High contribution of ammonia-oxidizing archaea (AOA) to ammonia oxidation related to a potential active AOA species in various arable land soils

Purpose

Ammonia oxidation is the limiting step in soil nitrification and critical in the global nitrogen cycle. The discovery of ammonia-oxidizing archaea (AOA) has improved our knowledge of microbial mechanisms for ammonia oxidation in complex soil environments. However, the relative contributions of AOA and ammonia-oxidizing bacteria (AOB) to ammonia oxidation remain unclear.

Materials and methods

In this study, through large geographical scale sampling in China, totally nine samples representing various types of arable land soils were selected for analyzing the ammonia oxidation activity. The AOA and AOB activities were separately determined by using the dicyandiamide and 1-octyne inhibition method. High-throughput pyrosequencing and DNA stable-isotope probing (DNA-SIP) analysis were applied to investigate the distribution and activity of Candidatus Nitrosocosmicus franklandus in the arable land soils.

Results and discussion

In this study, AOA abundance (3.2?×?10⁷–3.4?×?10⁹ copies g^?1) and activity (0.01–1.33 mg N kg^?1 dry soil day^?1) were evaluated for nine selected arable land soils and accounted for 4–100% of ammonia oxidation. By separately determining AOA and AOB rates, we observed that archaeal ammonia oxidation dominated the ammonia oxidation process in six soils, revealing a considerable contribution of AOA in ammonia oxidation in arable land soils. Based on high-throughput pyrosequencing analysis, the AOA species Ca. N. franklandus with relatively low abundance (0.6–13.5% in AOA) was ubiquitously distributed in all the tested samples. Moreover, according to the DNA-SIP analysis for Urumqi sample, the high activity and efficiency of Ca. N. franklandus in using CO₂ suggests that this species plays an important role in archaeal ammonia oxidation in arable land soils.

Conclusions

Through determining the AOA activity and analyzing the potential predominant functional AOA species, this study greatly improves our understanding of ammonia oxidation in arable land soils.

     

Potato cultivar type affects the structure of ammonia oxidizer communities in field soil under potato beyond the rhizosphere

The effects of plants on the microbiota involved in the oxidation of ammonia in soils have been controversial. Here, we investigated the dynamics in the abundances and community structures of the bacterial and archaeal ammonia oxidizers (AOB and AOA, respectively) in two fields that were cropped with potato. Six different potato cultivars were used, including a genetically-modified one, in a fourfold replicated experimental set-up. On the basis of bulk and rhizosphere soil extracted microbial community DNA, AOB and AOA quantitative PCR as well as PCR-DGGE were performed. In addition, samples were used for the production and analysis of amoA gene fragment based clone libraries. Regardless of sample type (bulk versus rhizosphere soil) and across soils, the population sizes of AOA (of the order 10⁴–10⁸ amoA gene copies g⁻¹ dry soil), were generally higher than those of AOB in the same samples (about 10⁴–10⁵ g⁻¹ dry soil), resulting in ratio's of log-transformed values > 1.0. Whereas the AOB numbers were generally raised in the rhizosphere versus bulk soils in both soils, the opposite was true for the AOA numbers. Moreover, significant effects of cultivar type on both the AOB and AOA community structures were found in both soils, and these extended to beyond the rhizospheres. The effects were found across the whole growth season. Soil type did not significantly affect the community structures of AOA, but had a small effect on the community structure of AOB. Analysis of the structures of the AOB communities revealed a prevalence of AOB subgroups 2, 3a, 3b and 4 in one field soil and of 2 and 4 in the other one. With respect to the AOA, soil/sediment clusters (SS) I, II, III and IV were found to prevail.     

Long-term fertilization effects on active ammonia oxidizers in an acidic upland soil in China

The effects of long-term fertilization of acidic soils on ammonia-oxidizing archaea (AOA) and bacteria (AOB) communities and its ecological implications remain poorly understood. We chose an acidic upland soil site under long-term (27-year) fertilization to investigate ammonia oxidizer communities under four different regimes: mineral N fertilizer (N), mineral NPK fertilizer (NPK), organic manure (OM) and an unfertilized control (CK). Soil net nitrification rates were significantly higher in OM soils than in CK, N or NPK soils. Quantitative analysis of the distribution of amoA genes by DNA-based stable isotope probing revealed that AOA dominate in CK, N and NPK soils, while AOB dominate in OM soils. Denaturing gradient gel electrophoresis and clone library analyses of amoA genes revealed that Group 1.1a-associated AOA (also referred to as Nitrosotalea) were the most dominant active AOA population (>92%), while Nitrosospira Cluster 3 and Cluster 9 were predominant among active AOB communities. The functional diversity of active ammonia oxidizers in acidic soils is affected by long-term fertilization practices, and the responses of active ammonia oxidizers to mineral fertilizer and organic manure are clearly different. Our results provide strong evidence that AOA are more highly adapted to growth at low pH and low substrate availability than AOB, and they suggest that the niche differentiation and metabolic diversity of ammonia oxidizers in acidic soils are more complex than previously thought.     

Fluctuations in the abundance of ammonia oxidizers and the contents of inorganic nitrogen in solarized soil

Solarization makes a great impact on the abundance of ammonia oxidizers and nitrifying activity in soil. To elucidate fluctuations in the abundance of ammonia oxidizers and nitrification in solarized soil, copy numbers of amoA gene of ammonia-oxidizing bacteria (AOB) and archaea (AOA), viable number of ammonia oxidizers and inorganic nitrogen contents were investigated in greenhouse experiments. The copy number of amoA gene and the viable number of ammonia oxidizers were determined by the quantitative polymerase chain reaction and most probable number methods, respectively. Abundance of AOB based on the estimation of amoA gene copy numbers and viable counts of ammonia oxidizers was decreased by the solarization treatment and increased during the tomato (Solanum lycopersicum L.) cultivation period following the solarization. Effect of solarization on the copy number of amoA gene of AOA was less evident than that on AOB. The proportion of nitrate in inorganic nitrogen contents was declined by the solarization and increased during the tomato cultivation period following the solarization. Positive correlations were found between the proportion of nitrate in inorganic nitrogen content and the copy number of bacterial or archaeal amoA gene or the viable number of ammonia oxidizers; the copy number of bacterial amoA gene showed a strong correlation with the viable number of ammonia oxidizers. The present study revealed influences of solarization on the fluctuation in the abundance of ammonia oxidizers and dynamics of inorganic nitrogen contents in soil and the results indicate that the determination of amoA gene of AOB is possibly a quick and useful diagnostic technique for evaluating suppression and restoration of nitrification following solarization.     

基于宏基因组学的酸性森林土壤氨氧化微生物群落特征研究

全球30%以上陆地面积是酸性土壤(pH5.5),而酸性土壤中氨氧化微生物群落特征研究是破译其硝化过程微生物学机理的基础。尤其随着完全硝化微生物(Complete ammonia oxidizer,comammox)的发现,亟需重新认知酸性土壤中氨氧化微生物类群。以酸性马尾松林为研究对象,综合利用荧光定量PCR(qPCR)、凝胶电泳半定量和宏基因组测序等技术研究土壤中氨氧化古菌(Ammonia-oxidizing archaea,AOA)、氨氧化细菌(Ammonia-oxidizing bacteria,AOB)和Comammox的相对丰度以及群落组成特征。研究发现AOA和AOB amoA基因丰度分别为2.61×106 copies·g~(-1)和1.45×106copies·g~(-1);而comammoxamoA基因qPCR结果存在显著的非特异性扩增,导致其丰度被高估,而经凝胶电泳半定量矫正后,约为(1.38～1.47)×106copies·g~(-1),该结果和土壤宏基因测序揭示的comammox相对丰度基本吻合。此外,宏基因组分析发现经典嗜酸group1.1a-associated仅占AOA总类群的12%,而group1.1b则占88%,尽管目前仍未有嗜酸group 1.1b AOA纯菌株的报道。AOB主要类群为Nitrosospira(约64%),而Nitrosomonas约占36%。Comammox主要类群为clade B(约64%),而clade A仅占36%且均隶属于clade A.1亚枝,这暗示clade B与已报道的嗜中性comammox clade A纯菌株有极大的生理代谢差异。总之,本研究提供了综合利用qPCR、半定量和宏基因组分析土壤氨氧化微生物群落的策略,并建议优化comammox的qPCR引物,同时本研究系统分析了酸性马尾松林土壤中氨氧化微生物的相对丰度和群落组成特征。     

Response of ammonia oxidizing archaea and bacteria to changing water filled pore space

This study examined the effect of water filled pore space (WFPS) on gross N fluxes and community structure and abundance of ammonia oxidizing archaea and bacteria in a semi-arid soil. Different WFPS altered the community structure of both AOA and AOB. Ammonia oxidizer communities (for both archaea and bacteria) from ‘wet’ soils (95, 85 and 75% WFPS) and ‘dry’ soils (25, 45 and 55% WFPS) were distinctly different from one another. Additionally there was a significant relationship between community structure and gross rates of nitrification. There was also a significant relationship between WFPS and bacterial amoA abundance but not archaeal amoA abundance suggesting that bacterial ammonia oxidizers are more responsive to changes in soil water availability. These results are in agreement with other studies suggesting that both groups of ammonia oxidizers have distinct physiological characteristics and ecological niches with consequences for nitrification in response to WFPS. Overall findings from this study indicate that nitrification, both in terms of process rates and populations responsible for nitrification activity, is highly responsive to soil water availability.     

两种农田土壤中的氨氧化细菌和氨氧化古菌对硝化抑制剂DCD和DMPP的响应-盆栽试验

Taking two important agricultural soils with different pH, brown soil （Hap-Udic Luvisol） and cinnamon soil （Hap-Ustic Luvisol）, from Northeast China, a pot culture experiment with spring maize （Zea mays L.） was conducted to study the dynamic changes in the abundance and diversity of soil ammonia-oxidizing bacteria （AOB） and ammonia-oxidizing archaea （AOA） populations during maize growth period in response to the additions of nitrification inhibitors dicyandiamide （DCD） and 3,4-dimethylpyrazole phosphate （DMPP） by the methods of real-time polymerase chain reaction （PCR） assay, PCR-denaturing gradient gel electrophoresis （DGGE）, and construction of clone library targeting the amoA gene. Four treatments were established, i.e., no urea （control）, urea, urea plus DCD, and urea plus DMPP. Both DCD and DMPP inhibited growth of AOB significantly, compared to applying urea alone. Soil bacterial amoA gene copies had a significant positive linear correlation with soil nitrate content, but soil archaeal amoA gene copies did not. In both soils, all AOB sequences fell within Nitrosospira or Nitrosospira-like groups, and all AOA sequences belonged to group 1.1b crenaxchaea. With the application of DCD or DMPP, community composition of AOB and AOA in the two soils had less change except that the AOB community composition in Hap-Udic Luvisol changed at the last two growth stages of maize under the application of DCD. AOB rather than AOA likely dominated soil ammonia oxidation in these two agricultural soils.     

Biochar addition affected the dynamics of ammonia oxidizers and nitrification in microcosms of a coastal alkaline soil

Biochar amendments have frequently been reported to alter microbial communities and biogeochemical processes in soils. However, the impact of biochar application on bacterial (AOB) and archaeal ammonia oxidizers (AOA) remains poorly understood. In this study, we investigated the responses of AOB and AOA to the application of biochar derived from cotton stalk at rates of 5, 10, and 20 % by weight to a coastal alkaline soil during a 12-week incubation. The results showed that the amoA gene of AOB consistently outnumbered that of AOA, whereas only the AOA amoA gene copy number was significantly correlated with the potential ammonia oxidation (PAO) rate (P?<?0.01). The significant decrease of PAO rates in biochar treatments occurred after incubation for 4–6 weeks, which were distinctly longer than that in the control (2 weeks). The PAO rates were significantly different among treatments during the first 4 weeks of incubation (P?<?0.05), with the highest usually in the 10 % treatment. Biochar application significantly increased the abundance of both nitrifiers in the 4 weeks of incubation (P?<?0.05). Biochar amendment also decreased AOA diversity, but increased AOB diversity, which resulted in different community structures of both nitrifiers (P?<?0.01), as shown by the differences between the 5 % biochar and the control treatments. We conclude that biochar application generally enhanced the abundance and altered the composition of ammonia oxidizers; the rate of biochar application also affected the rate and dynamics of nitrification, and the risk for increasing the alkalinity and N leaching of the studied soil was lower with a lower application rate.     

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

Purpose

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.

Materials and methods

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.

Results and discussion

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.

Conclusions

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.     

Response of ammonia oxidizing microbes to the stresses of arsenic and copper in two acidic alfisols

Soil pollution by elevated heavy metals exhibits adverse effects on soil microorganisms. Ammonia oxidizing bacteria and ammonia oxidizing archaea perform ammonia oxidative processes in acidic soils. However, influence of heavy metal stress on soil ammonia oxidizers distribution and diversity is inadequately addressed. This study investigated the responses of ammonia oxidizing bacteria and archaea to heavy metals, Cu and As during short-term laboratory experiment. Two different acidic alfisols named as Rayka and Hangzhou spiked with different concentrations of As, Cu and As + Cu were incubated for 10 weeks. Significant reduction in copy numbers of archaeal-16S rRNA, bacterial-16S rRNA and functional amoA genes was observed along elevated heavy metal concentrations. Ammonia oxidizing archaea was found to be more abundant than ammonia oxidizing bacteria in all the heavy metal treatments. The potential nitrification rate significantly decreased with increasing As and Cu concentrations in the two soils examined. Denaturing gradient gel electrophoresis analysis revealed no apparent community shift for ammonia oxidizing archaea even at higher concentrations of As and Cu. Phylogenetic analysis of archaeal amoA gene from 4 clone libraries indicated that all the archaeal amoA sequences were placed within 3 distinct clusters from soil and sediment group 1.1b of Thaumarchaeota. Our results could be useful for the better understanding of the ecological effects of heavy metals on the abundance and diversity of soil ammonia oxidizers.     

Management practices have a major impact on nitrifier and denitrifier communities in a semiarid grassland ecosystem

Purpose

Nitrification and denitrification, two of the key nitrogen (N) transformation processes in the soil, are carried out by a diverse range of microorganisms and catalyzed by a series of enzymes. Different management practices, such as continuous grazing, mowing, and periodic fencing off from grazing, dramatically influenced grassland ecosystems. This study aimed to examine the effects of management practices on the abundance and community structure of nitrifier and denitrifier communities in grassland ecosystems.

Materials and methods

Soil samples were collected from a semiarid grassland ecosystem in Xilingol region, Inner Mongolia, where long-term management practices including free-grazing, different periods of enclosure from grazing, and different frequencies of mowing were conducted. Real-time quantitative polymerase chain reaction (Q-PCR), denaturing gradient gel electrophoresis (DGGE), sequencing, and phylogenetic analysis were applied to estimate the abundance and composition of amoA, nirS, nirK, and nosZ genes.

Results and discussion

The ammonia-oxidizing archaea (AOA) amoA copies were in the range 5.99?×?10⁸ to 8.60?×?10⁸, while those of ammonia-oxidizing bacteria (AOB) varied from 3.02?×?10⁷ to 4.61?×?10⁷. The abundance of AOA was substantially higher in the light grazing treatment (LG) than in the mowing treatments. The quantity and intensity of DGGE bands of AOA varied with pasture management. In stark contrast, AOB population abundance and community structure remained largely unchanged in all the soils irrespective of the management practices. All these results suggested that ammonia oxidizers were dominated by AOA. The higher gene abundance and greater intensity of DGGE bands of nirS and nosZ under the enclosure treatments would suggest greater stimulated denitrification. The ratio of nosZ/(nirS?+?nirK) was higher in mowing treatments than in the free-grazing and enclosure treatments, possibly leading to more complete denitrification. Correlation analysis indicated that soil moisture and inorganic nitrogen content were the two main soil environmental variables that influence the community structure of nitrifiers and denitrifiers.

Conclusions

In this semiarid neutral to alkaline grassland ecosystem under low temperature conditions, AOA mainly affiliated with Nitrososphaera dominated nitrification. These results clearly demonstrate that grassland management practices can have a major impact on nitrifier and denitrifier communities in this semiarid grassland ecosystem, under low temperature conditions.

     

Linking plant identity and interspecific competition to soil nitrogen cycling through ammonia oxidizer communities

Both plants and microbes influence soil nutrient cycling. However, the links between plants, microbes and nutrient cycling are poorly understood. In this study, we investigated how plant identity and interspecific competition influence soil nitrogen cycling and attempted to link plant identity and interspecific competition to community structures of bacterial and archaeal ammonia oxidizers based on terminal restriction fragment length polymorphism analysis (T-RFLP) of bacterial and archaeal ammonia monooxygenase (amoA) genes. Faba bean and maize monocultures and a faba bean/maize mixture were planted with two nitrogen levels (0 and 100 mg N kg⁻¹ soil as urea). Soil mineral nitrogen, ammonia oxidizer function (potential nitrification activity, PNA) and community structures were measured 28 and 54 days after plant emergence. Faba bean and maize substantially differed in their influences on mineral nitrogen concentrations and PNA in rhizosphere soils. Soil mineral nitrogen and PNA in the rhizosphere soils of the faba bean/maize mixture were closer to those of the maize monoculture than to those of the faba bean monoculture. T-RFLP with restriction enzymes BsaJI and Hpy8I distinguished variations in bacterial and archaeal ammonia oxidizers community structure, respectively, and detected both between-cluster and within-cluster variations in bacterial ammonia oxidizers. T-RFLP data showed that nitrogen addition favored part of a Nitrosospira cluster 3b sequence type and suppressed part of a cluster Nitrosospira 3a sequence type of bacterial ammonia oxidizers, while it had no influence on the archaeal ammonia oxidizer community structure. Although multivariate analysis showed that the function and community structure of bacterial ammonia oxidizers were significantly correlated, plant species and interspecific competition did not significantly change the community structure of bacterial and archaeal ammonia oxidizers. These results indicate that plant species and interspecific competition regulate soil nitrogen cycling via a mechanism of other than alteration in the community structure of ammonia oxidizers as investigated by DNA based methods.     

Responses of ammonia-oxidizing bacteria and archaea to nitrogen fertilization and precipitation increment in a typical temperate steppe in Inner Mongolia

As the first and rate-limiting step of nitrification, ammonia oxidation can be realized either by ammonia-oxidizing bacteria (AOB) or archaea (AOA). However, the key factors driving the abundance, community structure and activity of ammonia oxidizers are still unclear, and the relative importance of AOA and AOB in ammonia oxidation is unresolved. In the present study, we examined the effects of long-term (6 years) nitrogen (N) addition and simulated precipitation increment on the abundance and community composition of AOA and AOB based on a field trial in a typical temperate steppe of northern China. We used combined approaches of quantitative PCR, terminal-restriction fragment length polymorphism (T-RFLP) and clone library analyses of amoA genes. The study objective was to determine (1) AOA and AOB diversity and activity in response to N addition and increased precipitation and (2) the relative contributions of AOA and AOB to soil ammonia oxidation in the typical temperate steppe. The results showed that the potential nitrification rate (PNR) increased with N addition, but decreased with increased precipitation. Both N addition and increased precipitation significantly increased AOB but not AOA abundance, and a significant correlation was only observed between PNR and AOB amoA gene copies. The T-RFLP analysis showed that both N and precipitation were key factors in shaping the composition of AOB, while AOA were only marginally influenced. Phylogenetic analysis indicated that all AOA clones fell within the soil and sediment lineage while all AOB clones fell within the Nitrosospira. The study suggested that AOA and AOB had distinct physiological characteristics and ecological niches. AOB were shown to be more sensitive to N and precipitation than AOA, and the ammonia oxidation process was therefore supposed to be mainly driven by AOB in this temperate steppe.     

pH-dependent distribution of soil ammonia oxidizers across a large geographical scale as revealed by high-throughput pyrosequencing

Purpose

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) are ubiquitous and important for nitrogen transformations in terrestrial ecosystems. However, the distribution patterns of these microorganisms as affected by the terrestrial environments across a large geographical scale are not well understood. This study was designed to gain insights into the ecological characteristics of AOA and AOB in 65 soils, collected from a wide range of soil and ecosystem types.

Materials and methods

Barcoded pyrosequencing in combination with quantitative PCR was employed to characterize the relative abundance, diversity, and community composition of archaeal 16S rRNA gene, and AOA and AOB amoA genes in 65 soil samples.

Results and discussion

The operational taxonomic unit richness and Shannon diversity of Thaumarchaeota, AOA, and AOB were highly variable among different soils, but their variations were best explained by soil pH. Soil pH was strongly correlated with the overall community composition of ammonia oxidizers, as measured by the pairwise Bray–Curtis dissimilarity across all sites. These findings were further corroborated by the evident pH-dependent distribution patterns of four thaumarchaeal groups (I.1a-associated, I.1b, I.1c, and I.1c-associated) and four AOB clusters (2, 3a.1, 10, and 12). The ratios of AOA to AOB amoA gene copy numbers significantly decreased with increasing pH, suggesting a competitive advantage of AOA over AOB in acidic soils.

Conclusions

These results suggest that the distribution of ammonia oxidizers across large-scale biogeographical settings can be largely predicted along the soil pH gradient, thus providing important indications for the ecological characteristics of AOA and AOB in different soils.     

Effects of inorganic fertilizer and manure on soil archaeal abundance at two experimental farms during three consecutive rotation-cropping seasons

Soil archaeal population dynamics at two experimental sites of the same clay-loam type in Ottawa and Woodslee, Ontario, were investigated to determine fertilizer and manure effects following their different long-term crop rotation and fertilization schemes. Phylogenetic analysis of cloned soil archaeal 16S rRNA gene libraries of both sites identified them with group 1.1b of Thaumarchaeota. The gene population dynamics subtly varied in the order of 10⁷ copies g⁻¹ soil when monitored by quantitative real-time PCR during three growing seasons (2007–2009). In Ottawa, where plots were amended with dairy-farm manure, soil thaumarchaeal gene abundance was double of the unamended plots. At the Woodslee N-P-K-fertilized plots, it remained at least 30% fewer than that of the unfertilized ones. These cultivated plots showed soil carbon limitation while the fertilized ones were low in soil pH (ca. 5.5). Surface soils from an unfertilized sod plot and an adjacent deciduous forest had higher total carbon content (C:N ratio of 9 and 11, respectively). Their thaumarchaeal gene abundance varied up to 4.8 × 10⁷ and 7.0 × 10⁷ copies g⁻¹ soil, respectively. The former value was also attained at the manure-amended plots in Ottawa, where the C:N ratio was just below 10. Where soil pH was above 6.0, there was a weak and positive correlation between soil total C and the estimated gene abundance. Such gene population dynamics consistently demonstrated the stimulating and suppressive effects of dairy-farm manure (Ottawa site) and inorganic fertilizers (Woodslee site), respectively, on soil thaumarchaea. At both sites archaeal amoA and 16S rRNA gene abundance were similarly affected. Archaeal amoA gene abundance also outnumbered bacterial amoA abundance, suggesting that ammonia-oxidizing archaea might be dominant in these soils. Only minor crop effects on gene population dynamics were detected.     

Soil particle surface electrochemical property effects on abundance of ammonia-oxidizing bacteria and ammonia-oxidizing archaea, NH4 activity, and net nitrification in an acid soil

Soil surface electrochemical properties may have a strong influence on nitrifying microorganisms, H⁺ and NH₄⁺ activities, and therefore on the nitrification process. A gradient of surface electrochemical parameters was obtained by amendment of a subtropical acid pine soil (Oxisol) with 0% (control), 3%, 5%, 8%, 10% and 12% pure Ca-Montmorillonite by weight. The H⁺ and NH₄⁺ activities, the abundance of the ammonia-oxidizing bacterial (AOB) and archaeal (AOA) amoA gene copies, and time-dependent kinetics of net nitrification were investigated. Soil particle surface specific area ranged from 53 to 103 m² g⁻¹ and increased with increasing montmorillonite application rate. Similar to specific area, surface charge quantity, surface charge density, electric field strength and surface potential increased after montmorillonite amendment. The H⁺ and NH₄⁺ activities decreased linearly after montmorillonite addition. AOB amoA gene copy number was 1.82 × 10⁵ copies g⁻¹ for unamended soil, and the highest AOB amoA gene copy numbers were found for the 10% montmorillonite amendment (3.11 × 10⁷ g⁻¹ soil), which was more than 150 times higher than unamended soil. AOA amoA gene copy numbers were 9.19 × 10³ copies g⁻¹ dry unamended soil, and the highest AOA amoA gene copy numbers were found in the 8% montmorillonite amendment (1.22 × 10⁵ g⁻¹ soil). Although pH significantly decreased during the first three weeks of incubation, no significant difference was observed between the unamended control and different rates of montmorillonite addition treatments during the whole incubation. The largest net nitrification (103 mg N kg⁻¹) was observed in the 10% montmorillonite amendment and the lowest in unamended soil (62 mg N kg⁻¹). While montmorillonite did not change the kinetic patterns of net nitrification, the highest nitrification potential (275 mg N kg⁻¹) for the 10% montmorillonite treatment was more than 3 times higher than unamended soil from simulation of time-dependent kinetics. Nitrification was significantly stimulated after montmorillonite amendment in acid soil mainly due to an increase in the quantity and activity of AOB and AOA. We concluded that soil particle surface parameters can significantly influence nitrification, especially in acid soils.     

Chronic Nitrogen Fertilization Modulates Competitive Interactions Among Microbial Ammonia Oxidizers in a Loess Soil

Nitrogen(N) application may lead to niche segregation of soil ammonia-oxidizing archaea(AOA) and bacteria(AOB), thereby reducing the competitive interactions between AOA and AOB due to higher ammonium substrate availability. However, the adaptive mechanisms of AOA and AOB under N enrichment remain poorly understood. Stable isotope probing(SIP) microcosm incubation was employed to reveal community changes of active AOA and AOB in a loess soil from a field experiment growing potatoes that received no N(control, CK), low N(LN, 75 kg N ha~(-1)), and high N(HN, 375 kg N ha~(-1)). The results showed that the soil potential nitrification rate(PNR) was measured by culturing of the soil samples from the field experiment. Soil PNR was significantly increased in HN by87.5% and 67.5% compared with CK and LN, respectively. Compared with CK, the~(13)C-amoA genes of soil AOA and AOB in HN had 2.58 × 10~4 and 1.55 × 10~6 copies, representing 1.6-and 16.2-fold increase respectively. It was indicated that AOB dominated soil ammonia oxidation. A phylogenetic analysis of the~(13)C-amoA gene showed that N application significantly increased the proportion of54 d9-like AOA up to 90% in HN, while the Nitrososphaera gargensis-like and Nitrososphaera viennensis-like AOA were inhibited and completely disappeared. Nitrogen application also resulted in the community shift of active AOB-dominant group from Nitrosospira briensis-like to Nitrosospira sp. TCH711-like. Our study provides compelling evidence for the emergence and maintenance of active nitrifying communities under the intensified N input to an agricultural ecosystem.     

Comparison of lipid biomarker and gene abundance characterizing the archaeal ammonia-oxidizing community in flooded soils

In the last years, archaea have been identified as key players in global N cycling, especially in nitrification. Ammonia-oxidizing archaea (AOA) are postulated to belong to the new phylum Thaumarchaeota for which the lipid crenarchaeol should be specific. The ratios between two independent markers for AOA, the ammonia monooxygenase gene and crenarchaeol have been studied in different aerated soils, but so far not in flooded soils. This study investigated ammonia-oxidizing archaea in four paddy soils and a tidal wetland. Ratios were significantly higher in the paddy soils compared to the tidal wetland and in general higher as in upland soils, leading to the assumption that archaeal ammonia oxidizers different from crenarchaeol-containing Thaumarchaeota may play an important role in paddy soils.     

Ammonia-oxidizing archaea are more important than ammonia-oxidizing bacteria in nitrification and NO3 −-N loss in acidic soil of sloped land

As part of a long-term sloped land use experiment established in 1995 at Taoyuan Agro-ecosystem Research Station (111°26′ E, 28°55′ N) in China, soil samples were collected from three land use types, including cropland (CL), natural forest, and tea plantation. Quantitative polymerase chain reaction and terminal restriction fragment length polymorphism were used to determine the abundance and community composition of amoA-containing bacteria (AOB) and archaea (AOA). The results indicate that land use type induced significant changes in soil potential nitrification rate and community composition, diversity, and abundance of AOB and AOA. Both AOB and AOA community compositions were generally similar between upper and lower slope positions (UP and LP), except within CL. The LP soils had significantly (p?<?0.05) higher diversity and abundance of both AOB and AOA than in the UP. Potential nitrification rate was significantly correlated (p?<?0.05) with diversity and abundance of AOA, but not with AOB. Among land use types, the NO₃ ^? and amoA-containing AOA runoff loss was greatest in CL. Nitrate-N runoff loss was significantly correlated (p?<?0.05) with the loss of AOA amoA copies in the runoff water. Furthermore, relationships between NO₃ ^?-N runoff loss and abundance of AOA but not of AOB at both slope positions were significantly correlated (p?<?0.05). These findings suggest that AOA are more important than AOB in nitrification and NO₃ ^?-N runoff loss in acidic soils across sloped land use types.     

Effects of 3,4-dimethylpyrazole phosphate (DMPP) on the abundance of ammonia oxidizers and denitrifiers in two different intensive vegetable cultivation soils

Purpose

Nitrification and denitrification in the N cycle are affected by various ammonia oxidizers and denitrifying microbes in intensive vegetable cultivation soils, but our current understanding of the effect these microbes have on N₂O emissions is limited. The nitrification inhibitor, 3,4-dimethylpyrazole phosphate (DMPP), acts by slowing nitrification and is used to improve fertilizer use efficiency and reduce N losses from agricultural systems; however, its effects on nitrifier and denitrifier activities in intensive vegetable cultivation soils are unknown.

Materials and methods

In this study, we measured the impacts of DMPP on N₂O emissions, ammonia oxidizers, and denitrifying microbes in two intensive vegetable cultivation soils: one that had been cultivated for a short term (1 year) and one that had been cultivated over a longer term (29 years). The quantitative PCR technique was used in this study. Three treatments, including control (no fertilizer), urea alone, and urea with DMPP, were included for each soil. The application rates of urea and DMPP were 1800 kg ha^?1 and 0.5% of the urea-N application rate.

Results and discussion

The application of N significantly increased N₂O emissions in both soils. The abundance of ammonia-oxidizing bacteria (AOB) increased significantly with high rate of N fertilizer application in both soils. Conversely, there was no change in the growth rate of ammonia-oxidizing archaea (AOA) in response to the applied urea despite the presence of larger numbers of AOA in these soils. This suggests AOB may play a greater role than AOA in the nitrification process, and N₂O emission in intensive vegetable cultivation soils. The application of DMPP significantly reduced soil NO₃^?-N content and N₂O emission, and delayed ammonia oxidation. It greatly reduced AOB abundance, but not AOA abundance. Moreover, the presence of DMPP was correlated with a significant decrease in the abundance of nitrite reductase (nirS and nirK) genes.

Conclusions

Long-term intensive vegetable cultivation with heavy N fertilization altered AOB and nirS abundance. In vegetable cultivation soils with high N levels, DMPP can be effective in mitigating N₂O emissions by directly inhibiting both ammonia oxidizing and denitrifying microbes.