amoA Copy Numbers Research Articles

Diversity and abundance of archaeal amoA genes in the permanent and temporary oxygen minimum zones of Indian Ocean

Oxygen minimum zones are results of oxygen consumption exceeding the oxygen availability in stratified water columns of the marine environment. We compared the ammonia monooxygenase subunit A (amoA) gene abundance and the diversity of ammonia-oxidising archaea (AOA) in the Arabian Sea (AS) with those of the Bay of Bengal (BoB). Three primer pairs targeting amoA genes of water column A (WCA), water column B (WCB) and total AOA (amoAt) captured different densities of gene copy numbers in both marginal seas. Water column A (WCA) ecotypes were more abundant in the AS than in the BoB. Core-OMZ depths of the BoB contained 10 times lower amoA copy numbers than those of the AS. Along with sampling depth, concentration of ammonia shapes the WCA/WCB ecotypes in AS/BoB. Among the total AOA populations, WCB ecotypes were more abundant. The amoA gene sequences were either of Nitrosopumilales or Ca. Nitrosotaleales members and belonged to NP-γ, NP-δ, NS-β, NS-γ and NS-ε sub-clades. Pairwise distance and nucleotide diversity index analysis reveals that BoB nurtures two times more diverse amoA sequences than the AS. The core OMZ region of the BoB contains a two-fold higher diversity of amoA gene sequences compared to the AS, whereas the AS contains 13 times more abundant amoA copies than the BoB.

Fertilization altered co-occurrence patterns and microbial assembly process of ammonia-oxidizing microorganisms

Ammonia-oxidizing archaea and bacteria (AOA and AOB, respectively) are important intermediate links in the nitrogen cycle. Apart from the AOA and AOB communities in soil, we further investigated co-occurrence patterns and microbial assembly processes subjected to inorganic and organic fertilizer treatments for over 35 years. The amoA copy numbers and AOA and AOB communities were found to be similar for the CK and organic fertilizer treatments. Inorganic fertilizers decreased the AOA gene copy numbers by 0.75–0.93-fold and increased the AOB gene copy numbers by 1.89–3.32-fold compared to those of the CK treatment. The inorganic fertilizer increased Nitrososphaera and Nitrosospira. The predominant bacteria in organic fertilizer was Nitrosomonadales. Furthermore, the inorganic fertilizer increased the complexity of the co-occurrence pattern of AOA and decreased the complexity pattern of AOB comparing with organic fertilizer. Different fertilizer had an insignificant effect on the microbial assembly process of AOA. However, great difference exists in the AOB community assembly process: deterministic process dominated in organic fertilizer treatment and stochastic processes dominated in inorganic fertilizer treatment, respectively. Redundancy analysis indicated that the soil pH, NO3−N, and available phosphorus contents were the main factors affecting the changes in the AOA and AOB communities. Overall, this findings expanded our knowledge concerning AOA and AOB, and ammonia‐oxidizing microorganisms were more disturbed by inorganic fertilizers than organic fertilizers.

Soil nitrification response to dairy digestate and inorganic ammonium sources depends on soil pH and nitrifier abundances

AbstractTo reduce CH4 emissions, some dairy operations use manure as feedstock for anaerobic digesters, after which the nonvolatilized portion forms a digestate effluent with high NH4+ content that is used as fertilizer. When applied to soil, N fertilizers are subject to nitrification, where NH3–oxidizing archaea (AOA) and bacteria (AOB) oxidize NH3 to NO2–, NO2––oxidizing bacteria (NOB) oxidize NO2– to NO3−, and N oxides are produced from biotic and abiotic reactions. This study used soil microcosms to evaluate the properties of nitrification in response to the addition of N as digestate vs. NH4Cl in soils from the different climatic regions of the western Oregon coastal plain (CP) and eastern Oregon Columbia River basin (CRB). In each soil, properties of NH3 oxidation or N oxide accumulation did not differ when N was provided via digestate or NH4Cl over the normal temperature range; however, nitrification rates, communities, and products differed between soils. The average nitrification rate in CP soil was almost twofold higher than in CRB soil (P ≤ .001), despite no significant differences in AOB activity or AOB amoA copy numbers. With N addition, AOA activity also increased in CP soil, where AOA amoA copy numbers were fivefold higher than CRB soil (P < .002). Accumulation of NO2– only in CRB soil was likely due to NOB inhibition by high NH3 concentrations (∼560 μM) present at the high pH of CRB soil (8.5). Overall, nitrification was dictated by differences in edaphic properties, temperature, and nitrifier communities more than N source.

Monthly distribution of ammonia-oxidizing microbes in a tropical bay.

Ammonia oxidation, performed by ammonia-oxidizing archaea (AOA) and bacteria (AOB), plays a critical role in the cycle of nitrogen in the ocean. For now, environmental variables controlling distribution of ammonia-oxidizing microbes are still largely unknown in oceanic environments. In this study, we used real-time quantitative PCR and high-throughput sequencing methods to investigate the abundance and diversity of AOA and AOB from sediment and water in Zhanjiang Bay. Phylogenic analysis revealed that the majority of AOA amoA sequences in water and sediment were affiliated with the genus Nitrosopumilus, whereas the Nitrosotalea cluster was only detected with low abundance in water. Nitrosomonas and Nitrosospira dominated AOB amoA sequences in water and sediment, respectively. The amoA copy numbers of both AOA and AOB varied significantly with month for both sediment and water. When water and sediment temperature dropped to 17-20°C in December and February, respectively, the copy number of AOB amoA genes increased markedly and was much higher than for AOA amoA genes. Also, AOA abundance in water peaked in December when water temperature was lowest (17-20°C). Stepwise multiple regression analyses revealed that temperature was the most key factor driving monthly changes of AOA or AOB abundance. It is inferred that low water temperature may inhibit growth of phytoplankton and other microbes and so reduce competition for a common substrate, ammonium.

Effects of sea animal colonization on the coupling between dynamics and activity of soil ammonia-oxidizing bacteria and archaea in maritime Antarctica

Abstract. The colonization by a large number of sea animals, including penguins and seals, plays an important role in the nitrogen cycle of the tundra ecosystem in coastal Antarctica. However, little is known about the effects of sea animal colonization on ammonia-oxidizing archaea (AOA) and bacteria (AOB) communities involved in nitrogen transformations. In this study, we chose active seal colony tundra soils (SSs), penguin colony soils (PSs), adjacent penguin-lacking tundra soils (PLs), tundra marsh soils (MSs), and background tundra soils (BSs) to investigate the effects of sea animal colonization on the abundance, activity, and diversity of AOA and AOB in maritime Antarctica. Results indicated that AOB dominated over AOA in PS, SS, and PL, whereas AOB and AOA abundances were similar in MS and BS. Penguin or seal activities increased the abundance of soil AOB amoA genes but reduced the abundance of AOA amoA genes, leading to very large ratios (1.5×102 to 3.2×104) of AOB to AOA amoA copy numbers. Potential ammonia oxidation rates (PAORs) were significantly higher (P=0.02) in SS and PS than in PL, MS, and BS and were significantly positively correlated (P<0.001) with AOB amoA gene abundance. The predominance of AOB over AOA and their correlation with PAOR suggested that AOB play a more important role in the nitrification in animal colony soils. Sequence analysis for gene clones showed that AOA and AOB in tundra soils were from the Nitrososphaera and Nitrosospira lineages, respectively. Penguin or seal activities led to a predominance of AOA phylotypes related to Nitrososphaera cluster I and AOB phylotypes related to Nitrosospira clusters I and II but very low relative abundances in AOA phylotypes related to cluster II, and AOB phylotypes related to clusters III and IV. The differences in AOB and AOA community structures were closely related to soil biogeochemical processes under the disturbance of penguin or seal activities: soil C : N alteration and sufficient input of NH4+–N and phosphorus from animal excrements. The results significantly enhanced the understanding of ammonia-oxidizing microbial communities in the tundra environment of maritime Antarctica.

Relationships between ammonia oxidizers and N2O and CH4 fluxes in agricultural fields with different soil types

Nitrous oxide (N2O) is a greenhouse gas that contributes to the destruction of stratospheric ozone, and agricultural soil is an important source of N2O. Aerobic soils are sinks for atmospheric methane (CH4), a greenhouse gas. Ammonia monooxygenase (AMO) can oxidize CH4, but CH4 is mostly oxidized by methane monooxygenase (MMO), and CH4 oxidation by AMO is generally negligible in the soil. We monitored the N2O and CH4 fluxes after urea application in fields containing different soils using an automated sampling system to determine the effects of environmental and microbial factors on the N2O and CH4 fluxes. The soil types were Low-humic Andosol (Gleyic Haplic Andosol), yellow soil (Gleyic Haplic Alisol) and gray lowland soil (Entric Fluvisol). Cumulative N2O emissions from the yellow soil were higher than those from other soil types, although the difference was not significant. The CH4 uptake level by Andosol was one order of magnitude higher than that by other soils. There were significant relationships between the ammonia oxidation potential, AOB and AOA amoA copy numbers, and the CH4 uptake. In contrast, the gene copy numbers of methane-oxidizing bacteria (MOB) pmoA were below the detection limit. Our results suggested that the AMOs of AOB and AOA may have more important roles than those previously considered during CH4 oxidation in agricultural soils treated with N fertilizers.

Abundance and diversity of ammonia-oxidizing archaea in response to various habitats in Pearl River Delta of China, a subtropical maritime zone

Ammonia-oxidizing archaea (AOA) are widely considered key to ammonia oxidation in various environments. However, little work has been conducted to simultaneously investigate the abundance and diversity of AOA as well as correlations between archaeal amoA genotypes and environmental parameters of different ecosystems at one district. To understand the abundance, diversity, and distribution of AOA in Pearl River Delta of China in response to various habitats, the archaeal amoA genes in soil, marine, river, lake, hot spring and wastewater treatment plant (WWTP) samples were investigated using real-time fluorescent quantitative PCR and clone libraries. Our analyses indicated that the diversity of AOA in various habitats was different and could be clustered into five major clades, i.e., estuary sediment, marine water/sediment, soil, hot spring and Cluster 1. Phylogenetic analyses revealed that the structure of AOA communities in similar ecological habitats exhibited strong relation. The canonical correspondence method indicated that the AOA community structure was strongly correlated to temperature, pH, total organic carbon, total nitrogen and dissolved oxygen variables. Assessing AOA amoA gene copy numbers, ranging from 6.84 × 106 to 9.45 × 107 copies/g in dry soil/sediment, and 6.06 × 106 to 2.41 × 107 copies/L in water samples, were higher than ammonia-oxidizing bacteria (AOB) by 1–2 orders of magnitude. However, AOA amoA copy numbers were much lower than AOB in WWTP activated sludge samples. Overall, these studies suggested that AOA may be a major contributor to ammonia oxidation in natural habitats but play a minor role in highly aerated activated sludge. The result also showed the ratio of AOA to AOB amoA gene abundance was positively correlated with temperature and less correlated with other environmental parameters. New data from our study provide increasing evidence for the relative abundance and diversity of ammonia-oxidizing archaea in the global nitrogen cycle.

Mimicking the oxygen minimum zones: stimulating interaction of aerobic archaeal and anaerobic bacterial ammonia oxidizers in a laboratory‐scale model system

In marine oxygen minimum zones (OMZs), ammonia-oxidizing archaea (AOA) rather than marine ammonia-oxidizing bacteria (AOB) may provide nitrite to anaerobic ammonium-oxidizing (anammox) bacteria. Here we demonstrate the cooperation between marine anammox bacteria and nitrifiers in a laboratory-scale model system under oxygen limitation. A bioreactor containing ‘Candidatus Scalindua profunda’ marine anammox bacteria was supplemented with AOA (Nitrosopumilus maritimus strain SCM1) cells and limited amounts of oxygen. In this way a stable mixed culture of AOA, and anammox bacteria was established within 200 days while also a substantial amount of endogenous AOB were enriched. ‘Ca. Scalindua profunda’ and putative AOB and AOA morphologies were visualized by transmission electron microscopy and a C18 anammox [3]-ladderane fatty acid was highly abundant in the oxygen-limited culture. The rapid oxygen consumption by AOA and AOB ensured that anammox activity was not affected. High expression of AOA, AOB and anammox genes encoding for ammonium transport proteins was observed, likely caused by the increased competition for ammonium. The competition between AOA and AOB was found to be strongly related to the residual ammonium concentration based on amoA gene copy numbers. The abundance of archaeal amoA copy numbers increased markedly when the ammonium concentration was below 30 μM finally resulting in almost equal abundance of AOA and AOB amoA copy numbers. Massive parallel sequencing of mRNA and activity analyses further corroborated equal abundance of AOA and AOB. PTIO addition, inhibiting AOA activity, was employed to determine the relative contribution of AOB versus AOA to ammonium oxidation. The present study provides the first direct evidence for cooperation of archaeal ammonia oxidation with anammox bacteria by provision of nitrite and consumption of oxygen.

Elevated Atmospheric CO2 Impacts Abundance and Diversity of Nitrogen Cycling Functional Genes in Soil

The concentration of CO(2) in the Earth's atmosphere has increased over the last century. Although this increase is unlikely to have direct effects on soil microbial communities, increased atmospheric CO(2) may impact soil ecosystems indirectly through plant responses. This study tested the hypothesis that exposure of plants to elevated CO(2) would impact soil microorganisms responsible for key nitrogen cycling processes, specifically denitrification and nitrification. We grew trembling aspen (Populus tremuloides) trees in outdoor chambers under ambient (360 ppm) or elevated (720 ppm) levels of CO(2) for 5 years and analyzed the microbial communities in the soils below the trees using quantitative polymerase chain reaction and clone library sequencing targeting the nitrite reductase (nirK) and ammonia monooxygenase (amoA) genes. We observed a more than twofold increase in copy numbers of nirK and a decrease in nirK diversity with CO(2) enrichment, with an increased predominance of Bradyrhizobia-like nirK sequences. We suggest that this dramatic increase in nirK-containing bacteria may have contributed to the significant loss of soil N in the CO(2)-treated chambers. Elevated CO(2) also resulted in a significant decrease in copy numbers of bacterial amoA, but no change in archaeal amoA copy numbers. The decrease in abundance of bacterial amoA was likely a result of the loss of soil N in the CO(2)-treated chambers, while the lack of response for archaeal amoA supports the hypothesis that physiological differences in these two groups of ammonia oxidizers may enable them to occupy distinct ecological niches and respond differently to environmental change.

Distribution of Ammonia-Oxidizing Archaea and Bacteria in the Surface Sediments of Matsushima Bay in Relation to Environmental Variables

Ammonia oxidization is the first and a rate-limiting step of nitrification, which is often a critical process in nitrogen removal from estuarine and coastal environments. To clarify the correlation of environmental conditions with the distribution of ammonia oxidizers in organic matter-rich coastal sediments, ammonia-oxidizing archaea (AOA) and bacteria (AOB) ammonia monooxygenase alpha subunit gene (amoA) abundance was determined in sediments of Matsushima Bay located in northeast Japan. The AOA and AOB amoA copy numbers ranged from 1.1×106 to 1.7×107 and from 7.1×105 to 7.6×106 copies g−1 sediment, respectively. AOA and AOB amoA abundance was negatively correlated with dissolved oxygen levels in the bottom water. AOA amoA abundance was also correlated with total phosphorus levels in the sediments. On the other hand, no significant relationship was observed between the amoA abundance and ammonium, organic matter (ignition loss), or acid volatile sulfide-sulfur levels in the sediments. These results show the heterogeneous distribution of ammonia oxidizers by the difference in environmental conditions within the bay. Moreover, AOA amoA diversity was relatively low in the area of high AOA amoA abundance, suggesting the variability of AOA community composition.

Dynamics of ammonia-oxidizing communities in barley-planted bulk soil and rhizosphere following nitrate and ammonium fertilizer amendment

Oxidation of ammonia by nitrifying microorganisms is a major pathway that fertilizer nitrogen (N) may take upon application to agricultural soils, but the relative roles of bacterial (AOB) vs. archaeal (AOA) ammonia oxidizers are controversial. We explored the effects of various forms of mineral N fertilizer on the AOB and AOA community dynamics in two different soils planted with barley. Ammonia oxidizers were monitored via real-time PCR and terminal restriction fragment length polymorphism analysis of bacterial and archaeal amoA genes following the addition of either [NH₄]₂SO₄, NH₄NO₃ or KNO₃. AOB and AOA communities were also studied specifically in the rhizospheres of two different barley varieties upon [NH₄]₂SO₄ vs. KNO₃ addition. AOB changed in community composition and increased in abundance upon ammonium amendment in bulk soil and rhizosphere, with changes in bacterial amoA copy numbers lagging behind relative to changes in soil ammonium. In both soils, only T-RFs corresponding to phylotypes related to Nitrosospira clade 3a underwent significant community changes. Increases in AOB abundance were generally stronger in the bulk soil than in the rhizosphere, implying significant ammonia uptake by plant roots. AOA underwent shifts in the community composition over time and fluctuated in abundance in all treatments irrespective of ammonia availability. AOB were thus considered as the main agents responsible for fertilizer ammonium oxidation, while the functions of AOA in soil N cycling remain unresolved.

Diversity of Archaea and detection of crenarchaeotal amoA genes in the rivers Rhine and Têt

Pelagic archaeal phylogenetic diversity and the potential for crenarchaeotal nitrification of Group 1.1a were determined in the rivers Rhine and Tet by 16S rRNA sequencing, catalyzed reported deposition-fluorescence in situ hybridization (CARD–FISH) and quantification of 16S rRNA and functional genes. Euryarchaeota were, for the first time, detected in temperate river water even though a net predominance of crenarchaeotal phylotypes was found. Differences in phylogenic distribution were observed between rivers and seasons. Our data suggest that a few archaeal phylotypes (Euryarchaeota Groups RC-V and LDS, Crenarchaeota Group 1.1a) are widely distributed in pelagic riverine environments whilst others (Euryarchaeota Cluster Sagma-1) may only occur seasonally in river water. Crenarchaeota Group 1.1a has recently been identified as a major nitrifier in the marine environment and phylotypes of this group were also present in both rivers, where they represented 0.3% of the total pelagic microbial community. Interestingly, a generally higher abundance of Crenarchaeota Group 1.1a was found in the Rhine than in the Tet, and crenarchaeotal ammonia monooxygenase gene (amoA) was also detected in the Rhine, with higher amoA copy numbers measured in February than in September. This suggests that some of the Crenarchaeota present in river waters have the ability to oxidize ammonia and that riverine crenarchaeotal nitrification of Group 1.1a may vary seasonally.

Spatial distribution of Bacteria and Archaea and amoA gene copy numbers throughout the water column of the Eastern Mediterranean Sea

Until recently, ammonia oxidation, a key process in the global nitrogen cycle, was thought to be mediated exclusively by a few bacterial groups. It has been shown now, that also Crenarchaeota are capable to perform this initial nitrification step. The abundance of ammonia oxidizing Bacteria and Archaea was determined using the bacterial and archaeal ammonia monooxygenase-alpha subunit (amoA) gene as functional markers in a quantitative PCR approach and related to the abundance of Bacteria and Archaea in the Eastern Mediterranean Sea. Archaeal amoA copy numbers decreased from 4000-5000 copies ml(-1) seawater from the 200-500 m depth layer to 20 copies ml(-1) at 1000 m depth. Beta-proteobacterial amoA genes were below the detection limit in all the samples. The archaeal amoA copy numbers were correlated with NO(2)(-) concentrations, suggesting that ammonia-oxidizing Archaea may play a significant role in the nitrification in the mesopelagic waters of the Eastern Mediterranean Sea. In the bathypelagic waters, however, archaeal amoA gene abundance was rather low although Crenarchaeota were abundant, indicating that Crenarchaeota might largely lack the amoA gene in these deep waters. Terminal restriction fragment length polymorphism analysis of the archaeal community revealed a distinct clustering with the mesopelagic community distinctly different from the archaeal communities of both, the surface waters and the 3000-4000 m layers. Hence, the archaeal community in the Eastern Mediterranean Sea appears to be highly stratified despite the absence of major temperature and density gradients between the meso- and bathypelagic waters of the Mediterranean Sea.

Comparative analysis of archaeal 16S rRNA and amoA genes to estimate the abundance and diversity of ammonia-oxidizing archaea in marine sediments

Considering their abundance and broad distribution, non-extremophilic Crenarchaeota are likely to play important roles in global organic and inorganic matter cycles. The diversity and abundance of archaeal 16S rRNA and putative ammonia monooxygenase alpha-subunit (amoA) genes were comparatively analyzed to study genetic potential for nitrification of ammonia-oxidizing archaea (AOA) in the surface layers (0-1 cm) of four marine sediments of the East Sea, Korea. After analysis of a 16S rRNA gene clone library, we found various archaeal groups that include the crenarchaeotal group (CG) I.1a (54.8%) and CG I.1b (5.8%), both of which are known to harbor ammonia oxidizers. Notably, the 16S rRNA gene of CG I.1b has only previously been observed in terrestrial environments. The 16S rRNA gene sequence data revealed a distinct difference in archaeal community among sites of marine sediments. Most of the obtained amoA sequences were not closely related to those of the clones retrieved from estuarine sediments and marine water columns. Furthermore, clades of unique amoA sequences were likely to cluster according to sampling sites. Using real-time PCR, quantitative analysis of amoA copy numbers showed that the copy numbers of archaeal amoA ranged from 1.1x10(7) to 4.9x10(7) per gram of sediment and were more numerous than those of bacterial amoA, with ratios ranging from 11 to 28. In conclusion, diverse CG I.1a and CG I.1b AOA inhabit surface layers of marine sediments and AOA, and especially, CG I.1a are more numerous than other ammonia-oxidizing bacteria.