Ammonia-oxidizing bacteria dominates over ammonia-oxidizing archaea in a saline nitrification reactor under low DO and high nitrogen loading

A continuous nitrification reactor treating saline wastewater was operated for almost 1 year under low dissolved oxygen (DO) levels (0.15-0.5 mg/L) and high nitrogen loadings (0.26-0.52 kg-N/(m(3) day)) in four phases. The diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) were analyzed by cloning, terminal restriction fragment length polymorphism (T-RFLP) and quantitative polymerase chain reaction (qPCR). The results showed that there were only one dominant AOA species and one dominant AOB species in the reactor in all of the four experimental phases. The amoA gene of the dominant AOA only had a similarity of 89.3% with the cultured AOA species Nitrosopumilus maritimus SCM1. All of the AOB species detected in the reactor belong to Nitrosomonas genus and it was found that the AOB populations changed with the ammonium loadings and DO levels. The abundance of AOB in the reactor was ～40 times larger than that of AOA, and the ratio of AOB to AOA increased significantly up to ～2,000 to ～4,000 with the increase of ammonium loading, indicating that AOB are much more competitive than AOA in high ammonium environments and probably AOA play a less important role than AOB in the nitrification reactors.     

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.     

亚高山/高山森林土壤有机层氨氧化细菌和氨氧化古菌丰度特征

为了解季节性冻融作用对川西亚高山/高山地区土壤氨氧化微生物群落的影响,采用qPCR技术,以氨单加氧酶基因的α亚基(amoA)为标记,在生长阶段、冻结阶段、融化阶段中的9个关键时期调查了该地区不同森林群落：岷江冷杉(Abies faxoniana)原始林(PF)、岷江冷杉(A. faxoniana)和红桦(Betula albosinensis)混交林(MF)、岷江冷杉次生林(SF)土壤有机层的氨氧化细菌(ammonia-oxidizing bacteria, AOB)和氨氧化古菌(ammonia-oxidizing archaea, AOA)丰度的特征。结果表明,三个森林群落土壤有机层中都具有相当数量的氨氧化细菌和古菌,均表现出从生长阶段至冻结阶段显著降低,在冻结阶段最低,但冻结阶段后显著增加,在融化阶段为全年最高的趋势。土壤氨氧化微生物类群结构(AOA/AOB)受负积温影响明显。冻结后期三个森林群落土壤负积温最大时,AOA数量明显高于AOB,但其他关键时期土壤氨氧化微生物类群结构与群落类型密切相关。高海拔的PF群落土壤有机层表现为AOA>AOB(冻结初期除外),低海拔的SF群落中表现为AOB>AOA(冻结后期除外),而MF群落则仅在融冻期和生长季节末期表现为AOB>AOA。这些结果为认识亚高山/高山森林及其相似区域的生态过程提供了一定的科学依据。     

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.     

Ammonia concentration determines differential growth of ammonia-oxidising archaea and bacteria in soil microcosms

The first step of nitrification, oxidation of ammonia to nitrite, is performed by both ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) in soil, but their relative contributions to ammonia oxidation and existence in distinct ecological niches remain to be determined. To determine whether available ammonia concentration has a differential effect on AOA and AOB growth, soil microcosms were incubated for 28 days with ammonium at three concentrations: native (control), intermediate (20 μg NH₄⁺-N per gram of soil) and high (200 μg NH₄⁺-N per gram of soil). Quantitative PCR demonstrated growth of AOA at all concentrations, whereas AOB growth was prominent only at the highest concentration. Similarly, denaturing gradient gel electrophoresis (DGGE) analysis revealed changes in AOA communities at all ammonium concentrations, whereas AOB communities changed significantly only at the highest ammonium concentration. These results provide evidence that ammonia concentration contributes to the definition of distinct ecological niches of AOA and AOB in soil.     

高含氮稻田深层土壤的氨氧化古菌和厌氧氨氧化菌共存及对氮循环影响

随着海洋生态系统中的厌氧氨氧化反应和氨氧化古菌的发现,自然生态系统的氮循环过程被重新认识,但是目前尚无在陆地深层的相关报道。结合同位素示踪与分子生物学技术探索了稻田深层土壤中anammox与AOA的存在及特性。结果表明,在沼渣处理废水浇灌的高含氮稻田深层土壤中,anammox与AOA共存。通过构建克隆文库发现,此土壤中厌氧氨氧化菌的生物多样性相对较低,35个克隆序列只分为4个独立操作单元(OTU),代表序列与Genebank数据库中已探明的厌氧氨氧化菌Candidatus 'Kuenenia stuttgartiensis’的同源性超过95%;对氨氧化古菌的分析发现,20个克隆子共得到5个OTU,其与基因库中土壤/沉积物进化分支关系最近,序列的同源性部分超过98%。同位素示踪的初步结果表明,anammox产生的氮气占此土壤总氮气生成量的24.1%-29.8%。AOA与anammox的共存为anammox反应的广泛存在与发生提供了新思路。     

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。     

污水短程硝化体系氨氧化菌与亚硝酸盐氧化菌的生态学研究进展

短程硝化(partial nitrification, PN)是一种绿色低碳的生物脱氮创新技术,伴随厌氧氨氧化(anaerobic ammonia oxidation, Anammox)污水脱氮技术的进一步推广,短程硝化作为提供其电子受体的重要环节,已成为了污水脱氮领域的研究热点。氨氧化菌(ammonia-oxidizing bacteria,AOB)和亚硝酸盐氧化菌(nitrite-oxidizing bacteria, NOB)是该技术的核心竞争微生物,掌握这两类微生物的生态学特征,借助生态学理论和手段调控AOB淘汰NOB,提高种群的可预测性,对于实现稳定高效的短程硝化具有重要意义。本文基于生态学角度介绍了AOB和NOB基础分类、生理性能及生态位分离,重点综述了短程硝化系统中AOB和NOB的生长动力学、群落构建、环境因素和相互作用,最后对这两类微生物的未来研究重点和研究方法进行了展望,为短程硝化工艺的快速启动和稳定运行提供理论指导。     

Diversity and abundance of ammonia-oxidizing archaea and bacteria in polluted mangrove sediment

Ammonia oxidation by microorganisms is a critical process in the nitrogen cycle. Recent research results show that ammonia-oxidizing archaea (AOA) are both abundant and diverse in a range of ecosystems. In this study, we examined the abundance and diversity of AOA and ammonia-oxidizing beta-proteobacteria (AOB) in estuarine sediments in Hong Kong for two seasons using the ammonia monooxygenase A subunit gene (amoA) as molecular biomarker. Relationships between diversity and abundance of AOA and AOB and physicochemical parameters were also explored. AOB were more diverse but less abundant than AOA. A few phylogenetically distinct amoA gene clusters were evident for both AOA and AOB from the mangrove sediment. Pearson moment correlation analysis and canonical correspondence analysis (CCA) were used to explore physicochemical parameters potentially important to AOA and AOB. Metal concentrations were proposed to contribute potentially to the distributions of AOA while total phosphorus (TP) was correlated to the distributions of AOB. Quantitative PCR estimates indicated that AOA were more abundant than AOB in all samples, but the ratio of AOA/AOB (from 1.8 to 6.3) was smaller than most other studies by one to two orders. The abundance of AOA or AOB was correlated with pH and temperature while the AOA/AOB ratio was with the concentrations of ammonium. Several physicochemical factors, rather than any single one, affect the distribution patterns suggesting that a combination of factors is involved in shaping the dynamics of AOA and AOB in the mangrove ecosystem.     

Comparison of the community structures of ammonia-oxidizing bacteria and archaea in rhizoplanes of floating aquatic macrophytes

Some common floating aquatic macrophytes could remove nutrients, such as nitrogen, from eutrophic water. However, the relationship between these macrophytes and the ammonia-oxidizing microorganisms on their rhizoplanes is still unknown. In this study, we examined communities of ammonia-oxidizing archaea (AOA) and bacteria (AOB) on the rhizoplanes of common floating aquatic macrophytes (Eichhornia crassipes, Pistia stratiotes and Ipomoea aquatic) in a eutrophic reservoir.The results show that AOB were the predominant ammonia-oxidizer on the three rhizoplanes. The principal AOB were Nitrosomonas europaea and Nitrosomonas ureae clades. The principal group of AOA was most similar to the clone from activated sludge. The ratio of AOB amoA gene copies to AOA varied from 1.36 (on E. crassipes) to 41.90 (on P. stratiotes). Diversity of AOA was much lower than that of AOB in most samples, with the exception of P. stratiotes.     

Altitude ammonia-oxidizing bacteria and archaea in soils of Mount Everest

To determine the abundance and distribution of bacterial and archaeal ammonia oxidizers in alpine and permafrost soils, 12 soils at altitudes of 4000–6550 m above sea level (m a.s.l.) were collected from the northern slope of the Mount Everest (Tibetan Plateau), where the permanent snow line is at 5800–6000 m a.s.l. Communities were characterized by real-time PCR and clone sequencing by targeting on amo A genes, which putatively encode ammonia monooxygenase subunit A. Archaeal amo A abundance was greater than bacterial amo A abundance in lower altitude soils (≤5400 m a.s.l.), but this situation was reversed in higher altitude soils (≥5700 m a.s.l.). Both archaeal and bacterial amo A abundance decreased abruptly in higher altitude soils. Communities shifted from a Nitrosospira amo A cluster 3a-dominated ammonia-oxidizing bacteria community in lower altitude soils to communities dominated by a newly designated Nitrosospira ME and cluster 2-related groups and Nitrosomonas cluster 6 in higher altitude soils. All archaeal amo A sequences fell within soil and sediment clusters, and the proportions of the major archaeal amo A clusters changed between the lower altitude and the higher altitude soils. These findings imply that the shift in the relative abundance and community structure of archaeal and bacterial ammonia oxidizers may result from selection of organisms adapted to altitude-dependent environmental factors in elevated soils.     

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.     

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...     

Relative contributions of archaea and bacteria to microbial ammonia oxidation differ under different conditions during agricultural waste composting

The aim of this study was to compare the relative contribution of ammonia-oxidizing archaea (AOA) and bacteria (AOB) to nitrification during agricultural waste composting. The AOA and AOB amoA gene abundance and composition were determined by quantitative PCR and denaturing gradient gel electrophoresis (DGGE), respectively. The results showed that the archaeal amoA gene was abundant throughout the composting process, while the bacterial amoA gene abundance decreased to undetectable level during the thermophilic and cooling stages. DGGE showed more diverse archaeal amoA gene composition when the potential ammonia oxidation (PAO) rate reached peak values. A significant positive relationship was observed between the PAO rate and the archaeal amoA gene abundance (R2=0.554; P<0.001), indicating that archaea dominated ammonia oxidation during the thermophilic and cooling stages. Bacteria were also related to ammonia oxidation activity (R2=0.503; P=0.03) especially during the mesophilic and maturation stages.     

Nitrification rates in Arctic soils are associated with functionally distinct populations of ammonia-oxidizing archaea

The functioning of Arctic soil ecosystems is crucially important for global climate, and basic knowledge regarding their biogeochemical processes is lacking. Nitrogen (N) is the major limiting nutrient in these environments, and its availability is strongly dependent on nitrification. However, microbial communities driving this process remain largely uncharacterized in Arctic soils, namely those catalyzing the rate-limiting step of ammonia (NH₃) oxidation. Eleven Arctic soils were analyzed through a polyphasic approach, integrating determination of gross nitrification rates, qualitative and quantitative marker gene analyses of ammonia-oxidizing archaea (AOA) and bacteria (AOB) and enrichment of AOA in laboratory cultures. AOA were the only NH₃ oxidizers detected in five out of 11 soils and outnumbered AOB in four of the remaining six soils. The AOA identified showed great phylogenetic diversity and a multifactorial association with the soil properties, reflecting an overall distribution associated with tundra type and with several physico-chemical parameters combined. Remarkably, the different gross nitrification rates between soils were associated with five distinct AOA clades, representing the great majority of known AOA diversity in soils, which suggests differences in their nitrifying potential. This was supported by selective enrichment of two of these clades in cultures with different NH₃ oxidation rates. In addition, the enrichments provided the first direct evidence for NH₃ oxidation by an AOA from an uncharacterized Thaumarchaeota–AOA lineage. Our results indicate that AOA are functionally heterogeneous and that the selection of distinct AOA populations by the environment can be a determinant for nitrification activity and N availability in soils.     

Interactions between Thaumarchaea,Nitrospira and methanotrophs modulate autotrophic nitrification in volcanic grassland soil

Ammonium/ammonia is the sole energy substrate of ammonia oxidizers, and is also an essential nitrogen source for other microorganisms. Ammonia oxidizers therefore must compete with other soil microorganisms such as methane-oxidizing bacteria (MOB) in terrestrial ecosystems when ammonium concentrations are limiting. Here we report on the interactions between nitrifying communities dominated by ammonia-oxidizing archaea (AOA) and Nitrospira-like nitrite-oxidizing bacteria (NOB), and communities of MOB in controlled microcosm experiments with two levels of ammonium and methane availability. We observed strong stimulatory effects of elevated ammonium concentration on the processes of nitrification and methane oxidation as well as on the abundances of autotrophically growing nitrifiers. However, the key players in nitrification and methane oxidation, identified by stable-isotope labeling using ¹³CO₂ and ¹³CH₄, were the same under both ammonium levels, namely type 1.1a AOA, sublineage I and II Nitrospira-like NOB and Methylomicrobium-/Methylosarcina-like MOB, respectively. Ammonia-oxidizing bacteria were nearly absent, and ammonia oxidation could almost exclusively be attributed to AOA. Interestingly, although AOA functional gene abundance increased 10-fold during incubation, there was very limited evidence of autotrophic growth, suggesting a partly mixotrophic lifestyle. Furthermore, autotrophic growth of AOA and NOB was inhibited by active MOB at both ammonium levels. Our results suggest the existence of a previously overlooked competition for nitrogen between nitrifiers and methane oxidizers in soil, thus linking two of the most important biogeochemical cycles in nature.