Graeme W. Nicol

Archaea predominate among ammonia-oxidizing prokaryotes in soils.

Ammonia oxidation is the first step in nitrification, a key process in the global nitrogen cycle that results in the formation of nitrate through microbial activity. The increase in nitrate availability in soils is important for plant nutrition, but it also has considerable impact on groundwater pollution owing to leaching. Here we show that archaeal ammonia oxidizers are more abundant in soils than their well-known bacterial counterparts. We investigated the abundance of the gene encoding a subunit of the key enzyme ammonia monooxygenase (amoA) in 12 pristine and agricultural soils of three climatic zones. amoA gene copies of Crenarchaeota (Archaea) were up to 3,000-fold more abundant than bacterial amoA genes. High amounts of crenarchaeota-specific lipids, including crenarchaeol, correlated with the abundance of archaeal amoA gene copies. Furthermore, reverse transcription quantitative PCR studies and complementary DNA analysis using novel cloning-independent pyrosequencing technology demonstrated the activity of the archaea in situ and supported the numerical dominance of archaeal over bacterial ammonia oxidizers. Our results indicate that crenarchaeota may be the most abundant ammonia-oxidizing organisms in soil ecosystems on Earth.

The influence of soil pH on the diversity, abundance and transcriptional activity of ammonia oxidizing archaea and bacteria.

Autotrophic ammonia oxidation occurs in acid soils, even though laboratory cultures of isolated ammonia oxidizing bacteria fail to grow below neutral pH. To investigate whether archaea possessing ammonia monooxygenase genes were responsible for autotrophic nitrification in acid soils, the community structure and phylogeny of ammonia oxidizing bacteria and archaea were determined across a soil pH gradient (4.9-7.5) by amplifying 16S rRNA and amoA genes followed by denaturing gradient gel electrophoresis (DGGE) and sequence analysis. The structure of both communities changed with soil pH, with distinct populations in acid and neutral soils. Phylogenetic reconstructions of crenarchaeal 16S rRNA and amoA genes confirmed selection of distinct lineages within the pH gradient and high similarity in phylogenies indicated a high level of congruence between 16S rRNA and amoA genes. The abundance of archaeal and bacterial amoA gene copies and mRNA transcripts contrasted across the pH gradient. Archaeal amoA gene and transcript abundance decreased with increasing soil pH, while bacterial amoA gene abundance was generally lower and transcripts increased with increasing pH. Short-term activity was investigated by DGGE analysis of gene transcripts in microcosms containing acidic or neutral soil or mixed soil with pH readjusted to that of native soils. Although mixed soil microcosms contained identical archaeal ammonia oxidizer communities, those adapted to acidic or neutral pH ranges showed greater relative activity at their native soil pH. Findings indicate that different bacterial and archaeal ammonia oxidizer phylotypes are selected in soils of different pH and that these differences in community structure and abundances are reflected in different contributions to ammonia oxidizer activity. They also suggest that both groups of ammonia oxidizers have distinct physiological characteristics and ecological niches, with consequences for nitrification in acid soils.

Growth, activity and temperature responses of ammonia-oxidizing archaea and bacteria in soil microcosms

Ammonia oxidation, as the first step in the nitrification process, plays a central role in the global cycling of nitrogen. Although bacteria are traditionally considered to be responsible for ammonia oxidation, a role for archaea has been suggested by data from metagenomic studies and by the isolation of a marine, autotrophic, ammonia-oxidizing, non-thermophilic crenarchaeon. Evidence for ammonia oxidation by non-thermophilic crenarchaea in marine and terrestrial environments is largely based on abundance of bacterial and archaeal ammonia monooxygenase (amo) genes, rather than activity. In this study, we have determined the influence of temperature on the response of ammonia-oxidizing bacteria and archaea in nitrifying soil microcosms using two approaches, involving analysis of transcriptional activity of 16S rRNA genes and of a key functional gene, amoA, which encodes ammonia monooxygenase subunit A. There was little evidence of changes in relative abundance or transcriptional activity of ammonia-oxidizing bacteria during nitrification. In contrast, denaturing gradient gel electrophoresis analysis of crenarchaeal 16S rRNA and crenarchaeal amoA genes provided strong evidence of changes in community structure of active archaeal ammonia oxidizers. Community structure changes were similar during incubation at different temperatures and much of the activity was due to a group of non-thermophilic crenarchaea associated with subsurface and marine environments, rather than soil. The findings suggest a role for crenarchaea in soil nitrification and that further information is required on their biogeography.

Relative contributions of archaea and bacteria to aerobic ammonia oxidation in the environment.

Traditionally, organisms responsible for major biogeochemical cycling processes have been determined by physiological characterization of environmental isolates in laboratory culture. Molecular techniques have, however, confirmed the widespread occurrence of abundant bacterial and archaeal groups with no cultivated representative, making it difficult to determine their ecosystem function. Until recently, ammonia oxidation, the first step in the globally important process of nitrification, was thought to be performed almost exclusively by bacteria. Metagenome studies, followed by laboratory isolation, then demonstrated the potential for significant ammonia oxidation by mesophilic crenarchaea, whose ecosystem function was previously unknown. Re-assessment of the role of bacteria in ammonia oxidation is now required and this article reviews the current evidence for the relative importance of bacteria and archaea. Much of this evidence is based on metagenomic analysis and molecular techniques for estimation of gene and gene transcript abundance, changes in ammonia oxidizer community structure during active nitrification and phylogeny of natural communities. These studies have been complemented by physiological characterization of a laboratory isolate and by incorporation of labelled substrates. Data from these studies provide increasingly convincing evidence for the importance of archaeal ammonia oxidizers in the global nitrogen cycle. They also highlight the need to re-assess the importance of ammonia-oxidizing bacteria, the requirement and limitations of molecular techniques in linking specific microbial groups to ecosystem function and the limitations of reliance on laboratory cultures.

Archaeal and bacterial ammonia- oxidisers in soil: the quest for niche specialisation and differentiation

Autotrophic archaeal and bacterial ammonia-oxidisers (AOA and AOB) drive soil nitrification. Ammonia limitation, mixotrophy, and pH have been suggested as factors providing niche specialisation and differentiation between soil AOA and AOB. However, current data from genomes, cultures, field studies, and microcosms suggest that no single factor discriminates between AOA and AOB. In addition, there appears to be sufficient physiological diversity within each group for growth and activity in all soils investigated, with the exception of acidic soils (pH <5.5), which are dominated by AOA. Future investigation of niche specialisation in ammonia-oxidisers, and other microbial communities, requires characterisation of a wider range of environmentally representative cultures, emphasis on experimental studies rather than surveys, and greater consideration of small-scale soil heterogeneity.

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

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

Cultivation of an obligate acidophilic ammonia oxidizer from a nitrifying acid soil

Nitrification is a fundamental component of the global nitrogen cycle and leads to significant fertilizer loss and atmospheric and groundwater pollution. Nitrification rates in acidic soils (pH < 5.5), which comprise 30% of the worlds soils, equal or exceed those of neutral soils. Paradoxically, autotrophic ammonia oxidizing bacteria and archaea, which perform the first stage in nitrification, demonstrate little or no growth in suspended liquid culture below pH 6.5, at which ammonia availability is reduced by ionization. Here we report the discovery and cultivation of a chemolithotrophic, obligately acidophilic thaumarchaeal ammonia oxidizer, “Candidatus Nitrosotalea devanaterra,” from an acidic agricultural soil. Phylogenetic analysis places the organism within a previously uncultivated thaumarchaeal lineage that has been observed in acidic soils. Growth of the organism is optimal in the pH range 4 to 5 and is restricted to the pH range 4 to 5.5, unlike all previously cultivated ammonia oxidizers. Growth of this organism and associated ammonia oxidation and autotrophy also occur during nitrification in soil at pH 4.5. The discovery of Nitrosotalea devanaterra provides a previously unsuspected explanation for high rates of nitrification in acidic soils, and confirms the vital role that thaumarchaea play in terrestrial nitrogen cycling. Growth at extremely low ammonia concentration (0.18 nM) also challenges accepted views on ammonia uptake and metabolism and indicates novel mechanisms for ammonia oxidation at low pH.

Archaea rather than bacteria control nitrification in two agricultural acidic soils

Nitrification is a central component of the global nitrogen cycle. Ammonia oxidation, the first step of nitrification, is performed in terrestrial ecosystems by both ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Published studies indicate that soil pH may be a critical factor controlling the relative abundances of AOA and AOB communities. In order to determine the relative contributions of AOA and AOB to ammonia oxidation in two agricultural acidic Scottish soils (pH 4.5 and 6), the influence of acetylene (a nitrification inhibitor) was investigated during incubation of soil microcosms at 20 °C for 1 month. High rates of nitrification were observed in both soils in the absence of acetylene. Quantification of respective amoA genes (a key functional gene for ammonia oxidizers) demonstrated significant growth of AOA, but not AOB. A significant positive relationship was found between nitrification rate and AOA, but not AOB growth. AOA growth was inhibited in the acetylene-containing microcosms. Moreover, AOA transcriptional activity decreased significantly in the acetylene-containing microcosms compared with the control, whereas no difference was observed for the AOB transcriptional activity. Consequently, growth and activity of only archaeal but not bacterial ammonia oxidizer communities strongly suggest that AOA, but not AOB, control nitrification in these two acidic soils.

Autotrophic ammonia oxidation by soil thaumarchaea

Nitrification plays a central role in the global nitrogen cycle and is responsible for significant losses of nitrogen fertilizer, atmospheric pollution by the greenhouse gas nitrous oxide, and nitrate pollution of groundwaters. Ammonia oxidation, the first step in nitrification, was thought to be performed by autotrophic bacteria until the recent discovery of archaeal ammonia oxidizers. Autotrophic archaeal ammonia oxidizers have been cultivated from marine and thermal spring environments, but the relative importance of bacteria and archaea in soil nitrification is unclear and it is believed that soil archaeal ammonia oxidizers may use organic carbon, rather than growing autotrophically. In this soil microcosm study, stable isotope probing was used to demonstrate incorporation of 13C-enriched carbon dioxide into the genomes of thaumarchaea possessing two functional genes: amoA, encoding a subunit of ammonia monooxygenase that catalyses the first step in ammonia oxidation; and hcd, a key gene in the autotrophic 3-hydroxypropionate/4-hydroxybutyrate cycle, which has been found so far only in archaea. Nitrification was accompanied by increases in archaeal amoA gene abundance and changes in amoA gene diversity, but no change was observed in bacterial amoA genes. Archaeal, but not bacterial, amoA genes were also detected in 13C-labeled DNA, demonstrating inorganic CO2 fixation by archaeal, but not bacterial, ammonia oxidizers. Autotrophic archaeal ammonia oxidation was further supported by coordinate increases in amoA and hcd gene abundance in 13C-labeled DNA. The results therefore provide direct evidence for a role for archaea in soil ammonia oxidation and demonstrate autotrophic growth of ammonia oxidizing archaea in soil.

Niche specialization of terrestrial archaeal ammonia oxidizers

Soil pH is a major determinant of microbial ecosystem processes and potentially a major driver of evolution, adaptation, and diversity of ammonia oxidizers, which control soil nitrification. Archaea are major components of soil microbial communities and contribute significantly to ammonia oxidation in some soils. To determine whether pH drives evolutionary adaptation and community structure of soil archaeal ammonia oxidizers, sequences of amoA, a key functional gene of ammonia oxidation, were examined in soils at global, regional, and local scales. Globally distributed database sequences clustered into 18 well-supported phylogenetic lineages that dominated specific soil pH ranges classified as acidic (pH <5), acido-neutral (5≤ pH <7), or alkalinophilic (pH ≥7). To determine whether patterns were reproduced at regional and local scales, amoA gene fragments were amplified from DNA extracted from 47 soils in the United Kingdom (pH 3.5–8.7), including a pH-gradient formed by seven soils at a single site (pH 4.5–7.5). High-throughput sequencing and analysis of amoA gene fragments identified an additional, previously undiscovered phylogenetic lineage and revealed similar pH-associated distribution patterns at global, regional, and local scales, which were most evident for the five most abundant clusters. Archaeal amoA abundance and diversity increased with soil pH, which was the only physicochemical characteristic measured that significantly influenced community structure. These results suggest evolution based on specific adaptations to soil pH and niche specialization, resulting in a global distribution of archaeal lineages that have important consequences for soil ecosystem function and nitrogen cycling.