Active Ammonia-oxidizing Bacteria Research Articles

Impact of dissolved oxygen levels on N2O emissions and metabolically active bacterial community in biological nitrogen removal

Biological nutrient removal (BNR) is an effective process for removing nitrogen from wastewater using microorganisms under different environmental conditions. However, these processes can result in nitrous oxide (N2O) emission. Dissolved oxygen (DO) levels shape bacterial communities and affect N2O emissions in BNR. Ammonia-oxidizing bacteria (AOB), the main N2O producers in the BNR process, are significantly affected by DO. The bacterial communities responding to DO at the DNA level in BNR have been elucidated. However, the metabolically active microbes are truly participant in the biodegradation process. Thus, it is worth exploring how metabolically active bulk and AOB microbial communities and their N2O production respond to the DO levels. In this study, N2O emission were measured in lab-scale sequencing batch reactors at varying DO levels. High-throughput sequencing of the metabolically active bacterial 16S rRNA and amoA genes was performed. The results indicate that increasing DO levels positively influence metabolically active bacterial biodiversity and AOB relative abundance without increasing N2O emissions. The carbon source significantly affected denitrification and nitrification in activated sludge. Optimal DO levels and adequate carbon sources can effectively reduce N2O accumulation. However, excessive aeration reduces the abundance of Zoogloea, leading to compromised sludge settling. This study revealed the effect of DO on N2O producing microorganisms based on cDNA levels, which provided an important basis for mitigating N2O emission in BNR.

Combined application of strong alkaline materials and specific organic fertilizer accelerates nitrification process of a rare earth mining soil

The extensive usage of ammonium sulfate as the leaching agent to extract rare earth elements led to widespread ammonia nitrogen (NH4+-N) pollution in the tailing soils of ion-adsorbed rare earth deposits in southern China. However, the cost-effective technologies to tackle with the long-term retention of NH4+-N in the rare earth mining soil have been largely unresolved. In this study, we developed a cost-effective approach to activate soil nitrification by the co-application of alkaline materials and organic fertilizer. The co-application of 0.3 % of organic fertilizer and 0.1 % ∼ 0.2 % of CaO or MgO or Mg(OH)2 stimulated a soil NH4+-N decrease rate of 2.01–7.58 mg kg−1 d−1 and a soil NO3−-N accumulation rate of 1.56–7.09 mg kg−1 d−1. Noting that only if the soil pH was elevated to 7.81–9.00, the NH4+-N decrease rate and NO3−-N accumulation rate were dependent on the proton consumption capacity of the alkaline materials. The application of CaCO3 could not stimulate soil nitrification possibly due to the soil pH was uncapable to be elevated to above 7.68. The qPCR, amplicon sequencing, and nitrification inhibitor batch incubation results demonstrated that organic fertilizer supplied active ammonia-oxidizing bacteria Nitrosomonas europaea. The proliferation of Nitrosomonas europaea in the alkaline materials and organic fertilizer co-applied soil was responsible for the soil nitrification. Furthermore, the application of commercial denitrifying bacteria inoculum promoted the removal of accumulated NO3−-N. The findings of this study provide a lost-cost technology to remove NH4+-N from the rare earth mining soil.

Activating soil nitrification by co-application of peanut straw biochar and organic fertilizer in a rare earth mining soil

The intensive mining activities to extract rare earth elements from ion-adsorption rare earth deposits have introduced massive amounts of ammonium into the tailing soils in southern China. Compared to the ubiquitous soil nitrification in cropland, forest, and grassland soils, however, there is no feasible strategy to alleviate the ammonium contamination in tailing soil. Herein, the feasibility to remove ammonium by adding ammonium adsorbents (e.g., biochar, activated carbon, and zeolite), alkaline materials, and organic fertilizer to the rare earth mining soil was explored. The amendment of rice straw biochar, activated carbon, or zeolite in combination with CaCO3 and organic fertilizer showed no significant effect on ammonium removal due to their limited capacity to elevate soil pH. However, the co-application of peanut straw biochar (PSBC), CaCO3, and organic fertilizer activated both the ammonia volatilization and soil nitrification processes. Specifically, the three components functioned as follows: organic fertilizer supplied active ammonia-oxidizing bacteria (AOB); PSBC stimulated AOB proliferation by elevating soil pH above 7.75; CaCO3 ameliorated soil acidity and reduced the lag time for activating soil nitrification. The soil ammonium removal and nitrate accumulation rates were positively correlated to the acid neutralization capacity of PSBC prepared at 400 °C–800 °C. The qPCR and microbial community analysis results indicated that Nitrosomonas europaea was the dominant AOB that was responsible for enhanced soil nitrification. Our findings pave the way for developing cost-effective strategies to remediate ammonium contamination in rare earth mining soils.

Reassembly of active ammonia oxidizing bacteria following invasion of exogenous microbiome in an acidic soil

Ammonia oxidation is the rate-limiting step of the global nitrogen cycle, and it is mainly catalyzed by ammonia-oxidizing bacteria (AOB) and archaea (AOA). AOA can carry out nitrification even in acidic soils, whereas AOB activity was not consistently observed in acidic soils. In this study, we manipulated two environmental variables, i.e., the microbial composition and ammonium source (urea and ammonium sulphate), in an acidic forest soil and tested their impacts on AOB abundance, diversity and activity. Soil microcosms were established either using the native forest soil (FS) or after amending the forest soil with 5% (w/w) agricultural soil (AFS) to inoculate new, exogenous microbes. Under the same microcosm incubation condition for 28 days, AOB did not grow in FS but they grew in AFS, suggesting that exogenous microbial invasion could stimulate AOB activity in the acidic soil. Urea amendment led to higher growth of AOB than ammonium sulphate amended in AFS microcosms. DNA-based stable isotope probing revealed that the active AOB lineages were dominated by Nitrosospira cluster 3a.2 and Nitrosospira cluster 10 in the ammonium sulphate supplied soil, but shifted to co-dominance of Nitrosospira cluster 3a.2 and Nitrosospira cluster 1 after urea amendment, demonstrating differential preferences of ammonia sources of phylogenetically distinct AOB lineages. Additionally, AOB abundance showed lower stability than AOA in soils subjected to these two environmental changes, suggesting AOB are more important in determining the fate of soil nitrification following short-term environmental disturbances.

Surface Ammonia-Oxidizer Abundance During the Late Summer in the West Antarctic Coastal System.

Marine ammonia oxidizers that oxidize ammonium to nitrite are abundant in polar waters, especially during the winter in the deeper mixed-layer of West Antarctic Peninsula (WAP) waters. However, the activity and abundance of ammonia-oxidizers during the summer in surface coastal Antarctic waters remain unclear. In this study, the ammonia-oxidation rates, abundance and identity of ammonia-oxidizing bacteria (AOB) and archaea (AOA) were evaluated in the marine surface layer (to 30 m depth) in Chile Bay (Greenwich Island, WAP) over three consecutive late-summer periods (2017, 2018, and 2019). Ammonia-oxidation rates of 68.31 nmol N L−1 day−1 (2018) and 37.28 nmol N L−1 day−1 (2019) were detected from illuminated 2 m seawater incubations. However, high ammonia-oxidation rates between 267.75 and 109.38 nmol N L−1 day−1 were obtained under the dark condition at 30 m in 2018 and 2019, respectively. During the late-summer sampling periods both stratifying and mixing events occurring in the water column over short timescales (February–March). Metagenomic analysis of seven nitrogen cycle modules revealed the presence of ammonia-oxidizers, such as the Archaea Nitrosopumilus and the Bacteria Nitrosomonas and Nitrosospira, with AOA often being more abundant than AOB. However, quantification of specific amoA gene transcripts showed number of AOB being two orders of magnitude higher than AOA, with Nitrosomonas representing the most transcriptionally active AOB in the surface waters. Additionally, Candidatus Nitrosopelagicus and Nitrosopumilus, phylogenetically related to surface members of the NP-ε and NP-γ clades respectively, were the predominant AOA. Our findings expand the known distribution of ammonium-oxidizers to the marine surface layer, exposing their potential ecological role in supporting the marine Antarctic system during the productive summer periods.

Reassembly of Active Ammonia Oxidizing Bacteria Following Invasion of Exogenous Microbiome in an Acidic Soil

Reassembly of Active Ammonia Oxidizing Bacteria Following Invasion of Exogenous Microbiome in an Acidic Soil

An RNA-based study of the distribution of ammonia-oxidizing microorganisms in vertical sediment

Ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) play crucial roles in ammonia nitrogen degradation in freshwater lake sediment. However, studies on ammonia-oxidizing microorganisms (AOM) in vertical sediment are still limited, especially with respect to the quantitative analysis of the vertical distribution of active AOM at the RNA level. Herein, by researching the AOM in a 75 cm vertical sediment from Lake Taihu, we found that although the total abundance of AOB throughout the whole vertical sediment surpassed that of AOA, active AOA had a closer correlation with the potential nitrification rate than active AOB. The active transcription abundance of AOM generally decreased with increasing depth, especially for AOA, while that of AOB decreased slightly. Compared to active transcription, the total abundance of AOM varied little. The community structures of AOA and AOB changed significantly with increasing depth, and Group I.1a and Nitrosomonas were the predominant clusters, respectively. Ultimately, our study demonstrated that research methods based on the RNA level can accurately reflect the vertical variation in AOM.

Active ammonia-oxidizing bacteria and archaea in wastewater treatment systems

Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are two microbial groups mediating nitrification, yet little is presently known about their abundances and community structures at the transcriptional level in wastewater treatment systems (WWTSs). This is a significant issue, as the numerical abundance of AOA or AOB at the gene level may not necessarily represent their functional role in ammonia oxidation. Using amoA genes as molecular markers, this study investigated the transcriptional abundance and community structure of active AOA and AOB in 14 WWTSs. Quantitative PCR results indicated that the transcriptional abundances of AOB amoA (averaged: 1.6 × 108 copies g−1 dry sludge) were higher than those of AOA (averaged: 3.4 × 107 copies g−1 dry sludge) in all WWTSs despite several higher abundances of AOA amoA at the gene level. Moreover, phylogenetic analysis demonstrated that Nitrosomonas europaea and unknown clusters accounted for 37.66% and 49.96% of the total AOB amoA transcripts, respectively, suggesting their dominant role in driving ammonia oxidation. Meanwhile, AOA amoA transcripts were only successfully retrieved from 3 samples, and the Nitrosospaera sister cluster dominated, accounting for 83.46%. Finally, the substrate utilization kinetics of different AOA and AOB species might play a fundamental role in shaping their niche differentiation, community composition, and functional activity. This study provides a basis for evaluating the relative contributions of ammonia-oxidizing microorganisms (AOMs) to nitrogen conversions in WWTSs.

Temperature-dependent changes in active nitrifying communities in response to field fertilization legacy

Using DNA-based stable isotope probing (SIP) in microcosms, we demonstrate the shifts in active nitrifying communities in soil from field plots imposed by 8 years of mineral fertilizer N (NPK) or manure (M) applications compared with an unfertilized control (CK), and associated shifts in their temperature sensitivity. DNA-SIP indicates that ammonia-oxidizing archaea (AOA) were labeled to a much greater extent in the CK soil (74.6%) than in the NPK-amended (19.8%) or M-amended (27.1%) soils at 28 °C. In contrast, NPK soil showed the highest labeling of ammonia-oxidizing bacteria (AOB) (23.3%) relative to M (4.03%) and CK (2.42%) soils. This is further supported by significant decreases in the 13C-amoA gene ratios of AOA/AOB in the NPK (4.14) and M (8.63) compared with CK (46.4) soils at 28 °C, while little difference was observed at 35 °C. The relative abundance of active AOA or AOB in the manure was between that in the CK and NPK soils. This may be attributable to the slower release of ammonium mineralized from manure than from urea. The active AOA in soil microcosms were dominated by Nitrososphaera viennensis-like groups regardless of fertilization history or temperature. Nitrosomonas communis-like AOB may be more adapted to the higher temperature than Nitrosospira cluster 3-like AOB. The labeling of nitrite oxidizers (nitrite-oxidizing bacteria) was lowest in NPK soil and was higher at 28 °C than 35 °C regardless of fertilization treatment. The finding indicates that intensified anthropogenic N inputs in the field may select for distinct active nitrifying communities that exhibited different temperature sensitivities.

Composition and activity of nitrifier communities in soil are unresponsive to elevated temperature and CO2, but strongly affected by drought

Nitrification is a fundamental process in terrestrial nitrogen cycling. However, detailed information on how climate change affects the structure of nitrifier communities is lacking, specifically from experiments in which multiple climate change factors are manipulated simultaneously. Consequently, our ability to predict how soil nitrogen (N) cycling will change in a future climate is limited. We conducted a field experiment in a managed grassland and simultaneously tested the effects of elevated atmospheric CO2, temperature, and drought on the abundance of active ammonia-oxidizing bacteria (AOB) and archaea (AOA), comammox (CMX) Nitrospira, and nitrite-oxidizing bacteria (NOB), and on gross mineralization and nitrification rates. We found that N transformation processes, as well as gene and transcript abundances, and nitrifier community composition were remarkably resistant to individual and interactive effects of elevated CO2 and temperature. During drought however, process rates were increased or at least maintained. At the same time, the abundance of active AOB increased probably due to higher NH4+ availability. Both, AOA and comammox Nitrospira decreased in response to drought and the active community composition of AOA and NOB was also significantly affected. In summary, our findings suggest that warming and elevated CO2 have only minor effects on nitrifier communities and soil biogeochemical variables in managed grasslands, whereas drought favors AOB and increases nitrification rates. This highlights the overriding importance of drought as a global change driver impacting on soil microbial community structure and its consequences for N cycling.

Nitrite production by ammonia-oxidizing bacteria mediates chloramine decay and resistance in a mixed-species community.

As water distribution centres increasingly switch to using chloramine to disinfect drinking water, it is of paramount importance to determine the interactions of chloramine with potential biological contaminants, such as bacterial biofilms, that are found in these systems. For example, ammonia-oxidizing bacteria (AOB) are known to accelerate the decay of chloramine in drinking water systems, but it is also known that organic compounds can increase the chloramine demand. This study expanded upon our previously published model to compare the decay of chloramine in response to alginate, Pseudomonas aeruginosa, Nitrosomonas europaea and a mixed-species nitrifying culture, exploring the contributions of microbial by-products, heterotrophic bacteria and AOBs to chloramine decay. Furthermore, the contribution of AOBs to biofilm stability during chloramination was investigated. The results demonstrate that the biofilm matrix or extracellular polymeric substances (EPS), represented by alginate in these experiments, as well as high concentrations of dead or inactive cells, can drive chloramine decay rather than any specific biochemical activity of P. aeruginosa cells. Alginate was shown to reduce chloramine concentrations in a dose-dependent manner at an average rate of 0.003mgl-1 h-1 per mgl-1 of alginate. Additionally, metabolically active AOBs mediated the decay of chloramine, which protected members of mixed-species biofilms from chloramine-mediated disinfection. Under these conditions, nitrite produced by AOBs directly reacted with chloramine to drive its decay. In contrast, biofilms of mixed-species communities that were dominated by heterotrophic bacteria due to either the absence of ammonia, or the addition of nitrification inhibitors and glucose, were highly sensitive to chloramine. These results suggest that mixed-species biofilms are protected by a combination of biofilm matrix-mediated inactivation of chloramine as well as the conversion of ammonia to nitrite through the activity of AOBs present in the community.

Land use pattern effects after 30 years of shifting cropland to fallow land on soil ammonia-oxidizer community

Sympatric differentiation of ammonia-oxidizing archaea and bacteria may occur due to differences in ammonia availability during agricultural activities such as fertilization, which shift key biological drivers of soil nitrification processes. Restoring native habitat from intensive agricultural activities (e.g. converting cropland into fallow land) reduces fertilizer inputs, which has the potential to change active ammonia-oxidizers and thus nitrification rates. We incubated soils from fields under 30 years fallow and under continuous cropping to understand shifts in ammonia-oxidizers and nitrification processes. Thirty years of land fallow practices sharply decreased the abundance and activity of soil ammonia-oxidizing bacteria compared to continuous cropping. Nitrification in cropland was driven mainly by ammonia-oxidizing bacteria while ammonia-oxidizing archaea were the main drivers of nitrification in fallow soil. Conversion of cropland into fallow land shifted the dominant active ammonia-oxidizers from ammonia-oxidizing bacteria to archaea, and the low nitrification rates in fallow soil were caused by the lack of active ammonia-oxidizing bacteria rather than N substrate availability.

Ammonia Oxidation in a Neutral Purple Soil Measured by the 13C-DNA-SIP Method

Increasing evidence suggests that ammonia oxidation in acidic soils is primarily catalyzed by ammonia-oxidizing archaea (AOA), while ammonia-oxidizing bacteria (AOB) drive ammonia oxidation in neutral and alkaline soils in which AOA overwhelmingly outnumber AOB. Therefore, neutral purple soil with a pH of 7.2 was selected to study the composition of the active ammoxidation microbial community with a stable isotope nucleic acid probe technique combined with cloning sequencing. Results showed that the nitrification rate was 9.68 mg·(kg·d)-1, and AOA and AOB were abundant in neutral purple soils. By using DNA-based stable isotope probing (SIP), we gathered strong evidence of archaeal ammonia oxidation by AOA and AOB. Phylogenetic analysis indicated that the Nitrosospira Cluster 3a.1 AOB was dominant in terms of quantity at 0 days, and the Nitrosospira Cluster 3a.2 only accounted for a small part. After 56 days of cultivation, the Nitrosospira Cluster 3a.2 replaced the Nitrosospira Cluster 3a.1 as the active AOB that dominated ammonia oxidation. The AOA that predominated quantitatively at day 0 was Nitrososphaera Subcluster 9, but after cultivation this became Nitrososphaera Subcluster 3.2/3.3. Thus, the community structure of AOA and AOB changed. Active autotrophic nitrification was found in this neutral purple soil. Sequencing analysis of the 13C-labeled DNA provided robust evidence that both archaea and bacteria played important roles in the nitrification and not all ammonia oxidizers in native soil were active in the nitrification. Phylogenetic analysis clearly showed that the dominant active archaea and bacteria during the incubation were affiliated with Nitrososphaera Subcluster 3.2/3.3 within the soil group 1.1b lineage and Nitrosospira Cluster 3a.2, respectively, which were different from the dominant ammonia oxidizers at the beginning of the incubation. These results suggest that the community structure of ammonia oxidizers can shift quickly upon changes in the substrate availability in soils.

Elevated temperature increased nitrification activity by stimulating AOB growth and activity in an acidic paddy soil

Global warming is predicted to alter the timing and magnitude of biogeochemical nitrogen cycling in paddy soils. However, little is known about its effect on active nitrifying populations. Here we investigated the responses of nitrification activity and active nitrifiers to elevated temperature in an acidic paddy soil. 13CO2-DNA-stable isotope probing (SIP), qPCR and high-throughput sequencing were used to determine active nitrifying phylotypes as well as difference in their abundance and community composition incubated at field temperature (15 °C) and elevated temperature (20 °C). Urea application led to significant production of nitrate and growth of ammonia-oxidizing bacteria (AOB) at both temperatures. Nitrification activity at elevated temperature was 148.3% and 18.5% higher than that of low temperature at day 28 and 56, respectively, accompanied by an increase in the extent of 13C-label incorporation by AOB. 13CO2-based SIP experiment indicated that both AOB and ammonia-oxidizing archaea (AOA) were involved in the nitrification activity and the active ammonia oxidizers changed from AOA to AOB with elevated temperature. Significant variation of AOA communities was observed under different temperatures. Dominant 13C-labeled nitrite-oxidizing bacteria (NOB) shifted from Nitrospira moscoviensis to Nitrospira japonica with higher temperature. Our findings emphasized that elevated temperature had pronounced effects on autotrophic nitrification which was mediated by altering relative abundance of active AOB and AOA, as well as the community composition of AOA and NOB. AOB were more adaptable than AOA with increasing abundance but no alteration of composition at elevated temperature.

Responses of Active Ammonia Oxidizers to Eutrophication and Oxygen Statuses in Taihu Freshwater Sediments

Here, we employed DNA-based stable isotope probing (SIP) and molecular biology methods to investigate active ammonia oxidizer communities in suboxic sediments (0 to –2 cm) at the micromolar oxygen level and layers (–2 to –5 cm) at nanomolar oxygen concentrations from meso-eutrophic and light-eutrophic locations in Taihu Lake. The results revealed that ammonia-oxidizing archaea (AOA) were less active in the anoxic layer of meso-eutrophic sites, while ammonia-oxidizing bacteria (AOB) were less active in suboxic sediments of light-eutrophic sites after 8 weeks of incubation. The active AOA in the meso- and light-eutrophic sediments belonged to the Nitrosopumilus, Nitrosotalea, and Nitrososphaera clusters and the Nitrosopumilus and Nitrososphaera clusters, respectively, with Nitrosopumilus cluster as the predominant AOA, which took up a higher ratio in the light-eutrophic and suboxic layers than their counterparts. The advantageous active AOB were numerically predominated by the Nitrosomonas cluster in the suboxic layers, and the Nitrosospira cluster in the anoxic layers, respectively, both of which were distributed in diverse frequencies in different eutrophication statuses. The role and community composition diversities of active ammonia oxidizers in freshwater sediments were attributed to the different eutrophication (including nitrogen and organic carbon content) and oxygen statuses.

Stimulating ammonia oxidizing bacteria (AOB) activity drives the ammonium oxidation rate in a constructed wetland (CW)

An integrated approach to document high ammonium oxidation rate in Guanjinggang constructed wetland (GJG-CW) was performed and the results showed that the substantial ammonium oxidation rate could be obtained by enhancing Ammonia Oxidizing Bacteria (AOB) activity rather than Ammonia Oxidizing Archaea (AOA) activity. In the plant-bed/ditch system, ditch center and plant-bed fringe were two active zones for NH4+-N removal with ammonium oxidation rate peaking at 2.98±0.04 and 2.15±0.02mgNkg−1d−1, respectively. The enhanced AOB activity were achieved by increasing water level fluctuations, extending hydraulic retention time (HRT) and stimulating substrate availability, which subsequently enhanced NH4+-N removal by 34.06% in GJG-CW. However, the high AOB activity was not correlated with high AOB abundance, but was instead mostly determined by specific AOB taxa, particularly Nitrosomonas, which dominated in the active AOB. The increased cell-specific AOA activity and high AOA diversity were also achieved using those engineering measures. Although the AOA activity decreased overall with extended HRT and increased NH4+-N contents in GJG-CW, AOA still played a major role on ammonium oxidation in plant-bed soil. The study illustrated that artificially enhancing AOB activity and certain species in anthropogenically polluted water ecosystems would be an effective strategy to improve NH4+-N removal.

Abundance and composition of ammonia oxidizers in response to degradation of root cap cells of rice in soil microcosms

Nitrification is a key process in the global nitrogen cycle, of which the first and rate-limiting step is catalyzed by ammonia monooxygenase. Root cap cells are one of substrates for microorganisms that thrive in the rhizosphere. The degradation of root cap cells brings about nitrification following ammonification of organic nitrogen derived from the root cap cells. This study was designed to gain insights into the response of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) to mineralized N from root cap cells and the composition of active bacterial and archaeal ammonia oxidizers in rice soil. Rice callus cells were used as a model for root cap cells, and unlabelled (12C) and 13C-labelled callus cells were allowed to decompose in aerobic soil microcosms. Real-time quantitative polymerase chain reaction (PCR), DNA-based stable isotope probing (SIP), and denaturing gradient gel electrophoresis (DGGE) were applied to determine the copy number of bacterial and archaeal amoA genes and the composition of active AOB and AOA. The growth of AOB was significantly stimulated by the addition of callus cells compared with the growth of AOA with a much lesser extent. AOB communities assimilated 13C derived from the callus cells, whereas no AOA communities grew on 13C-callus. Sequencing of the DGGE bands in the SIP experiments revealed that the AOB communities belonging to Nitrosospira spp. dominated microbial ammonia oxidation with rice callus amendment in soil. The present study suggests that root cap cells of rice significantly stimulated the growth of AOB, and the active members dominating microbial ammonia oxidation belonged to Nitrosospira spp. in rice rhizosphere.

Impact of Fungicides on the Diversity and Function of Non-target Ammonia-Oxidizing Microorganisms Residing in a Litter Soil Cover

Litter soil cover constitutes an important micro-ecosystem in sustainable viticulture having a key role in nutrient cycling and serving as a habitat of complex microbial communities. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are known to regulate nitrification in soil while little is known regarding their function and diversity in litter. We investigated the effects of two fungicides, penconazole and cyprodinil, commonly used in vineyards, on the function and diversity of total and active AOB and AOA in a microcosm study. Functional changes measured via potential nitrification and structural changes assessed via denaturating gradient gel electrophoresis (DGGE) at the DNA and RNA levels were contrasted with pesticide dissipation in the litter layer. The latter was inversely correlated with potential nitrification, which was temporarily inhibited at the initial sampling dates (0 to 21 days) when nearly 100 % of the applied pesticide amounts was still present in the litter. Fungicides induced changes in AOB and AOA communities with RNA-DGGE analysis showing a higher sensitivity. AOA were more responsive to pesticide application compared to AOB. Potential nitrification was less sensitive to the fungicides and was restored faster than structural changes, which persisted. These results support the theory of microbial redundancy for nitrification in a stressed litter environment.

三种利用方式对羊草草原土壤氨氧化细菌群落结构的影响

以内蒙古呼伦贝尔羊草草原为研究对象,利用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术及扩增产物序列分析方法比较研究了刈割、放牧和围栏封育3种利用方式对羊草草原土壤氨氧化细菌多样性和系统发育的影响,结果表明:羊草草原土壤中氨氧化细菌的优势类群为亚硝化螺旋菌属和亚硝化单孢菌属的细菌,分别占氨氧化细菌总数的72.4%和28.6%,Cluster 4和Cluster 6在各个样地中均为优势类群但所占比例不同,放牧样地中氨氧化细菌的多样性最高,逐步回归分析的结果表明,影响多样性指数的关键理化因子是土壤硝态氮含量。放牧样地中脲酶活性和硝化率都显著高于其他两种利用方式,其中,脲酶活性与土壤硝态氮和全磷含量表现出极显著的相关性,与速效磷含量表现出显著相关性;而硝化率仅与硝态氮含量表现出显著相关性。不同利用方式土壤氨氧化细菌群落与土壤理化因子的典范对应分析结果表明铵态氮的含量对氨氧化细菌群落的影响显著。;Available nitrogen is not only one of the most important limitation resources to manipulate primary productivity, but also a major factor to decide species composition in a grassland ecosystem. Chemolithotrophic nitrification is a two-step process, consisting of the conversion of ammonia to nitrite then further to nitrate. Those steps are involved two different groups of organisms, the ammonia-oxidizing bacteria (AOB) and the nitrite-oxidizing bacteria (NOB), respectively. In this study, the active AOB community was investigated by using approaches of polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and sequences analysis on the <em>Leymus chinensis</em> steppes with three of mowing, grazing and fencing treatments respectively in Hulunbeier steppe of Inner Mongolia. Sampling in August 2008 when vegetation was at its best condition. Samples were taken at depths of 0-0.20m. The community structure and diversity of the bacterial groups from the different samples was further analyzed by using different techniques, such as statistical analysis and diversity index evaluation of the band patterns etc. The results showed that grazing activity significantly increased the number of species and quantities of AOB, as well as the diversity. A total of 21 sequences representing 21 different sequence types were recovered from the DGGE gels after phylogenetic reconstructions. The results further revealed that most sequences were coming from <em>Nitrosospira</em> and <em>Nitrosomonas</em> of <em>β</em>-<em>Proteobacteria</em>, with accounting for 72.4% and 28.6% respectively. Cluster 4 of <em>Nitrosospira</em> existed in grazing, mowing and fencing plots with accounting for 25.0%, 27.2% and 26.3% respectively in their dominant AOB. And Cluster 6 of <em>Nitrosomonas</em> existed in grazing, mowing and fencing plots, those accounted for 33.1%, 18.1% and 21.1% respectively in their dominant AOB. Analyze of stepwise regression showed that the key soil physicochemical factor which influence the diversity index most is the content of nitrate nitrogen. Urease activity and nitrate rate in grazing plot were both higher than the other land use patterns. The content of nitrate nitrogen and total phosphorus were both extremely significant (<em>P</em><0.01) correlated with urease activity, and there is a significant (<em>P</em><0.05) relationship between urease activity and the content of available phosphorus; at the same time, there is also a significant relationship between nitrate rate and nitrate nitrogen contents. AOB communities in <em>Leymus chinensis</em> steppe were significantly (<em>P</em><0.05) influenced by the levels of ammonium nitrogen when canonical correspondence analysis was employed to identify relationship between AOB and soil physicochemical factors under different land use patterns. Based on the above findings, it seems that nitrogen cycles represent the key links between above and below ground ecosystems. Land use patterns in<em> Leymus chinensis</em> steppe have significant and long-term impacts on the size and the structure of the soil AOB community at both phylogenetic and functional levels, thus would further affect the transformation and utilization of nitrogen in the steppe ecosystem.

Autotrophic growth of nitrifying community in an agricultural soil

The two-step nitrification process is an integral part of the global nitrogen cycle, and it is accomplished by distinctly different nitrifiers. By combining DNA-based stable isotope probing (SIP) and high-throughput pyrosequencing, we present the molecular evidence for autotrophic growth of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) in agricultural soil upon ammonium fertilization. Time-course incubation of SIP microcosms indicated that the amoA genes of AOB was increasingly labeled by (13)CO(2) after incubation for 3, 7 and 28 days during active nitrification, whereas labeling of the AOA amoA gene was detected to a much lesser extent only after a 28-day incubation. Phylogenetic analysis of the (13)C-labeled amoA and 16S rRNA genes revealed that the Nitrosospira cluster 3-like sequences dominate the active AOB community and that active AOA is affiliated with the moderately thermophilic Nitrososphaera gargensis from a hot spring. The higher relative frequency of Nitrospira-like NOB in the (13)C-labeled DNA suggests that it may be more actively involved in nitrite oxidation than Nitrobacter-like NOB. Furthermore, the acetylene inhibition technique showed that (13)CO(2) assimilation by AOB, AOA and NOB occurs only when ammonia oxidation is not blocked, which provides strong hints for the chemolithoautotrophy of nitrifying community in complex soil environments. These results show that the microbial community of AOB and NOB dominates the nitrification process in the agricultural soil tested.