Ricardo J. Eloy Alves

Nitrification rates in Arctic soils are associated with functionally distinct populations of ammonia-oxidizing archaea

The functioning of Arctic soil ecosystems is crucially important for global climate, and basic knowledge regarding their biogeochemical processes is lacking. Nitrogen (N) is the major limiting nutrient in these environments, and its availability is strongly dependent on nitrification. However, microbial communities driving this process remain largely uncharacterized in Arctic soils, namely those catalyzing the rate-limiting step of ammonia (NH3) oxidation. Eleven Arctic soils were analyzed through a polyphasic approach, integrating determination of gross nitrification rates, qualitative and quantitative marker gene analyses of ammonia-oxidizing archaea (AOA) and bacteria (AOB) and enrichment of AOA in laboratory cultures. AOA were the only NH3 oxidizers detected in five out of 11 soils and outnumbered AOB in four of the remaining six soils. The AOA identified showed great phylogenetic diversity and a multifactorial association with the soil properties, reflecting an overall distribution associated with tundra type and with several physico-chemical parameters combined. Remarkably, the different gross nitrification rates between soils were associated with five distinct AOA clades, representing the great majority of known AOA diversity in soils, which suggests differences in their nitrifying potential. This was supported by selective enrichment of two of these clades in cultures with different NH3 oxidation rates. In addition, the enrichments provided the first direct evidence for NH3 oxidation by an AOA from an uncharacterized Thaumarchaeota–AOA lineage. Our results indicate that AOA are functionally heterogeneous and that the selection of distinct AOA populations by the environment can be a determinant for nitrification activity and N availability in soils.

Nitrososphaera viennensis gen. nov., sp. nov., an aerobic and mesophilic, ammonia-oxidizing archaeon from soil and a member of the archaeal phylum Thaumarchaeota

A mesophilic, neutrophilic and aerobic, ammonia-oxidizing archaeon, strain EN76T, was isolated from garden soil in Vienna (Austria). Cells were irregular cocci with a diameter of 0.6–0.9 µm and possessed archaella and archaeal pili as cell appendages. Electron microscopy also indicated clearly discernible areas of high and low electron density, as well as tubule-like structures. Strain EN76T had an S-layer with p3 symmetry, so far only reported for members of the Sulfolobales. Crenarchaeol was the major core lipid. The organism gained energy by oxidizing ammonia to nitrite aerobically, thereby fixing CO2, but growth depended on the addition of small amounts of organic acids. The optimal growth temperature was 42 °C and the optimal pH was 7.5, with ammonium and pyruvate concentrations of 2.6 and 1 mM, respectively. The genome of strain EN76T had a DNA G+C content of 52.7 mol%. Phylogenetic analyses of 16S rRNA genes showed that strain EN76T is affiliated with the recently proposed phylum Thaumarchaeota, sharing 85 % 16S rRNA gene sequence identity with the closest cultivated relative ‘Candidatus Nitrosopumilus maritimus’ SCM1, a marine ammonia-oxidizing archaeon, and a maximum of 81 % 16S rRNA gene sequence identity with members of the phyla Crenarchaeota and Euryarchaeota and any of the other recently proposed phyla (e.g. ‘Korarchaeota’ and ‘Aigarchaeota’). We propose the name Nitrososphaera viennensis gen. nov., sp. nov. to accommodate strain EN76T. The type strain of Nitrososphaera viennensis is strain EN76T ( = DSM 26422T = JMC 19564T). Additionally, we propose the family Nitrososphaeraceae fam. nov., the order Nitrososphaerales ord. nov. and the class Nitrososphaeria classis nov.

Physiological and genomic characterization of two novel marine thaumarchaeal strains indicates niche differentiation

Ammonia-oxidizing Archaea (AOA) are ubiquitous throughout the oceanic water column; however, our knowledge on their physiological and ecological diversity in different oceanic regions is rather limited. Here, we report the cultivation and characterization of two novel Nitrosopumilus strains, originating from coastal surface waters of the Northern Adriatic Sea. The combined physiological and genomic information revealed that each strain exhibits different metabolic and functional traits, potentially reflecting contrasting life modes. Strain NF5 contains many chemotaxis-related genes and is able to express archaella, suggesting that it can sense and actively seek favorable microenvironments such as nutrient-rich particles. In contrast, strain D3C is non-motile and shows higher versatility in substrate utilization, being able to use urea as an alternative substrate in addition to ammonia. Furthermore, it encodes a divergent, second copy of the AmoB subunit of the key enzyme ammonia monooxygenase, which might have an additional catalytic function and suggests further metabolic versatility. However, the role of this gene requires further investigation. Our results provide evidence for functional diversity and metabolic versatility among phylogenetically closely related thaumarchaeal strains, and point toward adaptations to free-living versus particle-associated life styles and possible niche differentiation among AOA in marine ecosystems.

Microbial nitrogen dynamics in organic and mineral soil horizons along a latitudinal transect in western Siberia.

Soil N availability is constrained by the breakdown of N-containing polymers such as proteins to oligopeptides and amino acids that can be taken up by plants and microorganisms. Excess N is released from microbial cells as ammonium (N mineralization), which in turn can serve as substrate for nitrification. According to stoichiometric theory, N mineralization and nitrification are expected to increase in relation to protein depolymerization with decreasing N limitation, and thus from higher to lower latitudes and from topsoils to subsoils. To test these hypotheses, we compared gross rates of protein depolymerization, N mineralization and nitrification (determined using 15N pool dilution assays) in organic topsoil, mineral topsoil, and mineral subsoil of seven ecosystems along a latitudinal transect in western Siberia, from tundra (67°N) to steppe (54°N). The investigated ecosystems differed strongly in N transformation rates, with highest protein depolymerization and N mineralization rates in middle and southern taiga. All N transformation rates decreased with soil depth following the decrease in organic matter content. Related to protein depolymerization, N mineralization and nitrification were significantly higher in mineral than in organic horizons, supporting a decrease in microbial N limitation with depth. In contrast, we did not find indications for a decrease in microbial N limitation from arctic to temperate ecosystems along the transect. Our findings thus challenge the perception of ubiquitous N limitation at high latitudes, but suggest a transition from N to C limitation of microorganisms with soil depth, even in high-latitude systems such as tundra and boreal forest. Key Points We compared soil N dynamics of seven ecosystems along a latitudinal transect Shifts in N dynamics suggest a decrease in microbial N limitation with depth We found no decrease in microbial N limitation from arctic to temperate zones

Effects of Soil Organic Matter Properties and Microbial Community Composition on Enzyme Activities in Cryoturbated Arctic Soils

Enzyme-mediated decomposition of soil organic matter (SOM) is controlled, amongst other factors, by organic matter properties and by the microbial decomposer community present. Since microbial community composition and SOM properties are often interrelated and both change with soil depth, the drivers of enzymatic decomposition are hard to dissect. We investigated soils from three regions in the Siberian Arctic, where carbon rich topsoil material has been incorporated into the subsoil (cryoturbation). We took advantage of this subduction to test if SOM properties shape microbial community composition, and to identify controls of both on enzyme activities. We found that microbial community composition (estimated by phospholipid fatty acid analysis), was similar in cryoturbated material and in surrounding subsoil, although carbon and nitrogen contents were similar in cryoturbated material and topsoils. This suggests that the microbial community in cryoturbated material was not well adapted to SOM properties. We also measured three potential enzyme activities (cellobiohydrolase, leucine-amino-peptidase and phenoloxidase) and used structural equation models (SEMs) to identify direct and indirect drivers of the three enzyme activities. The models included microbial community composition, carbon and nitrogen contents, clay content, water content, and pH. Models for regular horizons, excluding cryoturbated material, showed that all enzyme activities were mainly controlled by carbon or nitrogen. Microbial community composition had no effect. In contrast, models for cryoturbated material showed that enzyme activities were also related to microbial community composition. The additional control of microbial community composition could have restrained enzyme activities and furthermore decomposition in general. The functional decoupling of SOM properties and microbial community composition might thus be one of the reasons for low decomposition rates and the persistence of 400 Gt carbon stored in cryoturbated material.

The Phylum Thaumarchaeota

Thaumarchaeota represent a unique phylum within the domain Archaea that embraces ammonia-oxidizing organisms from soil, marine waters, and hot springs (currently two pure cultures and 13 enrichments), as well as many lineages represented only by environmental sequences from virtually every habitat that has been screened. All cultivated Thaumarchaeota perform the first step in nitrification, i.e., they oxidize ammonia to nitrite aerobically. They live under autotrophic conditions and fix CO2, but some are dependent on the presence of other bacteria or small amounts of organic material. Different from bacterial ammonia oxidizers, all cultivated Thaumarchaeota are adapted to comparably low amounts of substrate (ammonia) and inhabit not only moderate but also extreme environments, such as hot springs and acidic soils. All cultivated strains contain tetraether lipids with crenarchaeol, a Thaumarchaeota-specific core lipid. Taxonomy, Historical and Current The history of the phylum Thaumarchaeota, from its discovery until now, is relatively short but nevertheless very eventful. Thaumarchaeota first came into the view of scientists under the name ‘‘Group 1 Crenarchaeota’’ (DeLong 1998), or ‘‘mesophilic Crenarchaeota,’’ as a sister group of the hyperthermophilic Crenarchaeota. In 1992, two independent studies (DeLong 1992; Fuhrman et al. 1992) showed via molecular analyses the occurrence of Archaea in temperate marine environments that were related to hyperthermophilic Crenarchaeota. Since the discovery of the Archaea by Woese et al. in 1978 (Woese et al. 1978), and the definition of the three domains of life in 1990 (Woese et al. 1990), only methanogenic and halophilic Euryarchaeota were known to thrive under moderate temperatures. In the following years, the ‘‘mesophilic Crenarchaeota’’ (now Thaumarchaeota) were detected by molecular surveys in many other environments, including the ocean water column (DeLong et al. 1994; MacGregor et al. 1997; Massana et al. 1997; McInerney et al. 1997; Murray et al. 1998; Karner et al. 2001; Church et al. 2003), soils (Jurgens et al. 1997; Buckley et al. 1998; Ochsenreiter et al. 2003), freshwater sediments (Hershberger et al. 1996; Schleper et al. 1997a), marine holothurians (McInerney et al. 1995), and marine sponges (Preston et al. 1996), showing that they are widespread and very abundant across a great variety of ecosystems. The increasing number of environmental sequences obtained from ‘‘mesophilic Crenarchaeota’’ revealed distinct phylogenetic clusters, commonly referred to on the basis of their predominant environmental distribution. For example, group I.1a encompasses mainly sequences from marine or freshwater habitats and therefore is often referred to as ‘‘Marine Group I’’ (MGI) (DeLong 1992), whereas group I.1b is also known as the ‘‘Soil Group’’ (Schleper et al. 2005). Genome analyses of Ca. Cenarchaeum symbiosum (Schleper et al. 1997b; Hallam et al. 2006), an archaeon affiliated with Marine Group I that lives in a symbiotic association with a cold marine sponge (Preston et al. 1996), and molecular studies of environmental samples (Cubonova et al. 2005) indicated that ‘‘mesophilic Crenarchaeota’’ differed fundamentally from cultivated hyperthermophilic Crenarchaeota. Furthermore, metagenomic studies of soils (Treusch et al. 2005) and ocean waters (Venter et al. 2004) revealed genes that were distantly related to those encoding the bacterial ammonia monooxygenase (AMO), a key enzyme in ammonia oxidation. The direct link to Thaumarchaeota was given through the sequences of a large fosmid that carried both amoAB genes and rRNA genes (Treusch et al. 2005). In addition, increased levels of archaeal amoA transcripts were observed in soil slurries amended with ammonia (Treusch et al. 2005). The cultivation of strain Nitrosopumilus maritimus SCM1 gave the ultimate evidence for active growth through chemolithoautotrophic ammonia oxidation by a member of this phylum (Könneke et al. 2005). Furthermore, the high abundance of archaeal amoA genes (encoding subunit A of AMO) and their transcripts indicated that ammonia-oxidizing Archaea (AOA) outnumber E. Rosenberg et al. (eds.), The Prokaryotes – Other Major Lineages of Bacteria and the Archaea, DOI 10.1007/978-3-642-38954-2_338, # Springer-Verlag Berlin Heidelberg 2014 ammonia-oxidizing bacteria (AOB) in almost all environments (Leininger et al. 2006; Wuchter et al. 2006; He et al. 2007; Zhang et al. 2007; Adair and Schwartz 2008; Shen et al. 2008) and thus potentially play a major role in nitrogen cycling in the aquatic and terrestrial realm. The availability of the annotated genome of Ca. C. symbiosum (Hallam et al. 2006), assembled from a metagenomic library, allowed detailed phylogenetic anaysis (Brochier-Armanet et al. 2008) leading to the suggestion that ‘‘mesophilic Crenarchaeota’’ represent a distinct archaeal phylum, the Thaumarchaeota (Greek: thaumas, wonder). This proposal was based on (i) phylogenetic analyses with 53 concatenated R-proteins from complete archaeal genomes showing that Ca. C. symbiosum branches deeper, i.e., before the speciation of hyperthermophilic Crenarchaeota and Euryarchaeota; (ii) the finding that Ca. C. symbiosum shares more core genes with Euryarchaeota than with Crenarchaeota; and (iii) the fact that AOA represent a widespread and diverse group with a unique energy metabolism (Ochsenreiter et al. 2003; Könneke et al. 2005; Schleper et al. 2005; Leininger et al. 2006; Wuchter et al. 2006). A few years later, phylogenetic and comparative analyses including two additional genomes (from Ca. Nitrososphaera gargensis Ga9.2 and N. maritimus SCM1) supported the proposal of this phylum and revealed a Thaumarchaeota-specific set of information-processing genes (Spang et al. 2010). > Figure 26.1 shows an overview of the distribution of specific cellular features predicted from genome sequences, such as R-proteins, cell-division, and repair components, among the five known archaeal phyla, highlighting the Thaumarchaeota. More recently, another novel archaeal phylum, the ‘‘Aigarchaeota’’, was proposed by Nunoura et al., based on the analyses of the reconstructed genome from an environmental sample (‘‘Ca. Caldiarchaeum subterraneum’’ [Nunoura et al. 2011]). In 16S rRNA gene phylogenies, ‘‘Ca. C. subterraneum’’ indeed appears to represent a separate lineage beside the Thaumarchaeota and Crenarchaeota, whereas comparisons of orthologous genes revealed a closer relationship with the former (Brochier-Armanet et al. 2011, 2012). The resolution of the phylogenetic relationships between the two groups will require further genomic analyses of more representatives related to ‘‘Ca. C. subterraneum’’. The phylogenetic tree of 16S rRNA gene sequences in > Fig. 26.2 illustrates the known diversity of Thaumarchaeota, grouped according to the current nomenclature in the literature (groups I.1a, I.1a-associated (previously South Africa Gold Mine Crenarchaeotic Group, SAGMCG), I.1b, and ThAOA (thermophilic ammonia-oxidizing Archaea; previously Hot Water Crenarchaeotic Group III, HWCG III)), and highlights the strains characterized in culture. In addition to these lineages, several discrete clades (e.g., pSL12, ALOHA, group I.1c, and Marine Benthic Group B), which have solely been detected in environmental samples by 16S rRNA gene surveys, are also often referred to as Thaumarchaeota. This assumption derives not only from their phylogenetic proximity but also from correlation studies indicating that at least some of these organisms harbor amoA genes (Mincer et al. 2007), which is considered a distinctive feature for this phylum. However, the lack of cultured representatives and further genomic information regarding these organisms leaves their taxonomic assignment ambiguous, and thus they are here collectively referred to as ‘‘Thaumarchaeota-associated group’’ (> Fig. 26.2). The cultivation of Thaumarchaeota, and particularly their isolation in pure laboratory cultures, has proven extremely challenging, and only two strains have been characterized as pure isolates to date:Nitrosopumilus maritimus SMC1, from a marine aquarium and affiliated with group I.1a (Könneke et al. 2005), and Nitrososphaera viennensis EN76, isolated from a garden soil and affiliated with group I.1b (Tourna et al. 2011). Additionally, a number of enrichment cultures have been described for representatives of group I.1a (Muller et al. 2010; Park et al. 2010; Jung et al. 2011; Matsutani et al. 2011; Santoro and Casciotti 2011; French et al. 2012; Mosier et al. 2012c) and group I.1b (Hatzenpichler et al. 2008; Kim et al. 2012; Xu et al. 2012), as well as one representative for each of the I.1a-associated (LehtovirtaMorley et al. 2011) and ThAOA groups (de la Torre et al. 2008). Given the recent discovery of Thaumarchaeota and the notorious difficulty in obtaining pure cultures, there is no validly described and deposited type strain yet, but Nitrososphaera viennensis EN76 will become available soon (Stieglmeier et al. manuscript in review). Based on genomic analyses, and/or phenotypic characterization, several Candidatus species have been suggested for strains that have not yet been obtained in pure culture (in order to simplify the reading of this chapter, the term Candidatus will be omitted on the following pages): Cenarchaeum symbiosum (Preston et al. 1996), Nitrosopumilus sp. NM25 (Matsutani et al. 2011), Nitrosopumilus salaria BD31 (Mosier et al. 2012a),Nitrosoarchaeum limnia (strains BG20 and SFB1) (Blainey et al. 2011;Mosier et al. 2012b),Nitrosoarchaeum koreensis MY1 (Jung et al. 2011), Nitrosotalea devanaterra Nd1 (Lehtovirta-Morley et al. 2011), Nitrososphaera sp. JG1 (Kim et al. 2012),Nitrososphaera gargensis Ga9.2 (Hatzenpichler et al. 2008), Nitrososphaera viennensis (strain EN123) (Tourna et al. 2011), and Nitrosocaldus yellowstonii HL72 (de la Torre et al. 2008). Other enrichment cultures have been described although not suggested as Candida

Unifying the global phylogeny and environmental distribution of ammonia-oxidising archaea based on amoA genes

Ammonia-oxidising archaea (AOA) are ubiquitous and abundant in nature and play a major role in nitrogen cycling. AOA have been studied intensively based on the amoA gene (encoding ammonia monooxygenase subunit A), making it the most sequenced functional marker gene. Here, based on extensive phylogenetic and meta-data analyses of 33,378 curated archaeal amoA sequences, we define a highly resolved taxonomy and uncover global environmental patterns that challenge many earlier generalisations. Particularly, we show: (i) the global frequency of AOA is extremely uneven, with few clades dominating AOA diversity in most ecosystems; (ii) characterised AOA do not represent most predominant clades in nature, including soils and oceans; (iii) the functional role of the most prevalent environmental AOA clade remains unclear; and (iv) AOA harbour molecular signatures that possibly reflect phenotypic traits. Our work synthesises information from a decade of research and provides the first integrative framework to study AOA in a global context.Ammonia-oxidising archaea (AOA) were only discovered a little over a decade ago and remain poorly characterized despite their ubiquity and importance for nitrogen cycling. Here, the authors define a taxonomy of AOA based on a resolved amoA phylogeny and describe emergent global patterns in AOA diversity.

A plant–microbe interaction framework explaining nutrient effects on primary production

In most terrestrial ecosystems, plant growth is limited by nitrogen and phosphorus. Adding either nutrient to soil usually affects primary production, but their effects can be positive or negative. Here we provide a general stoichiometric framework for interpreting these contrasting effects. First, we identify nitrogen and phosphorus limitations on plants and soil microorganisms using their respective nitrogen to phosphorus critical ratios. Second, we use these ratios to show how soil microorganisms mediate the response of primary production to limiting and non-limiting nutrient addition along a wide gradient of soil nutrient availability. Using a meta-analysis of 51 factorial nitrogen–phosphorus fertilization experiments conducted across multiple ecosystems, we demonstrate that the response of primary production to nitrogen and phosphorus additions is accurately predicted by our stoichiometric framework. The only pattern that could not be predicted by our original framework suggests that nitrogen has not only a structural function in growing organisms, but also a key role in promoting plant and microbial nutrient acquisition. We conclude that this stoichiometric framework offers the most parsimonious way to interpret contrasting and, until now, unresolved responses of primary production to nutrient addition in terrestrial ecosystems.A stoichiometric framework predicts the contrasting results of nutrient effects on primary production, with predicted responses supported by a meta-analysis of N–P fertilization experiments.