Changes in soil ammonia oxidizers and potential nitrification after clear-cutting of boreal forests in China.

The Korean pine and broad-leaved mixed forests are the most typical and complete ecosystem among the global boreal forests, with extremely important ecological functions. However, few studies on the changes of soil ammonia oxidizers and potential nitrification after clear-cutting of forests are reported. In this study, in contrast to primary Korean pine forests, nitrate (NO3-) was significantly higher in secondary broad-leaved forests, while ammonium (NH4+) was on the contrary. The abundance of ammonia-oxidizing bacteria (AOB) was greatly higher in secondary broad-leaved forests, while levels of ammonia-oxidizing archaea (AOA) were not significantly different between them. The significant differences of community structure of AOA and AOB were observed in different forest types and soil layers. Compared with AOA, community compositions of AOB was more sensitive to forest type. The dominant groups of AOA were Nitrososphaera and Nitrosotalea, and the dominant group of AOB was Nitrosospira, of which Nitrosospira cluster 2 and 4 were functional groups with highly activity. Soil potential nitrification rate (PNR) was higher in secondary broad-leaved forests. Furthermore, PNR and AOB abundance had a significant positive correlation, but no significant correlation with AOA abundance. These results provide insights into the soil nitrogen balance and effects on forest restoration after clear-cutting.

Community diversity and abundance of ammonia-oxidizing archaea and bacteria in shrimp pond sediment at different culture stages.

AIMS: Ammonia oxidation is a significant process of nitrogen cycles in a lot of ecosystems sediments while there are few studies in shrimp culture pond (SCP) sediments. This paper attempted to explore the community diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in SCP sediments at different culture stages. METHODS AND RESULTS: We collected SCP sediments and analysed the community diversity and abundance of AOA and bacteria in shrimp pond sediment at different culture stages using the ammonia monooxygenase (amoA) gene with quantitative PCR (qPCR) and 16S rRNA gene sequencing. The AOB-amoA gene abundance was showed higher than AOA-amoA gene abundance in SCP sediments on Day 50 and Day 60 after shrimp larvae introducing into the pond, and the diversity of AOA in SCP sediments was higher than that of AOB. The phylogenetic tree revealed that the most of AOA were the member of Nitrosopumilus and Nitrososphaera, and the majority of AOB sequences were clustered into Nitrosospira, Nitrosomonas clusters 6a and 7. The AOA community has close relationship with total organic carbon (TOC), pH, total phosphorus (TP), nitrate reductase, urease, acid phosphatase and ß-glucosidase. The AOB community was related to TOC, C/N and nitrate reductase. CONCLUSIONS: AOA and AOB play the different ecological roles in SCP sediments at different culture stages. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggested that the different community diversity and abundance of AOA and AOB in SCP sediments, which may improve our ecological cognition of shrimp culture stages in SCP ecosystems.

Nitrosospira Cluster 8a Plays a Predominant Role in the Nitrification Process of a Subtropical Ultisol under Long-Term Inorganic and Organic Fertilization.

Long-term effects of inorganic and organic fertilization on nitrification activity (NA) and the abundances and community structures of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were investigated in an acidic Ultisol. Seven treatments applied annually for 27 years comprised no fertilization (control), inorganic NPK fertilizer (N), inorganic NPK fertilizer plus lime (CaCO3) (NL), inorganic NPK fertilizer plus peanut straw (NPS), inorganic NPK fertilizer plus rice straw (NRS), inorganic NPK fertilizer plus radish (NR), and inorganic NPK fertilizer plus pig manure (NPM). In nonfertilized soil, the abundance of AOA was 1 order of magnitude higher than that of AOB. Fertilization reduced the abundance of AOA but increased that of AOB, especially in the NL treatment. The AOA communities in the control and the N treatments were dominated by the Nitrososphaera and B1 clades but shifted to clade A in the NL and NPM treatments. Nitrosospira cluster 8a was found to be the most dominant AOB in all treatments. NA was primarily regulated by soil properties, especially soil pH, and the interaction with AOB abundance explained up to 73% of the variance in NA. When NL soils with neutral pH were excluded from the analysis, AOB abundance, especially the relative abundance of Nitrosospira cluster 8a, was positively associated with NA. In contrast, there was no association between AOA abundance and NA. Overall, our data suggest that Nitrosospira cluster 8a of AOB played an important role in the nitrification process in acidic soil following long-term inorganic and organic fertilization.IMPORTANCE The nitrification process is an important step in the nitrogen (N) cycle, affecting N availability and N losses to the wider environment. Ammonia oxidation, which is the first and rate-limiting step of nitrification, was widely accepted to be mainly regulated by AOA in acidic soils. However, in this study, nitrification activity was correlated with the abundance of AOB rather than that of AOA in acidic Ultisols. Nitrosospira cluster 8a, a phylotype of AOB which preferred warm temperatures, and low soil pH played a predominant role in the nitrification process in the test Ultisols. Our results also showed that long-term application of lime or pig manure rather than plant residues altered the community structure of AOA and AOB. Taken together, our findings contribute new knowledge to the understanding of the nitrification process and ammonia oxidizers in subtropical acidic Ultisol under long-term inorganic and organic fertilization.

Acyl-Homoserine Lactone Production in Nitrifying Bacteria of the Genera Nitrosospira, Nitrobacter, and Nitrospira Identified via a Survey of Putative Quorum-Sensing Genes.

The genomes of many bacteria that participate in nitrogen cycling through the process of nitrification contain putative genes associated with acyl-homoserine lactone (AHL) quorum sensing (QS). AHL QS or bacterial cell-cell signaling is a method of bacterial communication and gene regulation and may be involved in nitrogen oxide fluxes or other important phenotypes in nitrifying bacteria. Here, we carried out a broad survey of AHL production in nitrifying bacteria in three steps. First, we analyzed the evolutionary history of AHL synthase and AHL receptor homologs in sequenced genomes and metagenomes of nitrifying bacteria to identify AHL synthase homologs in ammonia-oxidizing bacteria (AOB) of the genus Nitrosospira and nitrite-oxidizing bacteria (NOB) of the genera Nitrococcus, Nitrobacter, and Nitrospira Next, we screened cultures of both AOB and NOB with uncharacterized AHL synthase genes and AHL synthase-negative nitrifiers by a bioassay. Our results suggest that an AHL synthase gene is required for, but does not guarantee, cell density-dependent AHL production under the conditions tested. Finally, we utilized mass spectrometry to identify the AHLs produced by the AOB Nitrosospira multiformis and Nitrosospira briensis and the NOB Nitrobacter vulgaris and Nitrospira moscoviensis as N-decanoyl-l-homoserine lactone (C10-HSL), N-3-hydroxy-tetradecanoyl-l-homoserine lactone (3-OH-C14-HSL), a monounsaturated AHL (C10:1-HSL), and N-octanoyl-l-homoserine lactone (C8-HSL), respectively. Our survey expands the list of AHL-producing nitrifiers to include a representative of Nitrospira lineage II and suggests that AHL production is widespread in nitrifying bacteria.IMPORTANCE Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite by nitrifying microorganisms, plays an important role in environmental nitrogen cycling from agricultural fertilization to wastewater treatment. The genomes of many nitrifying bacteria contain genes associated with bacterial cell-cell signaling or quorum sensing (QS). QS is a method of bacterial communication and gene regulation that is well studied in bacterial pathogens, but less is known about QS in environmental systems. Our previous work suggested that QS might be involved in the regulation of nitrogen oxide gas production during nitrite metabolism. This study characterized putative QS signals produced by different genera and species of nitrifiers. Our work lays the foundation for future experiments investigating communication between nitrifying bacteria, the purpose of QS in these microorganisms, and the manipulation of QS during nitrification.

Nitrosospira lacus sp. nov., a psychrotolerant, ammonia-oxidizing bacterium from sandy lake sediment.

A Gram-negative, spiral-shaped, chemolithotrophic, ammonia-oxidizing bacterium, designated APG3(T), was isolated into pure culture from sandy lake sediment collected from Green Lake, Seattle, WA, USA. Phylogenetic analyses based on the 16S rRNA gene sequence showed that strain APG3(T) belongs to cluster 0 of the genus Nitrosospira, which is presently not represented by described species, with Nitrosospira multiformis (cluster 3) as the closest species with a validly published name (identity of 98.6 % to the type strain). Strain APG3(T) grew at 4 °C but could not grow at 35 °C, indicating that this bacterium is psychrotolerant. Remarkably, the strain was able to grow over a wide range of pH (pH 5-9), which was greater than the pH range of any studied ammonia-oxidizing bacteria in pure culture. The DNA G+C content of the APG3(T) genome is 53.5 %, which is similar to that of Nitrosospira multiformis ATCC 25196(T) (53.9 %) but higher than that of Nitrosomonas europaea ATCC 19718 (50.7 %) and Nitrosomonas eutropha C71 (48.5 %). The average nucleotide identity (ANI) calculated for the genomes of strain APG3(T) and Nitrosospira multiformis ATCC 25196(T) was 75.45 %, significantly lower than the value of 95 % ANI that corresponds to the 70 % species-level cut-off based on DNA-DNA hybridization. Overall polyphasic taxonomy study indicated that strain APG3(T) represents a novel species in the genus Nitrosospira, for which the name Nitrosospira lacus sp. nov. is proposed (type strain APG3(T) = NCIMB 14869(T) = LMG 27536(T) = ATCC BAA-2542(T)).

Diversity, abundance, and spatial distribution of sediment ammonia-oxidizing Betaproteobacteria in response to environmental gradients and coastal eutrophication in Jiaozhou Bay, China.

Ongoing anthropogenic eutrophication of Jiaozhou Bay offers an opportunity to study the influence of human activity on bacterial communities that drive biogeochemical cycling. Nitrification in coastal waters appears to be a sensitive indicator of environmental change, suggesting that function and structure of the microbial nitrifying community may be associated closely with environmental conditions. In the current study, the amoA gene was used to unravel the relationship between sediment aerobic obligate ammonia-oxidizing Betaproteobacteria (Beta-AOB) and their environment in Jiaozhou Bay. Protein sequences deduced from amoA gene sequences grouped within four distinct clusters in the Nitrosomonas lineage, including a putative new cluster. In addition, AmoA sequences belonging to three newly defined clusters in the Nitrosospira lineage were also identified. Multivariate statistical analyses indicated that the studied Beta-AOB community structures correlated with environmental parameters, of which nitrite-N and sediment sand content had significant impact on the composition, structure, and distribution of the Beta-AOB community. Both amoA clone library and quantitative PCR (qPCR) analyses indicated that continental input from the nearby wastewater treatment plants and polluted rivers may have significant impact on the composition and abundance of the sediment Beta-AOB assemblages in Jiaozhou Bay. Our work is the first report of a direct link between a sedimentological parameter and the composition and distribution of the sediment Beta-AOB and indicates the potential for using the Beta-AOB community composition in general and individual isolates or environmental clones in the Nitrosomonas oligotropha lineage in particular as bioindicators and biotracers of pollution or freshwater or wastewater input in coastal environments.

Stable isotope probing analysis of interactions between ammonia oxidizers.

The response of natural microbial communities to environmental change can be assessed by determining DNA- or RNA-targeted changes in relative abundance of 16S rRNA gene sequences by using fingerprinting techniques such as denaturing gradient gel electrophoresis (DNA-DGGE and RNA-DGGE, respectively) or by stable isotope probing (SIP) of 16S rRNA genes following incubation with a (13)C-labeled substrate (DNA-SIP-DGGE). The sensitivities of these three approaches were compared during batch growth of communities containing two or three Nitrosospira pure or enriched cultures with different tolerances to a high ammonia concentration. Cultures were supplied with low, intermediate, or high initial ammonia concentrations and with (13)C-labeled carbon dioxide. DNA-SIP-DGGE provided the most direct evidence for growth and was the most sensitive, with changes in DGGE profiles evident before changes in DNA- and RNA-DGGE profiles and before detectable increases in nitrite and nitrate production. RNA-DGGE provided intermediate sensitivity. In addition, the three molecular methods were used to follow growth of individual strains within communities. In general, changes in relative activities of individual strains within communities could be predicted from monoculture growth characteristics. Ammonia-tolerant Nitrosospira cluster 3b strains dominated mixed communities at all ammonia concentrations, and ammonia-sensitive strains were outcompeted at an intermediate ammonia concentration. However, coexistence of ammonia-tolerant and ammonia-sensitive strains occurred at the lowest ammonia concentration, and, under some conditions, strains inhibited at high ammonia in monoculture were active at high ammonia in mixed cultures, where they coexisted with ammonia-tolerant strains. The results therefore demonstrate the sensitivity of SIP for detection of activity of organisms with relatively low yield and low activity and its ability to follow changes in the structure of interacting microbial communities.

The biogeography of ammonia-oxidizing bacterial communities in soil.

Although ammonia-oxidizing bacteria (AOB) are likely to play a key role in the soil nitrogen cycle, we have only a limited understanding of how the diversity and composition of soil AOB communities change across ecosystem types. We examined 23 soils collected from across North America and used sequence-based analyses to compare the AOB communities in each of the distinct soils. Using 97% 16S rRNA sequence similarity groups, we identified only 24 unique AOB phylotypes across all of the soils sampled. The majority of the sequences collected were in the Nitrosospira lineages (representing 80% of all the sequences collected), and AOB belonging to Nitrosospira cluster 3 were particularly common in our clone libraries and ubiquitous across the soil types. Community composition was highly variable across the collected soils, and similar ecosystem types did not always harbor similar AOB communities. We did not find any significant correlations between AOB community composition and measures of N availability. From the suite of environmental variables measured, we found the strongest correlation between temperature and AOB community composition; soils exposed to similar mean annual temperatures tended to have similar AOB communities. This finding is consistent with previous studies and suggests that temperature selects for specific AOB lineages. Given that distinct AOB taxa are likely to have unique functional attributes, the biogeographical patterns exhibited by soil AOB may be directly relevant to understanding soil nitrogen dynamics under changing environmental conditions.

Physiology, phylogeny and in situ evidence for bacterial and archaeal nitrifiers in the marine sponge Aplysina aerophoba.

The potential for nitrification in the Mediterranean sponge Aplysina aerophoba was assessed using a combined physiological and molecular approach. Nitrate excretion rates in whole sponges reached values of up to 344 nmol g(-1) dry weight (wt) h(-1) (unstimulated) and 1325 nmol g(-1) dry wt h(-1) (stimulated). Addition of nitrapyrin, a nitrification-specific inhibitor, effectively inhibited nitrate excretion. Ammonium was taken up by sponges in spring and excreted in fall, the sponges thus serving as either an ammonium sink or ammonium source. Nitrosospira cluster 1 and Crenarchaeota group I.1A 16S rRNA and amoA genes were recovered from A. aerophoba and other sponges from different world's oceans. The archaeal 16S rRNA genes formed a sponge-specific subcluster, indicating that their representatives are members of the stable microbial community of sponges. On the other hand, clustering was not evident for Nitrosospira rRNA genes which is consistent with their presence in sediment and seawater samples. The presence of both Nitrosospira cluster 1 and crenarchaeal group 1 phylotypes in sponge tissue was confirmed using fluorescently labelled 16S rRNA gene probes. This study contributes to an ongoing effort to link microbial diversity with metabolic functions in the phylogenetically diverse, elusive and so far uncultivated microbial communities of marine sponges.

[Nitrification performance and microbial community analysis in carbon membrane-aerated biofilm reactor].

A carbon membrane-aerated biofilm reactor was developed to treat nitrogenous inorganic wastewater. Influent NH; -N concentrations and HRT were changed to investigate nitrification performance of reactor,oxygen utilization and NH4+ -N's removal loading. Biofilm's surface characteristics and dominant bacteria of nitrifier were analyzed. The results show that under the conditions of intra-membrane pressure of 0.017 MPa, influent NH4+ -N of 50 mg/L and HRT of 8 h NH4+ -N removal efficiency reaches 96% and effluent average nitrite is 17 mg/L, which benefits short-cut nitrification to a certain extent. The bacteria within biofilm consume all oxygen supplied through carbon membrane. The maximum specific removal rate of NH4+ -N is 9.7 g/(m2 x d), which is limited by the amount of bacteria grown onto carbon membrane's surface. Fluorescent in situ hybridization analysis indicates that within the biofilm Nitrosomonas and Nitrosospira are main ammonia-oxidizing bacteria and occupy about 19% and 21% of the total bacteria number, respectively. The Nitrobacter are not observed and Nitrospira are dominant nitrite-oxidizing bacteria, the fraction of which is 20% of total bacteria.

PCR profiling of ammonia-oxidizer communities in acidic soils subjected to nitrogen and sulphur deposition.

Communities of ammonia-oxidizing bacteria (AOB) were characterized in two acidic soil sites experimentally subjected to varying levels of nitrogen and sulphur deposition. The sites were an acidic spruce forest soil in Deepsyke, Southern Scotland, with low background deposition, and a nitrogen-saturated upland grass heath in Pwllpeiran, North Wales. Betaproteobacterial ammonia-oxidizer 16S rRNA and ammonia monooxygenase (amoA) genes were analysed by cloning, sequencing and denaturing gradient gel electrophoresis (DGGE). DGGE profiles of amoA and 16S rRNA gene fragments from Deepsyke soil in 2002 indicated no effect of nitrogen deposition on AOB communities, which contained both Nitrosomonas europaea and Nitrosospira. In 2003, only Nitrosospira could be detected, and no amoA sequences could be retrieved. These results indicate a decrease in the relative abundance of AOB from the year 2002 to 2003 in Deepsyke soil, which may be the result of the exceptionally low rainfall in spring 2003. Nitrosospira-related sequences from Deepsyke soil grouped in all clusters, including cluster 1, which typically contains only sequences from marine environments. In Pwllpeiran soil, 16S rRNA gene libraries were dominated by nonammonia oxidizers and no amoA sequences were detectable. This indicates that autotrophic AOB play only a minor role in these soils even at high nitrogen deposition.

Ammonia-oxidizing beta-proteobacteria from the oxygen minimum zone off northern Chile.

The composition of ammonia-oxidizing bacteria from the beta-Proteobacteria subclass (betaAOB) was studied in the surface and upper-oxycline oxic waters (2- to 50-m depth, approximately 200 to 44 microM O(2)) and within the oxygen minimum zone (OMZ) suboxic waters (50- to 400-m depth, < or =10 microM O(2)) of the eastern South Pacific off northern Chile. This study was carried out through cloning and sequencing of genes coding for 16S rRNA and the ammonia monooxygenase enzyme active subunit (amoA). Sequences affiliated with Nitrosospira-like cluster 1 dominated the 16S rRNA gene clone libraries constructed from both oxic and suboxic waters. Cluster 1 consists exclusively of yet-uncultivated betaAOB from marine environments. However, a single clone, out of 224 obtained from the OMZ, was found to belong to Nitrosospira lineage cluster 0. To our knowledge, cluster 0 sequences have been derived from betaAOB isolated only from sand, soil, and freshwater environments. Sequences in clone libraries of the amoA gene from the surface and upper oxycline could be grouped in a marine subcluster, also containing no cultured representatives. In contrast, all 74 amoA sequences originating from the OMZ were either closely affiliated with cultured Nitrosospira spp. from clusters 0 and 2 or with other yet-uncultured betaAOB from soil and an aerated-anoxic Orbal process waste treatment plant. Our results reveal the presence of Nitrosospira-like betaAOB in both oxic and suboxic waters associated with the OMZ but with a clear community shift at the functional level (amoA) along the strong oxygen gradient.

Loss of diversity of ammonia-oxidizing bacteria correlates with increasing salinity in an estuary system.

Ammonia-oxidizing bacteria (AOB) play an important role in nitrogen cycling in estuaries, but little is known about AOB diversity, distribution and activity in relation to the chemical and physical changes encountered in estuary systems. Although estuarine salinity gradients are well recognized to influence microbial community structure, few studies have examined the influence of varying salinity on the diversity and stability of AOB populations. To investigate these relationships, we collected sediment samples from low-, mid- and high-salinity sites in Plum Island Sound estuary, MA, during spring and late summer over 3 years. Ammonia-oxidizing bacteria distribution and diversity were assessed by terminal restriction fragment length polymorphism (TRFLP) analysis of the ammonia monooxygenase (amoA) gene, and fragments were identified by screening amoA clone libraries constructed from each site. Most striking was the stability and low diversity of the AOB community at the high-salinity site, showing little variability over 3 years. Ammonia-oxidizing bacteria at the high-salinity site were not closely related to any cultured AOB, but were most similar to Nitrosospira spp. Ammonia-oxidizing bacteria at the mid- and low-salinity sites were distributed among Nitrosospira-like sequences and sequences related to Nitrosomonas ureae/oligotropha and Nitrosomonas sp. Nm143. Our study suggests that salinity is a strong environmental control on AOB diversity and distribution in this estuary.

Activity, diversity and population size of ammonia-oxidising bacteria in oil-contaminated landfarming soil.

Chemolithotrophic ammonia-oxidising bacteria (AOB) present in oil-contaminated landfarming soil were studied over two growing seasons in 1999 and 2000. The number of AOB (4-9 x 10(5) cellsg(-1) of dry soil) determined with the quantitative polymerase chain reaction (real-time PCR) and the rate of potential ammonium oxidation (0.05-0.28 microg NO2(-)-N g(-1) of dry soil h(-1)) indicated the presence of stable AOB populations. Denaturing gradient gel electrophoresis (DGGE) profiling and sequence analysis of PCR-amplified AOB 16S rRNA genes showed dominance of Nitrosospira-like sequences in clusters 2 and 3. The present results from the chronically oil-contaminated landfarming soil support the suggested importance of Nitrosospira-like AOB in terrestrial environments.

16S rRNA and amoA-based phylogeny of 12 novel betaproteobacterial ammonia-oxidizing isolates: extension of the dataset and proposal of a new lineage within the nitrosomonads.

The phylogenetic relationship of 12 ammonia-oxidizing isolates (eight nitrosospiras and four nitrosomonads), for which no gene sequence information was available previously, was investigated based on their genes encoding 16S rRNA and the active site subunit of ammonia monooxygenase (AmoA). Almost full-length 16S rRNA gene sequences were determined for the 12 isolates. In addition, 16S rRNA gene sequences of 15 ammonia-oxidizing bacteria (AOB) published previously were completed to allow for a more reliable phylogeny inference of members of this guild. Moreover, sequences of 453 bp fragments of the amoA gene were determined from 15 AOB, including the 12 isolates, and completed for 10 additional AOB. 16S rRNA gene and amoA-based analyses, including all available sequences of AOB pure cultures, were performed to determine the position of the newly retrieved sequences within the established phylogenetic framework. The resulting 16S rRNA gene and amoA tree topologies were similar but not identical and demonstrated a superior resolution of 16S rRNA versus amoA analysis. While 11 of the 12 isolates could be assigned to different phylogenetic groups recognized within the betaproteobacterial AOB, the estuarine isolate Nitrosomonas sp. Nm143 formed a separate lineage together with three other marine isolates whose 16S rRNA sequences have not been published but have been deposited in public databases. In addition, 17 environmentally retrieved 16S rRNA gene sequences not assigned previously and all originating exclusively from marine or estuarine sites clearly belong to this lineage.