Evidence that Ammonia-Oxidizing Archaea are More Abundant than Ammonia-Oxidizing Bacteria in Semiarid Soils of Northern Arizona,USA

Autotrophic ammonia-oxidizing communities, which are responsible for the rate-limiting step of nitrification in most soils, have not been studied extensively in semiarid ecosystems. Abundances of soil archaeal and bacterial amoA were measured with real-time polymerase chain reaction along an elevation gradient in northern Arizona. Archaeal amoA was the predominant form of amoA at all sites; however, ratios of archaeal to bacterial amoA ranged from 17 to more than 1,600. Although size of ammonia-oxidizing bacteria populations was correlated with precipitation, temperature, percent sand, and soil C/N, there were no significant relationships between ammonia-oxidizing archaea populations and any of the environmental parameters evaluated in this study. Our results suggest that in these soils, archaea may be the primary ammonia oxidizers, and that ammonia-oxidizing archaea and ammonia-oxidizing bacteria occupy different niches.     

Diversity and Abundance of Ammonia-Oxidizing Archaea and Bacteria in Diverse Chinese Paddy Soils

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) in three types of paddy soils of China before and after rice plantation were investigated by using an integrated approach including geochemistry, 454 pyrosequencing, and quantitative polymerase chain reaction (PCR). The abundances of AOA amoA gene were 1～2 orders of magnitude higher than AOB amoA gene. The types of paddy soils had important impacts on the diversities of both AOA and AOB via clay mineralogy (smectite or illite-rich) and bioavailability of ammonium. The Nitrososphaera subcluster 5 and Nitrosopumilis cluster of AOA, and Nitrosomonas subcluster 5 and Nitrosospira subcluster 3 of AOB were well adapted to soils with high ammonium concentrations. AOA and AOB community structures were different before and after rice plantation, likely due to changes of pH and ammonium fertilization. The Nitrosospira subclusters 2 and 9 were well adapted to acidic paddy soils. However, the sensitivity of AOA and AOB community structures to these factors may be complicated by other geochemical conditions. The results of this study collectively demonstrated that multiple environmental factors, such as clay mineralogy, ammonium content and total organic carbon as well as soil pH, shaped AOA and AOB community structure and abundance.     

Latitudinal Distribution of Ammonia-Oxidizing Bacteria and Archaea in the Agricultural Soils of Eastern China

The response of soil ammonia-oxidizing bacterial (AOB) and archaeal (AOA) communities to individual environmental variables (e.g., pH, temperature, and carbon- and nitrogen-related soil nutrients) has been extensively studied, but how these environmental conditions collectively shape AOB and AOA distributions in unmanaged agricultural soils across a large latitudinal gradient remains poorly known. In this study, the AOB and AOA community structure and diversity in 26 agricultural soils collected from eastern China were investigated by using quantitative PCR and bar-coded 454 pyrosequencing of the amoA gene that encodes the alpha subunit of ammonia monooxygenase. The sampling locations span over a 17° latitude gradient and cover a range of climatic conditions. The Nitrosospira and Nitrososphaera were the dominant clusters of AOB and AOA, respectively; but the subcluster-level composition of Nitrosospira-related AOB and Nitrososphaera-related AOA varied across the latitudinal gradient. Variance partitioning analysis showed that geography and climatic conditions (e.g., mean annual temperature and precipitation), as well as carbon-/nitrogen-related soil nutrients, contributed more to the AOB and AOA community variations (∼50% in total) than soil pH (∼10% in total). These results are important in furthering our understanding of environmental conditions influencing AOB and AOA community structure across a range of environmental gradients.     

Occurrence of Ammonia-Oxidizing Archaea in Wastewater Treatment Plant Bioreactors

We report molecular evidence that ammonia-oxidizing archaea (AOA) occur in activated sludge bioreactors used to remove ammonia from wastewater. Using PCR primers targeting archaeal ammonia monooxygenase subunit A (amoA) genes, we retrieved and compared 75 sequences from five wastewater treatment plants operating with low dissolved oxygen levels and long retention times. All of these sequences showed similarity to sequences previously found in soil and sediments, and they were distributed primarily in four major phylogenetic clusters. One of these clusters contained virtually identical amoA sequences obtained from all five activated sludge samples (from Oregon, Wisconsin, Pennsylvania, and New Jersey) and accounted for 67% of all the sequences, suggesting that this AOA phylotype may be widespread in nitrifying bioreactors.     

Vertical Distribution of Ammonia-Oxidizing Archaea and Bacteria in Sediments of a Eutrophic Lake

In order to characterize the vertical variation of abundance and community composition of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in sediments of a eutrophic lake, Lake Taihu, molecular techniques including real-time PCR, clone library, and sequencing were carried out in this study. Abundances of archaeal amoA gene (ranged from 2.34 × 10⁶ to 4.43 × 10⁷ copies [g dry sediment]^?1) were higher than those of bacterial amoA gene (ranged from 5.02 × 10⁴ to 6.91 × 10⁶ copies [g dry sediment]^?1) for all samples and both of them exhibited negative correlations with the increased depths. Diversities of archaeal and bacterial amoA gene increased with the elevated depths. There were no significant variations of AOB community structures derived from different sediment depths, whereas obvious differences were observed for the AOA community compositions. The information acquired in this study would be useful to elucidate the roles of AOA and AOB in the nitrogen cycling of freshwater ecosystems.     

The Molecular Composition of Dissolved Organic Matter in Forest Soils as a Function of pH and Temperature

We examined the molecular composition of forest soil water during three different seasons at three different sites, using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS). We examined oxic soils and tested the hypothesis that pH and season correlate with the molecular composition of dissolved organic matter (DOM). We used molecular formulae and their relative intensity from ESI-FT-ICR-MS for statistical analysis. Applying unconstrained and constrained ordination methods, we observed that pH, dissolved organic carbon (DOC) concentration and season were the main factors correlating with DOM molecular composition. This result is consistent with a previous study where pH was a main driver of the molecular differences between DOM from oxic rivers and anoxic bog systems in the Yenisei River catchment. At a higher pH, the molecular formulae had a lower degree of unsaturation and oxygenation, lower molecular size and a higher abundance of nitrogen-containing compounds. These characteristics suggest a higher abundance of tannin connected to lower pH that possibly inhibited biological decomposition. Higher biological activity at a higher pH might also be related to the higher abundance of nitrogen-containing compounds. Comparing the seasons, we observed a decrease in unsaturation, molecular diversity and the number of nitrogen-containing compounds in the course of the year from March to November. Temperature possibly inhibited biological degradation during winter, which could cause the accumulation of a more diverse compound spectrum until the temperature increased again. Our findings suggest that the molecular composition of DOM in soil pore waters is dynamic and a function of ecosystem activity, pH and temperature.     

Detection of Ammonia-Oxidizing Archaea in Fish Processing Effluent Treatment Plants

Ammonia oxidation is the rate limiting step in nitrification and thus have an important role in removal of ammonia in natural and engineered systems with participation of both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). However, their relative distribution and activity in fish processing effluent treatment plants (FPETPs) though significant, is hitherto unreported. Presence of AOA in sludge samples obtained from FPETPs was studied by amplification and sequencing of thaumarchaeal ammonia monooxygenase subunit A (AOA-amoA) gene. Different primer sets targeting 16S rRNA and AOA-amoA gene were used for the detection of AOA in FPETPs. Phylogenetic analysis of the gene revealed that the AOA was affiliated with thaumarchaeal group 1.1a lineage (marine cluster). Quantitative real time PCR of amoA gene was used to study the copy number of AOA and AOB in FPETPs. The AOA-amoA and AOB-amoA gene copy numbers of sludge samples ranged from 2.2 × 10⁶ to 4.2 × 10⁸ and 1.1 × 10⁷ to 8.5 × 10⁸ mg⁻¹ sludge respectively. Primer sets Arch-amoAF/Arch-amoAR and 340F/1000R were found to be useful for the sensitive detection of AOA-amoA and Archaeal 16S rRNA genes respectively in FPETPs. Their presence suggests the widespread occurrence and possible usefulness in removing ammonia from FPETPs which is in line with reports from other waste water treatment plants.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0484-6) contains supplementary material, which is available to authorized users.     

A Method for Cell Culture and Maintenance of Ammonia-Oxidizing Archaea in Agar Stab

Ammonia oxidizing archaea (AOA) are predominantly found and closely linked with geochemical cycling of nitrogen in non-extreme habitats. However, these strains have mainly been investigated using liquid cultures of enriched cells. Here, we provide an agar stab as a simple and reliable means of cultivating and maintaining AOA.     

Temporal and Spatial Stability of Ammonia-Oxidizing Archaea and Bacteria in Aquarium Biofilters

Nitrifying biofilters are used in aquaria and aquaculture systems to prevent accumulation of ammonia by promoting rapid conversion to nitrate via nitrite. Ammonia-oxidizing archaea (AOA), as opposed to ammonia-oxidizing bacteria (AOB), were recently identified as the dominant ammonia oxidizers in most freshwater aquaria. This study investigated biofilms from fixed-bed aquarium biofilters to assess the temporal and spatial dynamics of AOA and AOB abundance and diversity. Over a period of four months, ammonia-oxidizing microorganisms from six freshwater and one marine aquarium were investigated at 4–5 time points. Nitrogen balances for three freshwater aquaria showed that active nitrification by aquarium biofilters accounted for ≥81–86% of total nitrogen conversion in the aquaria. Quantitative PCR (qPCR) for bacterial and thaumarchaeal ammonia monooxygenase (amoA) genes demonstrated that AOA were numerically dominant over AOB in all six freshwater aquaria tested, and contributed all detectable amoA genes in three aquarium biofilters. In the marine aquarium, however, AOB outnumbered AOA by three to five orders of magnitude based on amoA gene abundances. A comparison of AOA abundance in three carrier materials (fine sponge, rough sponge and sintered glass or ceramic rings) of two three-media freshwater biofilters revealed preferential growth of AOA on fine sponge. Denaturing gel gradient electrophoresis (DGGE) of thaumarchaeal 16S rRNA genes indicated that community composition within a given biofilter was stable across media types. In addition, DGGE of all aquarium biofilters revealed low AOA diversity, with few bands, which were stable over time. Nonmetric multidimensional scaling (NMDS) based on denaturing gradient gel electrophoresis (DGGE) fingerprints of thaumarchaeal 16S rRNA genes placed freshwater and marine aquaria communities in separate clusters. These results indicate that AOA are the dominant ammonia-oxidizing microorganisms in freshwater aquarium biofilters, and that AOA community composition within a given aquarium is stable over time and across biofilter support material types.     

Ammonia-Oxidizing Bacteria and Archaea in Groundwater Treatment and Drinking Water Distribution Systems

The ammonia-oxidizing prokaryote (AOP) community in three groundwater treatment plants and connected distribution systems was analyzed by quantitative real-time PCR and sequence analysis targeting the amoA gene of ammonia-oxidizing bacteria (AOB) and archaea (AOA). Results demonstrated that AOB and AOA numbers increased during biological filtration of ammonia-rich anoxic groundwater, and AOP were responsible for ammonium removal during treatment. In one of the treatment trains at plant C, ammonia removal correlated significantly with AOA numbers but not with AOB numbers. Thus, AOA were responsible for ammonia removal in water treatment at one of the studied plants. Furthermore, an observed negative correlation between the dissolved organic carbon (DOC) concentration in the water and AOA numbers suggests that high DOC levels might reduce growth of AOA. AOP entered the distribution system in numbers ranging from 1.5 × 10³ to 6.5 × 10⁴ AOPs ml⁻¹. These numbers did not change during transport in the distribution system despite the absence of a disinfectant residual. Thus, inactive AOP biomass does not seem to be degraded by heterotrophic microorganisms in the distribution system. We conclude from our results that AOA can be commonly present in distribution systems and groundwater treatment, where they can be responsible for the removal of ammonia.Ammonia can be present in source water used for drinking water production or added to treated water with chlorine to form chloramines as a disinfectant. However, the presence of ammonia in drinking water is undesirable because nitrification might lead to toxic levels of nitrite (29) or adverse effects on water taste and odor (4) and might increase heterotrophic bacteria, including opportunistic pathogens (29). Two-thirds of the drinking water in The Netherlands is produced from groundwater. Most of the groundwater used for drinking water production is anoxic with relatively high concentrations of methane, iron, manganese, dissolved organic carbon (DOC), and ammonia. Treatment of anoxic groundwater aims at achieving biologically stable water, because drinking water in The Netherlands is distributed without a disinfectant residual. As a result, a highly efficient nitrification process during rapid medium filtration is required.Nitrification is the microbial oxidation of ammonia to nitrate and consists of two processes: the oxidation of ammonia to nitrite by ammonia-oxidizing prokaryotes (AOP) and the oxidation of nitrite to nitrate by nitrite-oxidizing bacteria (NOB). Recently it was shown that in addition to bacteria, archaea also are capable of ammonia oxidation (13). Since then, ammonia-oxidizing archaea (AOA) have been found in many different ecosystems, including wastewater treatment systems (10, 20, 24). However, it is currently unknown if AOA are present in drinking water treatment processes and distribution systems. Recent studies have focused on nitrification in drinking water treatment (16, 28). In those studies, AOB and NOB were enumerated by traditional most-probable-number (MPN) methods using selective liquid media. However, MPN methods are time-consuming and underestimate the numbers of AOP and NOB (3). Quantitative real-time PCR has been used to quantify AOB in drinking water (12) and might be a useful tool for quantifying AOB and AOA in drinking water.In our study, a real-time PCR method targeting the amoA gene of AOB or AOA was developed to quantify numbers of AOP in drinking water. This real-time PCR method was used together with a phylogenetic analysis of the amoA gene of AOB and AOA to do the following: (i) determine the treatment steps where AOP dominates in the groundwater treatment train of three drinking water production plants in The Netherlands, (ii) quantify the AOP entering the distribution system and determine the fate of AOP during transport in the distribution system, and (iii) elucidate the role of AOA in nitrification during drinking water treatment and in distribution systems.     

Altitudinal Distribution of Ammonia-Oxidizing Archaea and Bacteria in Alpine Grassland Soils Along the South-Facing Slope of Nyqentangula Mountains,Central Tibetan Plateau

Nitrogen is a major limiting nutrient for the net primary production of terrestrial ecosystems, especially on sentinel alpine ecosystem. Ammonia oxidation is the first and rate-limiting step on nitrification process and is thus crucial to nitrogen cycle. To decipher climatic influence on ammonia oxidizers, their communities were characterized by qPCR and clone sequencing by targeting amoA genes (encoding the alpha subunit of ammonia mono-oxygenase) in soils from 7 sites over an 800 m elevation transect (4400–5200 m a.s.l.), based on “space-to-time substitution” strategy, on a steppe-meadow ecosystem located on the central Tibetan Plateau (TP). Archaeal amoA abundance outnumbered bacterial amoA abundance at lower altitude (<4800 m a.s.l.), but bacterial amoA abundance was greater in surface soils at higher altitude (≥4800 m a.s.l.). Archaeal amoA abundance decreased with altitude in surface soil, while its abundance stayed relatively stable and was mostly greater than bacterial amoA abundance in subsurface soils. Conversely, bacterial amoA abundance gradually increased with altitude at all three soil depths. Statistical analysis indicated that altitude-dependent factors, in particular pH and precipitation, had a profound effect on the abundance and community of ammonia-oxidizing bacteria, but only on the community composition of ammonia-oxidizing archaea along the altitudinal gradient. These findings imply that the shifts in the relative abundance and/or community structure of ammonia-oxidizing bacteria and archaea may result from the precipitation variation along the altitudinal gradient. Thus, we speculate that altitude-related factors (mainly precipitation variation combing changed pH), would play a vital role in affecting nitrification process on this alpine grassland ecosystem located at semi-arid area on TP.     

High Abundance of Ammonia-Oxidizing Archaea in Coastal Waters,Determined Using a Modified DNA Extraction Method

Molecular characterizations of environmental microbial populations based on recovery and analysis of DNA generally assume efficient or unbiased extraction of DNA from different sample matrices and microbial groups. Appropriate controls to verify this basic assumption are rarely included. Here three different DNA extractions, performed with two commercial kits (FastDNA and UltraClean) and a standard phenol-chloroform method, and two alternative filtration methods (Sterivex and 25-mm-diameter polycarbonate filters) were evaluated, using the addition of Nitrosopumilus maritimus cells to track the recovery of DNA from marine Archaea. After the comparison, a simplified phenol-chloroform extraction method was developed and shown to be significantly superior, in terms of both the recovery and the purity of DNA, to other protocols now generally applied to environmental studies. The simplified and optimized method was used to quantify ammonia-oxidizing Archaea at different depth intervals in a fjord (Hood Canal) by quantitative PCR. The numbers of Archaea increased with depth, often constituting as much as 20% of the total bacterial community.Efficient DNA extraction from environmental samples is fundamental to many culture-independent characterizations (10). Thus, there was an early and concerted effort to establish appropriate methods of DNA extraction from different types of environmental samples (14, 19, 25, 30, 34, 43, 47). DNA extraction efficiency is particularly important for quantitative PCR (qPCR), because poor DNA extraction efficiency results in the underestimation of gene copy numbers in the samples examined (6, 42).Most methodological developments addressed DNA extraction from soil and sediment samples, with fewer comparative studies of the efficiency of collection and extraction from water samples (4, 13, 40). In part, a methodological focus on soils reflected the simplicity of filtration to collect aquatic populations and the generally good recovery of DNA from the Gram-negative bacteria making up a significant fraction of aquatic communities. However, small Archaea are now known to constitute a substantial fraction of the prokaryotic populations in marine and terrestrial systems (2, 7, 9, 20, 26, 31, 33, 45). Since the archaeal cell wall and membrane structures are distinct from those of bacteria, there is no assurance that commonly used extraction methods are adequate. With increasing reliance on commercially available bead-beating-type DNA extraction kits, these methods are now often used for different water samples (1, 5-7, 14, 19, 36). Although most protocols incorporate mechanical disruption to ensure more-uniform extraction than is possible by using methods that rely entirely on enzymatic digestion and/or chemical disruption (4, 13, 40), the suitability of these protocols for the concerted analysis of archaeal and bacterial populations has not been fully evaluated.In the studies reported here, the recently isolated marine archaeon Nitrosopumilus maritimus strain SCM1 (22) was therefore used as a reference standard for evaluation of the commonly employed DNA extraction methods by using qPCR. This archaeon was then used as a reference for the development of a simple, rapid, and efficient method of extracting DNA from both archaeal and bacterial cells. The modified protocol was subsequently employed to characterize the vertical distribution of ammonia-oxidizing Archaea in a fjord (Hood Canal) in Puget Sound (Washington State), revealing a high fractional representation of Archaea relative to Bacteria not observed previously in coastal waters.     

Lower Abundance of Ammonia-Oxidizing Archaea Than Ammonia-Oxidizing Bacteria Detected in the Subsurface Sediments of the Northern South China Sea

Diversity and abundance of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in samples of the northern South China Sea subsurface sediment were assessed by analyzing the amoA gene sequences retrieved from the samples. The microbial diversity was assessed using rarefaction and phylogenetic analyses. The deep-sea subsurface sediments harbored diverse and distinct AOA and AOB communities, but the abundance of AOA was lower than that of AOB, consistent with many other studies about bacteria and archaea in subsurface sediments. Diversity of AOA shown in the OTUs and Shannon index was correlated with the concentration of nitrite in the Pearson analysis, but no obvious relationships between the diversity or abundance of AOB and the physicochemical parameters could be identified in the present study, indicating the concentration of ammonium may not be an important factor to determine the diversity and abundance of ammonia-oxidizing prokaryotes in the subsurface sediments. Additionally, Nitrosomonas-like AOB was found to be dominant in subsurface sediments of the northern South China Sea showing a different adaption strategy comparing with some Nitrosospira-like AOB lineages. Concentration of nitrite was correlated with diversity of AOA, but no correlations between diversity and abundance of AOB and the physicochemical parameters were established in the study. Supplementary materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental files.     

Spatiotemporal Stability of an Ammonia-Oxidizing Community in a Nitrogen-Saturated Forest Soil

Elevated levels of nitrogen input into various terrestrial environments in recent decades have led to increases in soil nitrate production and leaching. However, nitrifying potential and nitrifying activity tend to be highly variable over space and time, making broad-scale estimates of nitrate production difficult. This study investigates whether the high spatiotemporal variation in nitrate production might be explained by differences in the structure of ammonia-oxidizing bacterial communities in nitrogen-saturated coniferous forest soils. The diversity of ammonia-oxidizing bacteria of the β-subgroup Proteobacteria was therefore investigated using two different PCR-based approaches. The first targeted the 16S rRNA gene and involved temporal temperature gradient electrophoresis (TTGE) of specifically amplified PCR products, with subsequent band excision and nucleotide sequence determination. The second approach involved the cloning and sequencing of PCR-amplified amoA gene fragments. All recovered 16S rDNA sequences were closely related to the culture strain Nitrosospira sp. AHB1, which was isolated from an acid soil and is affiliated with Nitrosospira cluster 2, a sequence group previously shown to be associated with acid environments. All amoA-like sequences also showed a close affinity with this acid-tolerant Nitrosospira strain, although greater sequence variation could be detected in the amoA analysis. The ammonia-oxidizing bacterial community in the nitrogen-saturated coniferous forest soil was determined to be very stable, showing little variation between different organic layers and throughout the year, despite large differences in the total Bacterial community structure as determined by 16S rDNA DGGE community fingerprinting. These results suggest that environmental heterogeneity affecting ammonia oxidizer numbers and activity, and not ammonia oxidizer community structure, is chiefly responsible for spatial and temporal variation in nitrate production in these acid forest soils.     

Cultivation of Autotrophic Ammonia-Oxidizing Archaea from Marine Sediments in Coculture with Sulfur-Oxidizing Bacteria

The role of ammonia-oxidizing archaea (AOA) in nitrogen cycling in marine sediments remains poorly characterized. In this study, we enriched and characterized AOA from marine sediments. Group I.1a crenarchaea closely related to those identified in marine sediments and “Candidatus Nitrosopumilus maritimus” (99.1 and 94.9% 16S rRNA and amoA gene sequence identities to the latter, respectively) were substantially enriched by coculture with sulfur-oxidizing bacteria (SOB). The selective enrichment of AOA over ammonia-oxidizing bacteria (AOB) is likely due to the reduced oxygen levels caused by the rapid initial growth of SOB. After biweekly transfers for ca. 20 months, archaeal cells became the dominant prokaryotes (>80%), based on quantitative PCR and fluorescence in situ hybridization analysis. The increase of archaeal 16S rRNA gene copy numbers was coincident with the amount of ammonia oxidized, and expression of the archaeal amoA gene was observed during ammonia oxidation. Bacterial amoA genes were not detected in the enrichment culture. The affinities of these AOA to oxygen and ammonia were substantially higher than those of AOB. [¹³C]bicarbonate incorporation and the presence and activation of genes of the 3-hydroxypropionate/4-hydroxybutyrate cycle indicated autotrophy during ammonia oxidation. In the enrichment culture, ammonium was oxidized to nitrite by the AOA and subsequently to nitrate by Nitrospina-like bacteria. Our experiments suggest that AOA may be important nitrifiers in low-oxygen environments, such as oxygen-minimum zones and marine sediments.Archaea have long been known as extremophiles, since most cultivated archaeal strains were cultivated from extreme environments, such as acidic, hot, and high-salt environments. The view of archaea as extremophiles (i.e., acidophiles, thermophiles, and halophiles) has radically changed by the application of molecular technologies, including PCR in environmental microbiology. Using Archaea-specific PCR primers, novel archaeal 16S rRNA gene sequences were discovered in seawater (23, 27). Following these discoveries, an ever-increasing and unexpectedly high variety of archaeal 16S rRNA gene sequences has been reported from diverse “nonextreme” environments (67). This indicates that archaea are, like bacteria, ubiquitous in the biosphere rather than exclusively inhabiting specific extreme niches. Archaea are abundant in water columns of some oceanic provinces (33, 36) and deep-subsea floor sediments (11, 12, 48). Despite the increasing number of reports of the diversity and abundance of these nonextreme archaea by molecular ecological studies, their physiology and ecological roles have remained enigmatic.Oxidation of ammonia, a trait long thought to be exclusive to the domain Bacteria (13), was recently suggested to be a trait of archaea of the crenarchaeal groups I.1a and I.1b, based on a metagenome analysis (79) and supported by the discovery of archaeal amoA-like genes in environmental shotgun sequencing studies of Sargasso Sea water (80) and genomic analysis of “Candidatus Cenarchaeum symbiosum,” a symbiont of a marine sponge (30). Molecular ecological studies indicated that these ammonia-oxidizing archaea (AOA) are often predominant over ammonia-oxidizing bacteria (AOB) in ocean waters (9, 53, 87), soils (17, 47), and marine sediments (61). Critical evidence for autotrophic archaeal ammonia oxidation was obtained by the characterization of the first cultivated mesophilic crenarchaeon (group I.1a), “Candidatus Nitrosopumilus maritimus SCM1,” from an aquarium (38), and a related archaeon from North Sea water (87) and subsequently by enrichment of thermophilic AOA (22, 31). Whole-genome-based phylogenetic studies recently indicated that the nonthermophilic crenarchaea, including the AOA, likely form a phylum separate from the Crenarchaeota and Euryarchaeota phyla (15, 16, 72). This proposed new phylum was called Thaumarchaeota (15).Microorganisms in marine sediments contribute significantly to global biogeochemical cycles because of their abundance (85). Nitrification is essential to the nitrogen cycle in marine sediments and may be metabolically coupled with denitrification and anaerobic ammonium oxidation, resulting in the removal of nitrogen as molecular nitrogen and the generation of greenhouse gases, such as nitrous oxide (19, 75). Compared with studies on archaeal nitrification in the marine water column, only limited information on archaeal nitrification in marine sediments is available so far. Archaeal amoA genes have been retrieved from marine and coastal sediments (8, 26, 61), and the potentially important role of AOA in nitrification has been suggested based on the abundance of archaeal amoA genes relative to that of bacterial amoA genes in surface marine sediments from Donghae (South Korea) (61). Cultivation of AOA, although difficult (38), remains essential to estimating the metabolic potential of archaea in environments such as soils (47) and marine sediments (61). Here, we report the successful enrichment of AOA of crenarchaeal group I.1a from marine sediments by employing a coculture with sulfur-oxidizing bacteria (SOB) which was maintained for ca. 20 months with biweekly transfers. In this way, we were able to characterize AOA from marine sediments, providing a clue for the role of AOA in the nitrogen cycle of marine sediments.     

Abundance and Diversity of Archaea in Heavy-Metal-Contaminated Soils

The impact of heavy-metal contamination on archaean communities was studied in soils amended with sewage sludge contaminated with heavy metals to varying extents. Fluorescent in situ hybridization showed a decrease in the percentage of Archaea from 1.3% ± 0.3% of 4′,6-diamidino-2-phenylindole-stained cells in untreated soil to below the detection limit in soils amended with heavy metals. A comparison of the archaean communities of the different plots by denaturing gradient gel electrophoresis revealed differences in the structure of the archaean communities in soils with increasing heavy-metal contamination. Analysis of cloned 16S ribosomal DNA showed close similarities to a unique and globally distributed lineage of the kingdom Crenarchaeota that is phylogenetically distinct from currently characterized crenarchaeotal species.     

Variation in pH Optima of Hydrolytic Enzyme Activities in Tropical Rain Forest Soils

Extracellular enzymes synthesized by soil microbes play a central role in the biogeochemical cycling of nutrients in the environment. The pH optima of eight hydrolytic enzymes involved in the cycles of carbon, nitrogen, phosphorus, and sulfur, were assessed in a series of tropical forest soils of contrasting pH values from the Republic of Panama. Assays were conducted using 4-methylumbelliferone-linked fluorogenic substrates in modified universal buffer. Optimum pH values differed markedly among enzymes and soils. Enzymes were grouped into three classes based on their pH optima: (i) enzymes with acidic pH optima that were consistent among soils (cellobiohydrolase, β-xylanase, and arylsulfatase), (ii) enzymes with acidic pH optima that varied systematically with soil pH, with the most acidic pH optima in the most acidic soils (α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminidase), and (iii) enzymes with an optimum pH in either the acid range or the alkaline range depending on soil pH (phosphomonoesterase and phosphodiesterase). The optimum pH values of phosphomonoesterase were consistent among soils, being 4 to 5 for acid phosphomonoesterase and 10 to 11 for alkaline phosphomonoesterase. In contrast, the optimum pH for phosphodiesterase activity varied systematically with soil pH, with the most acidic pH optima (3.0) in the most acidic soils and the most alkaline pH optima (pH 10) in near-neutral soils. Arylsulfatase activity had a very acidic optimum pH in all soils (pH ≤3.0) irrespective of soil pH. The differences in pH optima may be linked to the origins of the enzymes and/or the degree of stabilization on solid surfaces. The results have important implications for the interpretation of hydrolytic enzyme assays using fluorogenic substrates.Measurements of the activities of extracellular enzymes involved in the turnover of nutrients from organic compounds provide important information on biogeochemical cycles in tropical soils (13, 59, 63). In particular, they are the primary mechanism by which microbes decompose organic matter and can provide key information on the nutrient status of the ecosystem (45, 60). For example, changes in the activities of phosphatase and N-acetyl-β-glucosaminidase in soil chronosequences reflect long-term changes in nitrogen and phosphorus availability during pedogenesis in both tropical and temperate rain forests (3, 34).The pH of the soil solution exerts a strong control on enzyme activity, because it influences the conformation of the enzyme, its adsorption on solid surfaces, and the ionization and solubility of substrates and cofactors (38, 51). Although some studies have determined enzyme activity at the soil pH (e.g., references 19 and 63), assays are usually conducted at the optimum pH for the enzyme, which yields a measure of its maximum potential activity at a given temperature (7, 27). For example, the assay of acid phosphomonoesterase activity using the chromogenic substrate para-nitrophenyl phosphate is typically performed at pH 6.5 (51), based on the determination of the pH optima of this enzyme in a series of temperate agricultural soils (15, 21, 50).Once the optimum pH for a given enzyme has been determined, it is usually assumed that this will apply broadly to other soils, allowing the recommendation of a single buffer pH in standardized procedures (44, 51). However, the pH optima of some enzymes can vary markedly among soils. For example, Niemi and Vepsäläinen (33) reported soil-specific pH optima for three hydrolytic enzymes (acid phosphomonoesterase, phosphodiesterase, and N-acetyl-β-glucosaminidase) in six soils under contrasting land uses in Finland. Such soil-specific pH optima led Malcolm (27) to recommend, in a critique of soil enzyme assays, that the pH optimum of the enzyme under study should be determined for each soil.Differences among soils in relation to the pH optima of an individual enzyme might be due to a variety of factors, including the composition of the soil microbial community (i.e., if isoenzymes originating from different organisms have different pH optima) and the location of the enzyme in the soil matrix (e.g., intracellular, free in solution, or adsorbed on solid surfaces, etc.) (4). For example, the sorption of an enzyme on a clay surface can increase its optimum pH by one or two pH units relative to that of the same enzyme in solution (31, 40). This is due to “unfolding” of enzymes on solid surfaces, which is most likely to occur at soil pH values below the isoelectric point of the enzyme (26, 38).Information on the optimum pH of enzyme activity is of particular importance for studies that use fluorogenic substrates in multiwell plates to assay several enzymes simultaneously. Such studies are usually simplified by assaying all enzymes in a single buffer, such as acetate at pH 5.5 (57) or 2-(N-morpholino)ethanesulfonic acid (MES) at pH 6.1 (30). However, this may not coincide with the pH optima of all the enzymes involved, especially if the optimum pH for a given enzyme varies among soils. Further, most studies of the pH optima of hydrolytic enzymes have been conducted using chromogenic substrates linked to para-nitrophenol, and it is not clear whether the values correspond to the pH optima for fluorogenic substrates linked to 4-methylumbelliferone.Here I report the pH optima for eight hydrolytic enzymes involved in the cycles of carbon, nitrogen, phosphorus, and sulfur in a series of soils under a lowland tropical rain forest in the Republic of Panama. The aim was to determine the extent to which the pH optima of activity varied among enzymes and soils, in order to develop a method suitable for the measurement of enzyme activities in a broad range of tropical rain forest soils.     

Low Temperature Decreases the Phylogenetic Diversity of Ammonia-Oxidizing Archaea and Bacteria in Aquarium Biofiltration Systems

The phylogenetic diversity and species richness of ammonia-oxidizing archaea (AOA) and bacteria (AOB) were examined with aquarium biofiltration systems. Species richness, deduced from rarefaction analysis, and diversity indices indicated that the phylogenetic diversity and species richness of AOA are greater than those of AOB; the diversity of AOA and of AOB is minimized in cold-water aquaria. This finding implies that temperature is a key factor influencing the population structure and diversity of AOA and AOB in aquarium biofiltration systems.     

Characterisation of Archaea in Soils by Polar Lipid Analysis

While phospholipid fatty acid (PLFA) profiling is a well‐established method used for the determination of bacterial and eukaryotic organisms in soil ecology, phospholipid etherlipid (PLEL) analyses for the characterisation of Archaea is a rather new approach. Analyses of PLEL derived isoprenoid side chains by GC/MS provided a broad picture of the archaeal community in a mixed soil extract, as lipids previously identified in isolates belonging to the kingdoms Eury‐ and Crenarchaeota were covered. Furthermore, ether‐linked isoprenoid hydrocarbons, which have not been detected in archaeal isolates and monomethyl‐branched alkanes which have only been found in hyperthermophilic bacteria, were detected in these soil extracts. Monomethyl‐branched alkanes were the most dominant ones and accounted for 43.4% of the total identified ether‐linked hydrocarbons, followed by straight chain (unbranched) and isoprenoid hydrocarbons, which accounted for 34.6 and 15.5%, respectively.