川西高山/亚高山森林代表性植物康定柳、方枝柏、红桦和岷江冷杉凋落物在分解第一年(2011年11月-2012年10月)不同关键时期质量损失的贡献.结果表明: 在凋落物第一年的分解过程中, 不同物种凋落物的分解速率大小依次为康定柳＞红桦＞岷江冷杉＞方枝柏,且均为生长季节大于冻融季节.土壤动物对凋落物分解的贡献率(P_fau)为方枝柏(26.7%)＞岷江冷杉(18.8%)＞红桦(15.7%)＞康定柳(13.2%),其中康定柳和方枝柏的P_fau在生长季节大于冻融季节,而红桦和岷江冷杉的P_fau为冻融季节大于生长季节.冻融季节土壤动物的作用与凋落物初始C、P和N/P显著相关,而生长季节则与N、C/N、木质素、木质素/纤维素显著相关.     

Effects of simulated warming on soil ammonia-oxidizing bacteria and archaea communities in an alpine forest of western Sichuan,China

Ongoing climate change, characterized by winter warming, snow cover decline and extreme weather events, is changing terrestrial ecosystem processes in high altitude and latitude regions. Winter soil processes could be particularly sensitive to climate change. In fact, winter warming and snow cover decline are interdependent in cold biomes, and have a synergistic effect on soil processes. Soil microorganisms not only play crucial roles in material cycling and energy flow, but also act as sensitive bio-indicators of climate change. However, little information is available on the effect of winter warming on forest soil ammonia-oxidizing bacteria (AOB) and archaea (AOA). The alpine and subalpine forest ecosystems on the eastern Tibet Plateau have important roles in conserving soil, holding water, and maintaining biodiversity. To understand the changes in AOB and AOA communities under climate change scenarios, an altitudinal gradient experiment in combination with soil column transplanting was conducted at the Long-term Research Station of Alpine Forest Ecosystems, which is situated in the Bipeng Valley of Lixian County, Sichuan, China. Thirty intact soil columns under an alpine forest at an altitude of 3582 m were transplanted and incubated at 3298 m and 3023 m forest sites, respectively. Compared with the 3582 m, we expected air temperature increases of 2 °C and 4 °C at the 3298 m and 3023 m, respectively. However, the temperatures in the soil organic layer (OL) and mineral soil layer (ML) increased by 0.27 °C and 0.13 °C, respectively, at 3023 m and ? 0.36 °C and ? 0.35 °C at 3298 m. Based on a previous study and with simultaneous monitoring of soil temperature, the abundances of AOB and AOA communities in both the OL and ML were measured by qPCR in December 2010 (i.e., the onset of the frozen soil period) and March 2011 (i.e., the late frozen soil period). The soil columns incubated at 3023 m had relatively higher AOB abundances and lower AOA/AOB ratios than those at 3298 m, while higher AOA abundances and AOA/AOB ratios were observed at 3298 m. The abundance of the microbial community at the late frozen period was higher than that at the onset of frozen soil, and the changes in microbial community abundance at the late frozen period were more substantial. Furthermore, the nitrate nitrogen (N) concentrations in both the OL and ML were significantly higher than ammonia N concentrations, implying that soil nitrate N is the primary component of the inorganic N pool in the alpine forest ecosystem. Additionally, the responses of AOA and AOB in the soil OL to soil column transplanting were more sensitive than the responses of those in ML. In conclusion, climate warming alters the abundance of the ammonia-oxidizing microbial community in the alpine forest ecosystem, which, in turn, might affect N cycling.     

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.     

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.     

Dynamics of ammonia-oxidizing archaea and bacteria populations and contributions to soil nitrification potentials

It is well known that the ratio of ammonia-oxidizing archaea (AOA) and bacteria (AOB) ranges widely in soils, but no data exist on what might influence this ratio, its dynamism, or how changes in relative abundance influences the potential contributions of AOA and AOB to soil nitrification. By sampling intensively from cropped-to-fallowed and fallowed-to-cropped phases of a 2-year wheat/fallow cycle, and adjacent uncultivated long-term fallowed land over a 15-month period in 2010 and 2011, evidence was obtained for seasonal and cropping phase effects on the soil nitrification potential (NP), and on the relative contributions of AOA and AOB to the NP that recovers after acetylene inactivation in the presence and absence of bacterial protein synthesis inhibitors. AOB community composition changed significantly (P⩽0.0001) in response to cropping phase, and there were both seasonal and cropping phase effects on the amoA gene copy numbers of AOA and AOB. Our study showed that the AOA:AOB shifts were generated by a combination of different phenomena: an increase in AOA amoA abundance in unfertilized treatments, compared with their AOA counterparts in the N-fertilized treatment; a larger population of AOB under the N-fertilized treatment compared with the AOB community under unfertilized treatments; and better overall persistence of AOA than AOB in the unfertilized treatments. These data illustrate the complexity of the factors that likely influence the relative contributions of AOA and AOB to nitrification under the various combinations of soil conditions and NH₄⁺-availability that exist in the field.