Autotrophic ammonia-oxidizing bacteria contribute minimally to nitrification in a nitrogen-impacted forested ecosystem

Deposition rates of atmospheric nitrogenous pollutants to forests in the San Bernardino Mountains range east of Los Angeles, California, are the highest reported in North America. Acidic soils from the west end of the range are N-saturated and have elevated rates of N-mineralization, nitrification, and nitrate leaching. We assessed the impact of this heavy nitrogen load on autotrophic ammonia-oxidizing communities by investigating their composition, abundance, and activity. Analysis of 177 cloned beta-Proteobacteria ammonia oxidizer 16S rRNA genes from highly to moderately N-impacted soils revealed similar levels of species composition; all of the soils supported the previously characterized Nitrosospira clusters 2, 3, and 4. Ammonia oxidizer abundance measured by quantitative PCR was also similar among the soils. However, rates of potential nitrification activity were greater for N-saturated soils than for soils collected from a less impacted site, but autotrophic (i.e., acetylene-sensitive) activity was low in all soils examined. N-saturated soils incubated for 30 days with ammonium accumulated additional soluble ammonium, whereas less-N-impacted soils had a net loss of ammonium. Lastly, nitrite production by cultivated Nitrosospira multiformis, an autotrophic ammonia-oxidizing bacterium adapted to relatively high ammonium concentrations, was significantly inhibited in pH-controlled slurries of sterilized soils amended with ammonium despite the maintenance of optimal ammonia-oxidizing conditions. Together, these results showed that factors other than autotrophic ammonia oxidizers contributed to high nitrification rates in these N-impacted forest soils and, unlike many other environments, differences in nitrogen content and soil pH did not favor particular autotrophic ammonia oxidizer groups.     

Ammonia-oxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils

Increasing evidence demonstrated the involvement of ammonia-oxidizing archaea (AOA) in the global nitrogen cycle, but the relative contributions of AOA and ammonia-oxidizing bacteria (AOB) to ammonia oxidation are still in debate. Previous studies suggest that AOA would be more adapted to ammonia-limited oligotrophic conditions, which seems to be favored by protonation of ammonia, turning into ammonium in low-pH environments. Here, we investigated the autotrophic nitrification activity of AOA and AOB in five strongly acidic soils (pH<4.50) during microcosm incubation for 30 days. Significantly positive correlations between nitrate concentration and amoA gene abundance of AOA, but not of AOB, were observed during the active nitrification. ¹³CO₂-DNA-stable isotope probing results showed significant assimilation of ¹³C-labeled carbon source into the amoA gene of AOA, but not of AOB, in one of the selected soil samples. High levels of thaumarchaeal amoA gene abundance were observed during the active nitrification, coupled with increasing intensity of two denaturing gradient gel electrophoresis bands for specific thaumarchaeal community. Addition of the nitrification inhibitor dicyandiamide (DCD) completely inhibited the nitrification activity and CO₂ fixation by AOA, accompanied by decreasing thaumarchaeal amoA gene abundance. Bacterial amoA gene abundance decreased in all microcosms irrespective of DCD addition, and mostly showed no correlation with nitrate concentrations. Phylogenetic analysis of thaumarchaeal amoA gene and 16S rRNA gene revealed active ¹³CO₂-labeled AOA belonged to groups 1.1a-associated and 1.1b. Taken together, these results provided strong evidence that AOA have a more important role than AOB in autotrophic ammonia oxidation in strongly acidic soils.     

Autotrophic Ammonia Oxidation at Low pH through Urea Hydrolysis

Ammonia oxidation in laboratory liquid batch cultures of autotrophic ammonia oxidizers rarely occurs at pH values less than 7, due to ionization of ammonia and the requirement for ammonium transport rather than diffusion of ammonia. Nevertheless, there is strong evidence for autotrophic nitrification in acid soils, which may be carried out by ammonia oxidizers capable of using urea as a source of ammonia. To determine the mechanism of urea-linked ammonia oxidation, a ureolytic autotrophic ammonia oxidizer, Nitrosospira sp. strain NPAV, was grown in liquid batch culture at a range of pH values with either ammonium or urea as the sole nitrogen source. Growth and nitrite production from ammonium did not occur at pH values below 7. Growth on urea occurred at pH values in the range 4 to 7.5 but ceased when urea hydrolysis was complete, even though ammonia, released during urea hydrolysis, remained in the medium. The results support a mechanism whereby urea enters the cells by diffusion and intracellular urea hydrolysis and ammonia oxidation occur independently of extracellular pH in the range 4 to 7.5. A proportion of the ammonia produced during this process diffuses from the cell and is not subsequently available for growth if the extracellular pH is less than 7. Ureolysis therefore provides a mechanism for nitrification in acid soils, but a proportion of the ammonium produced is likely to be released from the cell and may be used by other soil organisms.     

Nitrification of archaeal ammonia oxidizers in acid soils is supported by hydrolysis of urea

The hydrolysis of urea as a source of ammonia has been proposed as a mechanism for the nitrification of ammonia-oxidizing bacteria (AOB) in acidic soil. The growth of Nitrososphaera viennensis on urea suggests that the ureolysis of ammonia-oxidizing archaea (AOA) might occur in natural environments. In this study, ¹⁵N isotope tracing indicates that ammonia oxidation occurred upon the addition of urea at a concentration similar to the in situ ammonium content of tea orchard soil (pH 3.75) and forest soil (pH 5.4) and was inhibited by acetylene. Nitrification activity was significantly stimulated by urea fertilization and coupled well with abundance changes in archaeal amoA genes in acidic soils. Pyrosequencing of 16S rRNA genes at whole microbial community level demonstrates the active growth of AOA in urea-amended soils. Molecular fingerprinting further shows that changes in denaturing gradient gel electrophoresis fingerprint patterns of archaeal amoA genes are paralleled by nitrification activity changes. However, bacterial amoA and 16S rRNA genes of AOB were not detected. The results strongly suggest that archaeal ammonia oxidation is supported by hydrolysis of urea and that AOA, from the marine Group 1.1a-associated lineage, dominate nitrification in two acidic soils tested.     

Autotrophic ammonia oxidation at low pH through urea hydrolysis.

Ammonia oxidation in laboratory liquid batch cultures of autotrophic ammonia oxidizers rarely occurs at pH values less than 7, due to ionization of ammonia and the requirement for ammonium transport rather than diffusion of ammonia. Nevertheless, there is strong evidence for autotrophic nitrification in acid soils, which may be carried out by ammonia oxidizers capable of using urea as a source of ammonia. To determine the mechanism of urea-linked ammonia oxidation, a ureolytic autotrophic ammonia oxidizer, Nitrosospira sp. strain NPAV, was grown in liquid batch culture at a range of pH values with either ammonium or urea as the sole nitrogen source. Growth and nitrite production from ammonium did not occur at pH values below 7. Growth on urea occurred at pH values in the range 4 to 7.5 but ceased when urea hydrolysis was complete, even though ammonia, released during urea hydrolysis, remained in the medium. The results support a mechanism whereby urea enters the cells by diffusion and intracellular urea hydrolysis and ammonia oxidation occur independently of extracellular pH in the range 4 to 7.5. A proportion of the ammonia produced during this process diffuses from the cell and is not subsequently available for growth if the extracellular pH is less than 7. Ureolysis therefore provides a mechanism for nitrification in acid soils, but a proportion of the ammonium produced is likely to be released from the cell and may be used by other soil organisms.     

Growth of ammonia-oxidizing archaea in soil microcosms is inhibited by acetylene

Autotrophic ammonia-oxidizing bacteria were considered to be responsible for the majority of ammonia oxidation in soil until the recent discovery of the autotrophic ammonia-oxidizing archaea. To assess the relative contributions of bacterial and archaeal ammonia oxidizers to soil ammonia oxidation, their growth was analysed during active nitrification in soil microcosms incubated for 30 days at 30 °C, and the effect of an inhibitor of ammonia oxidation (acetylene) on their growth and soil nitrification kinetics was determined. Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial ammonia oxidizer 16S rRNA genes did not detect any change in their community composition during incubation, and quantitative PCR (qPCR) analysis of bacterial amoA genes indicated a small decrease in abundance in control and acetylene-containing microcosms. DGGE fingerprints of archaeal amoA and 16S rRNA genes demonstrated changes in the relative abundance of specific crenarchaeal phylotypes during active nitrification. Growth was also indicated by increases in crenarchaeal amoA gene copy number, determined by qPCR. In microcosms containing acetylene, nitrification and growth of the crenarchaeal phylotypes were suppressed, suggesting that these crenarchaea are ammonia oxidizers. Growth of only archaeal but not bacterial ammonia oxidizers occurred in microcosms with active nitrification, indicating that ammonia oxidation was mostly due to archaea in the conditions of the present study.     

Nitrogen Cycling and Community Structure of Proteobacterial β-Subgroup Ammonia-Oxidizing Bacteria within Polluted Marine Fish Farm Sediments

A multidisciplinary approach was used to study the effects of pollution from a marine fish farm on nitrification rates and on the community structure of ammonia-oxidizing bacteria in the underlying sediment. Organic content, ammonium concentrations, nitrification rates, and ammonia oxidizer most-probable-number counts were determined in samples of sediment collected from beneath a fish cage and on a transect at 20 and 40 m from the cage. The data suggest that nitrogen cycling was significantly disrupted directly beneath the fish cage, with inhibition of nitrification and denitrification. Although visual examination indicated some slight changes in sediment appearance at 20 m, all other measurements were similar to those obtained at 40 m, where the sediment was considered pristine. The community structures of proteobacterial β-subgroup ammonia-oxidizing bacteria at the sampling sites were compared by PCR amplification of 16S ribosomal DNA (rDNA), using primers which target this group. PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE) and with oligonucleotide hybridization probes specific for different ammonia oxidizers. A DGGE doublet observed in PCR products from the highly polluted fish cage sediment sample was present at a lower intensity in the 20-m sample but was absent from the pristine 40-m sample station. Band migration, hybridization, and sequencing demonstrated that the doublet corresponded to a marine Nitrosomonas group which was originally observed in 16S rDNA clone libraries prepared from the same sediment samples but with different PCR primers. Our data suggest that this novel Nitrosomonas subgroup was selected for within polluted fish farm sediments and that the relative abundance of this group was influenced by the extent of pollution.     

Links between ammonia oxidizer species composition, functional diversity and nitrification kinetics in grassland soils

Molecular approaches have revealed considerable diversity and uncultured novelty in natural prokaryotic populations, but not direct links between the new genotypes detected and ecosystem processes. Here we describe the influence of the structure of communities of ammonia-oxidizing bacteria on nitrogen cycling in microcosms containing natural and managed grasslands and amended with artificial sheep urine, a major factor determining local ammonia concentrations in these environments. Nitrification kinetics were assessed by analysis of changes in urea, ammonia, nitrite and nitrate concentrations and ammonia oxidizer communities were characterized by analysis of 16S rRNA genes amplified from extracted DNA using ammonia oxidizer-specific primers. In natural soils, ammonia oxidizer community structure determined the delay preceding nitrification, which depended on the relative abundance of two Nitrosospira clusters, termed 3a and 3b. In batch cultures, pure culture and enrichment culture representatives of Nitrosospira 3a were sensitive to high ammonia concentration, while Nitrosospira cluster 3b representatives and Nitrosomonas europaea were tolerant. Delays in nitrification occurred in natural soils dominated by Nitrosospira cluster 3a and resulted from the time required for growth of low concentrations of Nitrosospira cluster 3b. In microcosms dominated by Nitrosospira cluster 3b and Nitrosomonas, no substantial delays were observed. In managed soils, no delays in nitrification were detected, regardless of initial ammonia oxidizer community structure, most probably resulting from higher ammonia oxidizer cell concentrations. The data therefore demonstrate a direct link between bacterial community structure, physiological diversity and ecosystem function.     

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

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

Response of Nitrosospira sp. Strain AF-Like Ammonia Oxidizers to Changes in Temperature, Soil Moisture Content, and Fertilizer Concentration

Very little is known regarding the ecology of Nitrosospira sp. strain AF-like bacteria, a unique group of ammonia oxidizers within the Betaproteobacteria. We studied the response of Nitrosospira sp. strain AF-like ammonia oxidizers to changing environmental conditions by applying molecular methods and physiological measurements to Californian grassland soil manipulated in the laboratory. This soil is naturally high in Nitrosospira sp. strain AF-like bacteria relative to the much-better-studied Nitrosospira multiformis-like ammonia-oxidizing bacteria. Increases in temperature, soil moisture, and fertilizer interacted to reduce the relative abundance of Nitrosospira sp. strain AF-like bacteria, although they remained numerically dominant. The overall abundance of ammonia-oxidizing bacteria increased with increasing soil moisture and decreased with increasing temperature. Potential nitrification activity was altered by interactions among temperature, soil moisture, and fertilizer, with activity tending to be higher when soil moisture and temperature were increased. The increase in potential nitrification activity with increased temperature was surprising, given that the overall abundance of ammonia-oxidizing bacteria decreased significantly under these conditions. This observation suggests that (i) Nitrosospira sp. strain AF-like bacteria may respond to increased temperature with an increase in activity, despite a decrease in abundance, or (ii) that potential nitrification activity in these soils may be due to organisms other than bacteria (e.g., archaeal ammonia oxidizers), at least under conditions of increased temperature.     

High abundance of ammonia-oxidizing archaea in acidified subtropical forest soils in southern China after long-term N deposition

Nitrification has been believed to be performed only by autotrophic ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) until the recent discovery of ammonia-oxidizing archaea (AOA). Meanwhile, it has been questioned whether AOB are significantly responsible for NH(3) oxidation in acidic forest soils. Here, we investigated nitrifying communities and their activity in highly acidified soils of three subtropical forests in southern China that had received chronic high atmospheric N deposition. Nitrifying communities were analyzed using PCR- and culture (most probable number)-based approaches. Nitrification activity was analyzed by measuring gross soil nitrification rates using a (15) N isotope dilution technique. AOB were not detected in the three forest soils: neither via PCR of 16S rRNA and ammonia monooxygenase (amoA) genes nor via culture-based approaches. In contrast, an extraordinary abundance of the putative archaeal amoA was detected (3.2?×?10(8) -1.2?×?10(9) g?soil(-1) ). Moreover, this abundance was correlated with gross soil nitrification rates. This indicates that amoA-possessing archaea rather than bacteria were predominantly responsible for nitrification of the soils. Furthermore, sequences of the genus Nitrospira, a dominant group of soil NOB, were detected. Thus, nitrification of acidified subtropical forest soils in southern China could be performed by a combination of AOA and NOB.     

Habitat Specialization Along a Wetland Moisture Gradient Differs Between Ammonia-oxidizing and Denitrifying Microorganisms

Gradients in abiotic parameters, such as soil moisture, can strongly influence microbial community structure and function. Denitrifying and ammonia-oxidizing microorganisms, in particular, have contrasting physiological responses to abiotic factors such as oxygen concentration and soil moisture. Identifying abiotic factors that govern the composition and activity of denitrifying and ammonia-oxidizing communities is critical for understanding the nitrogen cycle. The objectives of this study were to (i) examine denitrifier and archaeal ammonia oxidizer community composition and (ii) assess the taxa occurring within each functional group related to soil conditions along an environmental gradient. Soil was sampled across four transects at four locations along a dry to saturated environmental gradient at a restored wetland. Soil pH and soil organic matter content increased from dry to saturated plots. Composition of soil denitrifier and ammonia oxidizer functional groups was assessed by terminal restriction fragment length polymorphism (T-RFLP) community analysis, and local soil factors were also characterized. Microbial community composition of denitrifiers and ammonia oxidizers differed along the moisture gradient (denitrifier: ANOSIM R?=?0.739, P?<?0.001; ammonia oxidizers: ANOSIM R?=?0.760, P?<?0.001). Individual denitrifier taxa were observed over a larger range of moisture levels than individual archaeal ammonia oxidizer taxa (Wilcoxon rank sum, W?=?2413, P value?=?0.0002). Together, our data suggest that variation in environmental tolerance of microbial taxa have potential to influence nitrogen cycling in terrestrial ecosystems.     

Interactions between Methane Oxidation and Nitrification in Coastal Sediments

Galveston Bay sediments exhibit substantial spatial and seasonal variability in rates of nitrification and aerobic methane oxidation. We examined the biogeochemical and microbiological controls on these processes using aerobic enrichment slurries. Potential aerobic methane and ammonia oxidation rates from unamended control slurries were compared to rates in slurries amended with methane, ammonium, or methane + ammonium. Bacterial community composition was monitored using denaturing gradient gel electrophoresis (DGGE) analysis of PCR amplified ribosomal and functional gene DNA. Potential methane and ammonia oxidation rates increased over time in sediments amended with methane and ammonium, respectively. The highest potential methane oxidation rates occurred in treatments receiving both ammonium and methane suggesting that methanotrophs in the enrichment cultures were nitrogen limited. The highest ammonia oxidation rates occurred in treatments amended with ammonium only. Treatments receiving both ammonium and methane exhibited ammonia oxidation rates and porewater ammonium concentrations similar to those measured in the unamended control suggesting that methanotrophs may have inhibited ammonia oxidation by sequestering available ammonia. Sequence analysis revealed a decrease in general bacterial community diversity over time and a shift in ammonia-oxidizing bacterial composition corresponding with methane availability. However, methanotroph community composition similarities between treatments with different relative methane oxidation rates suggest that changes in physiological activity, as well as shifts in community composition, contributed to the observed patterns in potential rates.     

河口生态系统氨氧化菌生态学研究进展

由amoA基因编码的氨单加氧酶(AMO)所调控的氨氧化作用,是硝化作用的限速步骤和中心环节,而含有amoA基因的氨氧化细菌(AOB)和氨氧化古菌(AOA)多样性与环境因子关系密切,对缓解河口生态系统因人类活动造成的富营养化等环境问题具有特别重要的意义。水、陆和海交汇形成高度变异的具环境因子梯度的河口生态系统,是研究AOA和AOB生态学的天然实验室。河口AOA与AOB的群落组成、丰富度特征和生物有效性,与河口主要环境因子盐度、富营养化程度、植被、温度、碳、氮、硫、铁等,尤其是对盐度和富营养化有着较为强烈的响应。AOA和AOB多样性变化规律及其与河口特有的环境因子之间的相关性,应当是今后我国河口氨氧化菌研究的方向和重点。包括:(1)建立有效的氨氧化菌活性评价方法;(2)研究AOA的同化作用方式;(3)依据氨氧化菌分类和组成对河口环境变化的适应进化机制,建议可作为指示河口环境质量变化的生物标记;(4)将传统的分离培养方法与现代分子生物学研究方法相结合,筛选我国河口高效的氨氧化菌,并将其应用于生产。     

Ammonia-oxidizing archaea possess a wide range of cellular ammonia affinities

Nitrification, the oxidation of ammonia to nitrate, is an essential process in the biogeochemical nitrogen cycle. The first step of nitrification, ammonia oxidation, is performed by three, often co-occurring guilds of chemolithoautotrophs: ammonia-oxidizing bacteria (AOB), archaea (AOA), and complete ammonia oxidizers (comammox). Substrate kinetics are considered to be a major niche-differentiating factor between these guilds, but few AOA strains have been kinetically characterized. Here, the ammonia oxidation kinetic properties of 12 AOA representing all major cultivated phylogenetic lineages were determined using microrespirometry. Members of the genus Nitrosocosmicus have the lowest affinity for both ammonia and total ammonium of any characterized AOA, and these values are similar to previously determined ammonia and total ammonium affinities of AOB. This contrasts previous assumptions that all AOA possess much higher substrate affinities than their comammox or AOB counterparts. The substrate affinity of ammonia oxidizers correlated with their cell surface area to volume ratios. In addition, kinetic measurements across a range of pH values supports the hypothesis that—like for AOB—ammonia and not ammonium is the substrate for the ammonia monooxygenase enzyme of AOA and comammox. Together, these data will facilitate predictions and interpretation of ammonia oxidizer community structures and provide a robust basis for establishing testable hypotheses on competition between AOB, AOA, and comammox.Subject terms: Archaeal physiology, Metabolism, Microbial ecology     

Stimulation of thaumarchaeal ammonia oxidation by ammonia derived from organic nitrogen but not added inorganic nitrogen

Ammonia oxidation, the first step in nitrification, is performed by autotrophic bacteria and thaumarchaea, whose relative contributions vary in different soils. Distinctive environmental niches for the two groups have not been identified, but evidence from previous studies suggests that activity of thaumarchaea, unlike that of bacterial ammonia oxidizers, is unaffected by addition of inorganic N fertilizer and that they preferentially utilize ammonia generated from the mineralization of organic N. This hypothesis was tested by determining the influence of both inorganic and organic N sources on nitrification rate and ammonia oxidizer growth and community structure in microcosms containing acidic, forest soil in which ammonia oxidation was dominated by thaumarchaea. Nitrification rate was unaffected by the incubation of soil with inorganic ammonium but was significantly stimulated by the addition of organic N. Oxidation of ammonia generated from native soil organic matter or added organic N, but not added inorganic N, was accompanied by increases in abundance of the thaumarchaeal amoA gene, a functional gene for ammonia oxidation, but changes in community structure were not observed. Bacterial amoA genes could not be detected. Ammonia oxidation was completely inhibited by 0.01% acetylene in all treatments, indicating ammonia monooxygenase-dependent activity. The findings have implications for current models of soil nitrification and for nitrification control strategies to minimize fertilizer loss and nitrous oxide production.