Population size and specific nitrification potential of soil ammonia-oxidizing bacteria under long-term fertilizer management

Population size of soil ammonia-oxidizing bacteria (AOB) was quantified by real-time PCR in a long-term (16 years) field experiment under different fertilizer managements. AOB population sizes in mineral nitrogen-fertilized soils and organic manure-fertilized soil were 10.3 and 3.1 times, respectively, that of the control, while phosphorus and potassium fertilization had no significant effect. On the other hand, the AOB specific nitrification potential (soil nitrification potential per AOB cell) was significantly higher (P < 0.05) in organic manure-fertilized soil than in mineral-fertilized soils and the control, indicating that AOB was likely more metabolically active in organic manure-fertilized soils than in mineral nitrogen-fertilized soils after long-term application.     

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.     

Humic acids buffer the effects of urea on soil ammonia oxidizers and potential nitrification

Humic acids (HAs) play an important role in the global nitrogen cycle by influencing the distribution, bioavailability, and ultimate fate of organic nitrogen. Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in ecosystems and is limited, in part, by the availability of NH₄⁺. We evaluated the impact of HAs on soil AOB in microcosms by applying urea (1.0%, equal to 10 mg urea/g soil) with 0.1% bHA (biodegraded lignite humic acids, equal to 1 mg/g soil), 0.1% cHA (crude lignite humic acids) or no amendment. AOB population size, ammonium and nitrate concentrations were monitored for 12 weeks after urea and HA application. AOB densities (quantified by real-time PCR targeting the amoA) in the Urea treatments increased about ten-fold (the final abundance: 5.02 × 10⁷ copies (g of dry soil)⁻¹) after one week of incubation and decreased to the initial density after 12 weeks incubation; the population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) decreased from 1.12 × 10¹⁰ to 2.59 × 10⁹ copies (g of dry soil)⁻¹ at week one and fluctuated back to the initial copy number at week 12. In the Urea + bHA and Urea + cHA treatments, the AOB densities were 4 and 6 times higher, respectively, than the initial density of approximately 5.07 × 10⁶ copies (g of dry soil)⁻¹ at week 1 and did not change much up to week 4; the total bacteria density changed little over time. The AOB and total bacteria density of the controls changed little during the 12 weeks of incubation. The microbial community composition of the Urea treatment, based on T-RFLP using CCA (canonical correspondence analysis) and pCCA (partial CCA) analysis, was clearly different from those of other treatments, and suggested that lignite HAs buffered the change in diversity and quantity of total bacteria caused by the application of urea to the soil. We hypothesize that HAs can inhibit the change in microbial community composition and numbers, as well as AOB population size by reducing the hydrolysis rate from urea to ammonium in soils amended with urea.     

Net nitrification rate and presence of Nitrosospira cluster 2 in acid coniferous forest soils appear to be tree species specific

The impact of four coniferous tree species and their corresponding soil factors on N transformation rates and presence of ammonia-oxidising bacteria (AOB) was studied in an acid pine forest soil (Appelscha, The Netherlands). Pine soil had a relatively low net nitrification rate, while spruce, fir and larch soils showed high net nitrification rates. 16S rRNA and amoA sequences were only found in soils with high nitrification rates and belonged solely to Nitrosospira cluster 2. We conclude that tree species, possibly through their effects on soil C/N ratios, determines the presence of Nitrosospira cluster 2. Whenever AOB are present, however, the AOB community composition appears to be similar.     

Impacts of continuous excessive fertilization on soil potential nitrification activity and nitrifying microbial community dynamics in greenhouse system

Purpose

Sampling and analysis of greenhouse soils were conducted in Shouguang, China, to study continuous excessive fertilization effect on nitrifying microbial community dynamics in greenhouse environment.

Materials and methods

Potential nitrification activity (PNA), abundance, and structure of nitrifying microbial communities as well as the correlations with soil properties were investigated.

Results and discussion

Short-term excessive fertilization increased soil nutrient contents and the diversity of nitrifying microbial communities under greenhouse cultivation. However, the abundance and diversity of nitrifying communities decreased greatly due to the increase of soil acidity and salinity after 14 years of high fertilization in greenhouse. There was a significant positive correlation between soil PNA and the abundance of ammonia-oxidizing bacteria (AOB) but not that of ammonia-oxidizing archaea (AOA) in topsoil (0–20 cm) when pH ≥7. Soil PNA and AOB were strongly influenced by soil pH. The groups of Nitrososphaeraceae, Nitrosomonadaceae, and Nitrospiraceae were predominant in the AOA, AOB, and nitrite-oxidizing bacteria (NOB) communities, respectively. Nitrifying community structure was significantly correlated with soil electrical salinity (EC), organic carbon (OC), and nitrate nitrogen (NO₃ ^?–N) content by redundancy analysis (RDA).

Conclusions

Nitrification was predominated by AOB in greenhouse topsoil with high fertilizer loads. Soil salinity, OC, NO₃ ^?–N content, and pH affected by continuous excessive fertilization were the major edaphic factors in shaping nitrifying community structure in greenhouse soils.

     

Shift in abundance and structure of soil ammonia-oxidizing bacteria and archaea communities associated with four typical forest vegetations in subtropical region

Purpose

Nitrogen (N) is one of the most important elements that can limit plant growth in forest ecosystems. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are considered as the key drivers of global N biogeochemical cycling. Soil ammonia-oxidizing microbial communities associated with subtropical vegetation remain poorly characterized. The aim of this study was to determine how AOA and AOB abundance and community structure shift in response to four typical forest vegetations in subtropical region.

Materials and methods

Broad-leaved forest (BF), Chinese fir forest (CF), Pinus massoniana forest (PF), and moso bamboo forest (MB) were widely distributed in the subtropical area of southern China and represented typical vegetation types. Four types of forest stands of more than 30 years grew adjacent to each other on the same soil type, slope, and elevation, were chosen for this experiment. The abundance and community structure of AOA and AOB were characterized by using real-time PCR and denaturing gradient gel electrophoresis (DGGE). The impact of soil properties on communities of AOA and AOB was tested by canonical correspondence analysis (CCA).

Results and discussion

The results indicated that AOB dominated in numbers over AOA in both BF and MB soils, while the AOA/AOB ratio shifted with different forest stands. The highest archaeal and bacterial amoA gene copy numbers were detected in CF and BF soils, respectively. The AOA abundance showed a negative correlation with soil pH and organic C but a positive correlation with NO₃ ^??N concentration. The structures of AOA communities changed with vegetation types, but vegetation types alone would not suffice for shaping AOB community structure among four forest soils. CCA results revealed that NO₃ ^??N concentration and soil pH were the most important environmental gradients on the distribution of AOA community except vegetation type, while NO₃ ^??N concentration, soil pH, and organic C significantly affected the distribution of the AOB communities.

Conclusions

These results revealed the differences in the abundance and structure of AOA and AOB community associated with different tree species, and AOA was more sensitive to vegetation and soil chemical properties than AOB. N bioavailability could be directly linked to AOA and AOB community, and these results are useful for management activities, including forest tree species selection in areas managed to minimize N export to aquatic systems.     

pH regulates key players of nitrification in paddy soils

Increasing lines of evidence have suggested the functional importance of ammonia-oxidizing archaea (AOA) rather than bacteria (AOB) for nitrification in upland soils with low pH. However, it remains unclear whether niche specialization of AOA and AOB occurs in rice paddy wetlands constrained by oxygen availability. Using DNA-based stable isotope probing, we conclude that AOA dominated nitrification activity in acidic paddy soils (pH 5.6) while AOB dominated in alkaline soils (pH 8.2). Nitrification activity was stimulated by urea fertilization and accompanied by a significant increase of AOA in acid soils and AOB in alkaline soils. DNA-based stable isotope probing indicated significant assimilation of ¹³CO₂ for AOA only in acidic paddy soil, while AOB was the solely responsible for ammonia oxidation in the alkaline paddy soil. Phylogenetic analysis further indicated that AOA members within the soil group 1.1b lineage dominated nitrification in acid soils. Ammonia oxidation in the alkaline soil was catalyzed by Nitrosospira cluster 3-like AOB, suggesting that the physiological diversity of AOA is more complicated than previously thought, and soil pH plays important roles in shaping the community structures of ammonia oxidizers in paddy field.     

Relationship between ammonia oxidizing bacteria and bioavailable nitrogen in harvested forest soils of central Alberta

Forest soils are commonly limited in nitrogen (N), and the removal of aboveground biomass in harvesting operations can exacerbate the problem. Thus, the soil organisms that facilitate the rate-limiting step in the N cycle, the oxidation of ammonium (NH₄⁺), are of special interest in harvested environments. The objective of this study was to investigate the changes in ammonia oxidizing bacteria (AOB) communities that occurred in the years following clear cutting, and link those community shifts to availability of inorganic N forms NH₄⁺ and nitrate (NO₃^?). Genetic fingerprinting targeting the amoA gene coupled with denaturing gel gradient electrophoresis was carried out over two summers on forest floor (LFH) and mineral (Ae) soils of three similar cutblocks harvested during different years. In-situ NH₄⁺ and NO₃^? availability was measured over the growing seasons of 2009 and 2010, as well as a suite of physical soil characteristics. Results indicated that the AOB community composition differed in younger vs. older cutblocks, but not by soil horizon. The changes seen in the AOB paralleled the change in N bioavailability across sites, soil horizons, and sampling years, thus indicating that N bioavailability may be directly linked to AOB community composition. This link may provide the basis for the use of AOB as indicators of nutrient availability in the future.     

Community structure and functional diversity of indigenous fluorescent Pseudomonas of long-term swine compost applied maize rhizosphere

The community structure and catabolic functional diversity of indigenous rhizosphere fluorescent pseudomonad isolates recovered from un-amended soil (control treated) and following long-term (6 years) swine compost application (compost treated) were studied. Soils collected for the pot experiments were from a Taiwanese experimental farm established in 1995 to study the long-term effects of crop rotation and organic amendment. In the pot experiments, corn seed was cultured; after the elongation stage, 13 and 32 indigenous rhizosphere fluorescent pseudomonad isolates were collected from compost-treated (n=13) and control-treated (n=32) soils. All isolates were classified by 16S rDNA restriction fragment length polymorphism (RFLP) and unweighted pair-group method using arithmetic averages analyses. Most (84.4%) of the control-treated soil isolates belonged to genotype A, which corresponded to Pseudomonas putida based on 16S rDNA sequences analysis. However, only 61.5% of compost-treated soil isolates were from genotype A; 30.7% of the isolates belonged to genotype B, whose 16S rDNA profile corresponded to Stenotrophomonas maltophilia. Differing catabolic potentials of the P. putida isolates from control- and compost-treated soils were evident; isolates from control-treated soils utilized organic acids including citric acid, cis-aconitic acid, and formic acid more actively than compost-treated soil isolates. Moreover, the latter isolates more efficiently utilized carbohydrates of i-erythritol and l-rhamnose than P. putida isolates from control-treated soil, consistent with the alteration in the catabolic functions of indigenous rhizosphere P. putida by long-term compost application.     

Hydrolase activities, microbial biomass and bacterial community in a soil after long-term amendment with different composts

The use of composts in agricultural soils is a widespread practice and the positive effects on soil and plants are known from numerous studies. However, there have been few attempts to compare the effects of different kinds of composts in one single study. The aim of this paper is to investigate to what extent and to which soil depth four major types of composts would affect the soil and its microbiota.In a crop-rotation field experiment, composts produced from (i) urban organic wastes, (ii) green wastes, (iii) manure and (iv) sewage sludge were applied at a rate equivalent to 175 kg N ha⁻¹ yr⁻¹ for 12 years. General (total organic C (C_org), total N (N_t), microbial biomass C (C_mic), and basal respiration), specific (enzyme activities related to C, N and P cycles), biochemical properties and bacterial genetic diversity (based on DGGE analysis of 16S rDNA) were analyzed at different depths (0-10, 10-20 and 20-30 cm).Compost treatment increased C_org at all depths from 11 g kg⁻¹ for control soil to 16.7 g kg⁻¹ for the case of sewage sludge compost. Total N increased with compost treatment at 0-10 cm and 10-20 cm depths, but not at 20-30 cm. Basal respiration and C_mic declined with depth, and the composts resulted in an increase of C_mic and basal respiration. Enzyme activities were different depend on the enzyme and among compost treatments, but in general, the enzyme activities were higher in the upper layers (0-10 and 10-20 cm) than in the 20-30 cm layer. Diversity of ammonia oxidizers and bacteria was lower in the control than in the compost soils. The type of compost had less influence on the composition of the microbial communities than did soil depth.Some of the properties were sensitive enough to distinguish between different compost, while others were not. This stresses the need of multi-parameter approaches when investigating treatment effects on the soil microbial community. In general, with respect to measures of activity, biomass and community diversity, differences down the soil profile were more pronounced than those due to the compost treatments.     

Response of soil microbial communities to compost amendments

Soil organic matter is considered as a major component of soil quality because it contributes directly or indirectly to many physical, chemical and biological properties. Thus, soil amendment with composts is an agricultural practice commonly used to improve soil quality and also to manage organic wastes. We evaluated in laboratory scale experiments the response of the soilborne microflora to the newly created soil environments resulting from the addition of three different composts in two different agricultural soils under controlled conditions. At a global level, total microbial densities were determined by classical plate count methods and global microbial activities were assessed by measuring basal respiration and substrate induced respiration (SIR). Soil suppressiveness to Rhizoctonia solani diseases was measured through bioassays performed in greenhouses. At a community level, the modifications of the metabolic and molecular structures of bacterial and fungal communities were assessed. Bacterial community level physiological profiles (CLPP) were determined using Biolog™ GN microtiter plates. Bacterial and fungal community structures were investigated using terminal restriction fragment length polymorphism (T-RFLP) fingerprinting. Data sets were analyzed using analysis of variance and ordination methods of multivariate data. The impact of organic amendments on soil characteristics differed with the nature of the composts and the soil types. French and English spent mushroom composts altered all the biological parameters evaluated in the clayey soil and/or in the sandy silty clay soil, while green waste compost did not modify either bacterial and fungal densities, SIR values nor soil suppressiveness in any of the soils. The changes in bacterial T-RFLP fingerprints caused by compost amendments were not related to the changes in CLPP, suggesting the functional redundancy of soil microorganisms. Assessing the density, the activity and the structure of the soil microflora allowed us not only to detect the impact of compost amendment on soil microorganisms, but also to evaluate its effect at a functional level through the variation of soil disease suppressiveness. Differences in disease suppressiveness were related to differences in chemical composition, in availability of nutrients at short term and in microbial composition due to both incorporation and stimulation of microorganisms by the compost amendments.     

Responses of soil nitrous oxide production and abundances and composition of associated microbial communities to nitrogen and water amendment

Soil moisture and nitrogen (N) are two important factors influencing N₂O emissions and the growth of microorganisms. Here, we carried out a microcosm experiment to evaluate effects of soil moisture level and N fertilizer type on N₂O emissions and abundances and composition of associated microbial communities in the two typical arable soils. The abundances and community composition of functional microbes involved in nitrification and denitrification were determined via quantitative PCR (qPCR) and terminal restriction length fragment polymorphism (T-RFLP), respectively. Results showed that N₂O production was higher at 90% water-filled pore (WFPS) than at 50% WFPS. The N₂O emissions in the two soils amended with ammonium were higher than those amended with nitrate, especially at relatively high moisture level. In both soils, increased soil moisture stimulated the growth of ammonia-oxidizing bacteria (AOB) and nitrite reducer (nirK). Ammonium fertilizer treatment increased the population size of AOB and nirK genes in the alluvial soil, while reduced the abundances of ammonia-oxidizing archaea (AOA) and denitrifiers (nirK and nosZ) in the red soil. Nitrate addition had a negative effect on AOA abundance in the red soil. Total N₂O emissions were positively correlated to AOB abundance, but not to other functional genes in the two soils. Changed soil moisture significantly affected AOA rather than AOB community composition in both soils. The way and extent of N fertilizers impacted on nitrifier and denitrifier community composition varied with N form and soil type. These results indicate that N₂O emissions and the succession of nitrifying and denitrifying communities are selectively affected by soil moisture and N fertilizer form in the two contrasting types of soil.     

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.     

Short-term population dynamics of ammonia oxidizing bacteria in an agricultural soil

Ammonia oxidizing bacteria (AOB) control the rate limiting step of nitrification, the conversion of ammonia (NH₄⁺) to nitrite (NO₂⁻). The AOB therefore have an important role to play in regulating soil nitrogen cycling. Tillage aerates the soil, stimulating rapid changes in soil N cycling and microbial communities. Here we report results of a study of the short term responses of AOB and net nitrification to simulated tillage and NH₄⁺ addition to soil. The intensively farmed vegetable soils of the Salinas Valley, California, provide the context for this study. These soils are cultivated frequently, receive large N fertilizer inputs and there are regional concerns about groundwater N concentrations. An understanding of N dynamics in these systems is therefore important. AOB population sizes were quantified using a real-time PCR approach. In a 15 day experiment AOB populations, increased rapidly following tillage and NH₄⁺ addition and persisted after the depletion of soil NH₄⁺. AOB population sizes increased to a similar degree, over a 1.5-day period, irrespective of the amount of NH₄⁺ supplied. These data suggest selection of an AOB community in this intensively farmed and C-limited soil, that rapidly uses NH₄⁺ that becomes available. These data also suggest that mineralization may play an especially important role in regulating AOB populations where NH₄⁺ pool sizes are very low. Methodological considerations in the study of soil AOB communities are also discussed.     

不同施肥方式下土壤氨氧化细菌的群落特征

为了研究长期定位施肥对棕壤中氨氧化细菌（ammonia-oxidizing bacteria,AOB）种群结构多样性和垂直分布特征的影响,本研究采用化学分析、荧光定量PCR（qPCR）和变性梯度凝胶电泳（PCR-DGGE）技术,针对沈阳农业大学试验区不同施肥方式（不施肥、低量无机氮肥、高量无机氮肥、无机氮肥与有机肥配施）下不同土壤深度（0~20 cm、20~40 cm、40~60 cm）的土壤理化性质、AOB丰度及种群多样性进行分析,比较不同施肥方式对土壤AOB种群的影响。结果显示,与不施肥相比,施肥会降低土壤pH,增加土壤铵态氮（70.5%~939.21%）和硝态氮（253.20%~625.48%）含量。随土壤深度增加,土壤pH升高,铵态氮和硝态氮含量除低量无机氮肥处理外,多呈降低趋势。土壤增施氮肥可提高AOB丰度,降低总细菌丰度。其中,0~20 cm土层中AOB丰度较高,且高量无机氮肥处理的AOB数量最高,为9.65×10⁵拷贝数·g^-1（干土）。DGGE图谱分析显示,不同处理下,AOB群落结构多样性指数存在明显差异（P<0.05）,各多样性指数均在表层（0~20 cm）最高,增施氮肥则显著降低AOB的多样性。聚类分析表明,4个施肥处理中,高量无机氮肥处理聚为一类,其他处理则因土壤深度不同而异;3个土壤深度中,除不施肥处理外,所有施肥处理均表现为0~20 cm、20~40 cm土层发生聚类,40~60 cm则明显与其他两层分开。冗余梯度分析（RDA）显示,硝态氮（P=0.027）是造成影响AOB群落结构差异的主要原因。上述研究结果表明,长期定位施肥土壤AOB的数量和群落结构多样性受施肥方式显著影响,并表现出明显的垂直分布特征。与无机氮肥相比,有机无机配施处理有助于改善土壤pH,维持不同土壤深度下AOB群落结构多样性。     

Co-tolerance to zinc and copper of the soil nitrifying community and its relationship with the community structure

Soil microbial communities can develop trace metal tolerance upon soil contamination with corresponding metals. A few studies have reported co-tolerance in such cases, i.e. tolerance to other metals than those to which the microbial community had been exposed to. This study was set-up to test for co-tolerance of nitrifying communities to zinc (Zn) and copper (Cu) and to relate tolerances to shifts in community structure using amoA AOB (ammonia oxidizing bacteria) DGGE. Seven sets of soils, each representing a Cu or Zn contamination gradient were sampled from four locations. At two locations, both Cu and Zn had been added as single contaminants. Increased Zn and Cu tolerance of the nitrifying communities was consistently observed in response to corresponding soil contamination. Co-tolerance to Zn was obtained in two of the three Cu gradients and that to Cu in one of the four Zn gradients. DGGE analysis and sequencing showed that contamination with either Zn or Cu selected for identical AOB phylotypes in soils at one location but not at the other location. The nitrifying community structures in soils from different locations did not become more similar upon Zn exposure than those in corresponding uncontaminated soils. Hence, trace metal tolerance development was not due to the emergence of specific AOB phylotypes, but due to the emergence of different AOB phylotypes bearing tolerance mechanisms for Zn, Cu or both metals.     

Responses of bacterial and archaeal ammonia oxidisers of an acidic luvisols soil to different nitrogen fertilization rates after 9?years

It is still not clear which group of ammonia-oxidizing microorganisms plays the most important roles in nitrification in soils. Change in abundances and community compositions of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) under long-term different nitrogen (N) fertilization rates were investigated in an acidic luvisols soil using real-time polymerase chain reaction and denaturing gradient gel electrophoresis, respectively, based on the ammonia monooxygenase a-subunit gene. The experimental plan included the following treatments: control without N fertilization (N_CK), low N fertilization rate, middle N fertilization rate, and high N fertilization rate as 0, 100, 150, and 250?kg urea-N?ha^?1, respectively. Long-term different N fertilization rates did not significantly alter the total C and N contents of soil while it significantly decreased soil pH, which ranged from 5.60 to 5.20. The AOB abundance was more abundant in the N fertilization treatments than the N_CK treatment; the AOA abundance decreased by the increasing N fertilization rates, as did the ratios of AOA/AOB. The large differences in the potential nitrification rates among four treatments depended on the changes in AOA abundance but not to changes in AOB abundance. Phylogenetic analysis showed that the AOB communities were dominated by Nitrosospira clusters 1, 3, and 9 while all AOA sequences were grouped into soil/sediment cluster except for one sequence. Taken together, these results indicated that AOB and AOA preferred different soil N conditions and AOA were functionally more important in the nitrification than AOB in the acidic luvisols soil.     

Quantification of ammonia-oxidising bacteria in limed and non-limed acidic coniferous forest soil using real-time PCR

Ammonia-oxidising bacteria (AOB) in limed and non-limed acidic coniferous forest soil were investigated using real-time PCR. Two sites in southern Sweden were studied, 244 Åled and Oxafällan. The primers and probe used earlier appeared to be specific to the 16S rRNA gene of AOB belonging to the β-subgroup of the Proteobacteria [Appl. Environ. Microbiol. 67 (2001) 972]. Plots treated with two different doses of lime, 3 or 6 t ha⁻¹, were compared with non-limed control plots on two occasions during a single growing season. Three different soil depths were analysed to elucidate possible differences in the density of their AOB communities. The only clear effect of liming on the AOB was recorded in the beginning of the growing season at 244 Åled. In samples taken in April from this site, the numbers of AOB were higher in the limed plots than in the control plots. At the end of the growing season the AOB communities were all of a similar size in the different plots at both sites, irrespective of liming. The number of AOB, determined using real-time PCR, ranged between 6×10⁶ and 1×10⁹ cells g⁻¹ soil (dw) at the two sites, and generally decreased with increasing soil depth. The results showed no correlation between community density and potential nitrification. This may indicate a partly inactive AOB community. Furthermore, more than 10⁷ cells g⁻¹ soil (dw) were recorded using real-time PCR in the control plot at 244 Åled, although Bäckman et al. [Soil Biol. Biochem. 35 (2003) 1337] detected no AOB like sequences in the same plots using PCR followed by DGGE. Taken together our results strongly suggest that the primers and probe set used are not well suited for quantifying AOB in acidic forest soils, which is probably due to an insufficient specificity. This shows that it is extremely important to re-evaluate any primers and probe set when used in a new environment. Consideration should be given to the specificity and sensitivity, both empirically and using bioinformatic tools.     

Long-term impact of fertilization on activity and composition of bacterial communities and metabolic guilds in agricultural soil

To explore long-term impact of organic and inorganic fertilizers on microbial communities, we targeted both the total bacterial community and the autotrophic ammonia oxidizing bacteria (AOB) in soil from six treatments at an experimental field site established in 1956: cattle manure, sewage sludge, Ca(NO₃)₂, (NH₄)₂SO₄, unfertilized and unfertilized without crops. All plots, except the bare fallows, were cropped with maize. Effects on activity were assessed by measuring the basal respiration and substrate induced respiration (SIR) rates, and the potential activity of the AOB. To determine the bacterial community composition, 16S rRNA genes were used to fingerprint total soil communities by terminal restriction fragment length polymorphism analysis and AOB communities by denaturing gradient gel electrophoresis. The fertilization regimes had clear effects on both activity and composition of the soil communities. Basal respiration and r, which was kinetically derived as the exponentially growing fraction of the SIR-response, correlated well with the soil organic C content (r=0.93 and 0.66, respectively). Soil pH ranged from 3.97 to 6.26 in the treatments and was found to be an important factor influencing all microbial activities. pH correlated negatively with the ratio between basal respiration and SIR (r=0.90), indicating a decreased efficiency of heterotrophic microorganisms to convert organic carbon into microbial biomass in the most acid soils with pH 3.97 and 4.68 ((NH₄)₂SO₄ and sewage sludge fertilized plots, respectively). The lowest SIR and ammonia oxidation rates were also found in these treatments. In addition, these treatments exhibited individually different community fingerprints, showing that pH affected the composition of AOB and total bacterial communities. The manure fertilized plots harbored the most diverse AOB community and the pattern was linked to a high potential ammonia oxidation activity. Thus, the AOB community composition appeared to be more strongly linked to the activity than the total bacterial communities were, likely explained by physiological differences in the populations present.     

Inhibition of ammonia-oxidizing bacteria promotes the growth of ammonia-oxidizing archaea in ammonium-rich alkaline soils

Disparities in the substrate affinity and tolerance threshold for ammonia have been believed to play a key role in driving niche differentiation between ammonia-oxidizing archaea (AOA) and bacteria (AOB); however, recent surveys argue that direct competition between AOA and AOB is also important in this phenomenon. Accordingly, it is reasonable to predict that diverse AOA lineages would grow in ammonium (NH₄⁺)-rich alkaline arable soils if AOB growth is suppressed. To test this hypothesis, a microcosm study was established using three different types of alkaline arable soils, in which a high NH₄⁺ concentration (200 μg N g^-1 dry soil) was maintained by routinely replenishing urea and the activities of AOB were selectively inhibited by 1-octyne or 3,4-dimethylpyrazole phosphate (DMPP). Compared with amendment with urea alone, 1-octyne partially retarded AOB growth, while DMPP completely inhibited AOB. Both inhibitors accelerated the growth of AOA, with significantly higher ratios of abundance of AOA to AOB observed with DMPP amendment across soils. Nonmetric multidimensional scaling analysis (NMDS) indicated that different treatments significantly altered the community structures of both AOA and AOB and AOA OTUs enriched by high-NH₄⁺ amendment were taxonomically constrained across the soils tested and closely related to Nitrososphaera viennensis EN76 and N. garnensis. Given that these representative strains have been demonstrated to be sensitive to high ammonia concentrations, our results suggest that it is the competitiveness for ammonia, rather than disparities in substrate affinity and tolerance threshold for ammonia, that drives niche differentiation between these phylotypes and AOB in NH₄⁺-rich alkaline soils.