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.

Physical mixing in coastal waters controls and decouples nitrification via biomass dilution.

Nitrification is a central process of the aquatic nitrogen cycle that controls the supply of nitrate used in other key processes, such as phytoplankton growth and denitrification. Through time series observation and modeling of a seasonally stratified, eutrophic coastal basin, we demonstrate that physical dilution of nitrifying microorganisms by water column mixing can delay and decouple nitrification. The findings are based on a 4-y, weekly time series in the subsurface water of Bedford Basin, Nova Scotia, Canada, that included measurement of functional (amoA) and phylogenetic (16S rRNA) marker genes. In years with colder winters, more intense winter mixing resulted in strong dilution of resident nitrifiers in subsurface water, delaying nitrification for weeks to months despite availability of ammonium and oxygen. Delayed regrowth of nitrifiers also led to transient accumulation of nitrite (3 to 8 µmol · kgsw-1) due to decoupling of ammonia and nitrite oxidation. Nitrite accumulation was enhanced by ammonia-oxidizing bacteria (Nitrosomonadaceae) with fast enzyme kinetics, which temporarily outcompeted the ammonia-oxidizing archaea (Nitrosopumilus) that dominated under more stable conditions. The study reveals how physical mixing can drive seasonal and interannual variations in nitrification through control of microbial biomass and diversity. Variable, mixing-induced effects on functionally specialized microbial communities are likely relevant to biogeochemical transformation rates in other seasonally stratified water columns. The detailed study reveals a complex mechanism through which weather and climate variability impacts nitrogen speciation, with implications for coastal ecosystem productivity. It also emphasizes the value of high-frequency, multiparameter time series for identifying complex controls of biogeochemical processes in aquatic systems.

Nitrification inhibitors effectively target N2 O-producing Nitrosospira spp. in tropical soil.

The nitrification inhibitors (NIs) 3,4-dimethylpyrazole (DMPP) and dicyandiamide (DCD) can effectively reduce N2 O emissions; however, which species are targeted and the effect of these NIs on the microbial nitrifier community is still unclear. Here, we identified the ammonia oxidizing bacteria (AOB) species linked to N2 O emissions and evaluated the effects of urea and urea with DCD and DMPP on the nitrifying community in a 258 day field experiment under sugarcane. Using an amoA AOB amplicon sequencing approach and mining a previous dataset of 16S rRNA sequences, we characterized the most likely N2 O-producing AOB as a Nitrosospira spp. and identified Nitrosospira (AOB), Nitrososphaera (archaeal ammonia oxidizer) and Nitrospira (nitrite-oxidizer) as the most abundant, present nitrifiers. The fertilizer treatments had no effect on the alpha and beta diversities of the AOB communities. Interestingly, we found three clusters of co-varying variables with nitrifier operational taxonomic units (OTUs): the N2 O-producing AOB Nitrosospira with N2 O, NO3 - , NH4 + , water-filled pore space (WFPS) and pH; AOA Nitrososphaera with NO3 - , NH4 + and pH; and AOA Nitrososphaera and NOB Nitrospira with NH4 + , which suggests different drivers. These results support the co-occurrence of non-N2 O-producing Nitrososphaera and Nitrospira in the unfertilized soils and the promotion of N2 O-producing Nitrosospira under urea fertilization. Further, we suggest that DMPP is a more effective NI than DCD in tropical soil under sugarcane.

Long-term fertilization regimes change soil nitrification potential by impacting active autotrophic ammonia oxidizers and nitrite oxidizers as assessed by DNA stable isotope probing.

Chemoautotrophic ammonia-oxidizers and nitrite-oxidizers are responsible for a significant amount of soil nitrate production. The identity and composition of these active nitrifiers in soils under different long-term fertilization regimes remain largely under-investigated. Based on that soil nitrification potential significantly decreased in soils with chemical fertilization (CF) and increased in soils with organic fertilization (OF), a microcosm experiment with DNA stable isotope probing was further conducted to clarify the active nitrifiers. Both ammonia-oxidizing archaea (AOA) and bacteria (AOB) were found to actively respond to urea addition in soils with OF and no fertilizer (CK), whereas only AOB were detected in soils with CF. Around 98% of active AOB were Nitrosospira cluster 3a.1 in all tested soils, and more than 90% of active AOA were Nitrososphaera subcluster 1.1 in unfertilized and organically fertilized soils. Nitrite oxidation was performed only by Nitrospira-like bacteria in all soils. The relative abundances of Nitrospira lineage I and VI were 32% and 61%, respectively, in unfertilized soils, and that of Nitrospira lineage II was 97% in fertilized soils, indicating long-term fertilization shifted the composition of active Nitrospira-like bacteria in response to urea. This finding indicates that different fertilizer regimes impact the composition of active nitrifiers, thus, impacting soil nitrification potential.

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.

A patchwork pathway for oxygenase-independent degradation of side chain containing steroids.

The denitrifying betaproteobacterium Sterolibacterium denitrificans serves as model organism for studying the oxygen-independent degradation of cholesterol. Here, we demonstrate its capability of degrading various globally abundant side chain containing zoo-, phyto- and mycosterols. We provide the complete genome that empowered an integrated genomics/proteomics/metabolomics approach, accompanied by the characterization of a characteristic enzyme of steroid side chain degradation. The results indicate that individual molybdopterin-containing steroid dehydrogenases are involved in C25-hydroxylations of steroids with different isoprenoid side chains, followed by the unusual conversion to C26-oic acids. Side chain degradation to androsta-1,4-diene-3,17-dione (ADD) via aldolytic C-C bond cleavages involves acyl-CoA synthetases/dehydrogenases specific for the respective 26-, 24- and 22-oic acids/-oyl-CoAs and promiscuous MaoC-like enoyl-CoA hydratases, aldolases and aldehyde dehydrogenases. Degradation of rings A and B depends on gene products uniquely found in anaerobic steroid degraders, which after hydrolytic cleavage of ring A, again involves CoA-ester intermediates. The degradation of the remaining CD rings via hydrolytic cleavage appears to be highly similar in aerobic and anaerobic bacteria. Anaerobic cholesterol degradation employs a composite repertoire of more than 40 genes partially known from aerobic degradation in gammaproteobacteria/actinobacteria, supplemented by unique genes that are required to circumvent oxygenase-dependent reactions.

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.

Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization.

Molybdenum is an essential nutrient for metabolism in plant, bacteria, and animals. Molybdoenzymes are involved in nitrogen assimilation and oxidoreductive detoxification, and bioconversion reactions of environmental, industrial, and pharmaceutical interest. Molybdoenzymes contain a molybdenum cofactor (Moco), which is a pyranopterin heterocyclic compound that binds a molybdenum atom via a dithiolene group. Because Moco is a large and complex compound deeply buried within the protein, molybdoenzymes are accompanied by private chaperone proteins responsible for the cofactor's insertion into the enzyme and the enzyme's maturation. An efficient recombinant expression and purification of both Moco-free and Moco-containing molybdoenzymes and their chaperones is of paramount importance for fundamental and applied research related to molybdoenzymes. In this work, we focused on a D1 protein annotated as a chaperone of steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S. The D1 protein is presumably involved in the maturation of S25DH engaged in oxygen-independent oxidation of sterols. As this chaperone is thought to be a crucial element that ensures the insertion of Moco into the enzyme and consequently, proper folding of S25DH optimization of the chaperon's expression is the first step toward the development of recombinant expression and purification methods for S25DH. We have identified common E. coli strains and conditions for both expression and purification that allow us to selectively produce Moco-containing and Moco-free chaperones. We have also characterized the Moco-containing chaperone by EXAFS and HPLC analysis and identified conditions that stabilize both forms of the protein. The protocols presented here are efficient and result in protein quantities sufficient for biochemical studies.

Microbial community composition of a household sand filter used for arsenic, iron, and manganese removal from groundwater in Vietnam.

Household sand filters are used in rural areas of Vietnam to remove As, Fe, and Mn from groundwater for drinking water purposes. Currently, it is unknown what role microbial processes play in mineral oxide formation and As removal during water filtration. We performed most probable number counts to quantify the abundance of physiological groups of microorganisms capable of catalyzing Fe- and Mn-redox transformation processes in a household sand filter. We found up to 10(4) cells g(-1) dry sand of nitrate-reducing Fe(II)-oxidizing bacteria and Fe(III)-reducing bacteria, and no microaerophilic Fe(II)-oxidizing bacteria, but up to 10(6) cells g(-1) dry sand Mn-oxidizing bacteria. 16S rRNA gene amplicon sequencing confirmed MPN counts insofar as only low abundances of known taxa capable of performing Fe- and Mn-redox transformations were detected. Instead the microbial community on the sand filter was dominated by nitrifying microorganisms, e.g. Nitrospira, Nitrosomonadales, and an archaeal OTU affiliated to Candidatus Nitrososphaera. Quantitative PCR for Nitrospira and ammonia monooxygenase genes agreed with DNA sequencing results underlining the numerical importance of nitrifiers in the sand filter. Based on our analysis of the microbial community composition and previous studies on the solid phase chemistry of sand filters we conclude that abiotic Fe(II) oxidation processes prevail over biotic Fe(II) oxidation on the filter. Yet, Mn-oxidizing bacteria play an important role for Mn(II) oxidation and Mn(III/IV) oxide precipitation in a distinct layer of the sand filter. The formation of Mn(III/IV) oxides contributes to abiotic As(III) oxidation and immobilization of As(V) by sorption to Fe(III) (oxyhydr)oxides.

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)).

A novel method for RNA extraction from Andosols using casein and its application to amoA gene expression study in soil.

The lack of a universal method to extract RNA from soil hinders the progress of studies related to nitrification in soil, which is an important step in the nitrogen cycle. It is particularly difficult to extract RNA from certain types of soils such as Andosols (volcanic ash soils), which is the dominant agricultural soil in Japan, because of RNA adsorption by soil. To obtain RNA from these challenging soils to study the bacteria involved in nitrification, we developed a soil RNA extraction method for gene expression analysis. Autoclaved casein was added to an RNA extraction buffer to recover RNA from soil, and high-quality RNA was successfully extracted from eight types of agricultural soils that were significantly different in their physicochemical characteristics. To detect bacterial ammonia monooxygenase subunit A gene (amoA) transcripts, bacterial genomic DNA and messenger RNA were co-extracted from two different types of Andosols during incubation with ammonium sulfate. Polymerase chain reaction-denaturing gradient gel electrophoresis and reverse transcription polymerase chain reaction-denaturing gradient gel electrophoresis analyses of amoA in soil microcosms revealed that only few amoA, which had the highest similarities to those in Nitrosospira multiformis, were expressed in these soils after treatment with ammonium sulfate, although multiple amoA genes were present in the soil microcosms examined.

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.

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.

Community composition of ammonia-oxidizing bacteria and archaea in rice field soil as affected by nitrogen fertilization.

Little information is available on the ecology of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in flooded rice soils. Consequently, a microcosm experiment was conducted to determine the effect of nitrogen fertilizer on the composition of AOB and AOA communities in rice soil by using molecular analyses of ammonia monooxygenase gene (amoA) fragments. Experimental treatments included three levels of N (urea) fertilizer, i.e. 50, 100 and 150 mgNkg(-1) soil. Soil samples were operationally divided into four fractions: surface soil, bulk soil deep layer, rhizosphere and washed root material. NH(4)(+)-N was the dominant form of N in soil porewater and increased with N fertilization. Cloning and sequencing of amoA gene fragments showed that the AOB community in the rice soil consisted of three major groups, i.e. Nitrosomonas communis cluster, Nitrosospira cluster 3a and cluster 3b. The sequences related to Nitrosomonas were predominant. There was a clear effect of N fertilizer and soil depth on AOB community composition based on terminal restriction fragment length polymorphism fingerprinting. Nitrosomonas appeared to be more abundant in the potentially oxic or micro-oxic fractions, including surface soil, rhizosphere and washed root material, than the deep layer of anoxic bulk soil. Furthermore, Nitrosomonas increased relatively in the partially oxic fractions and that of Nitrosospira decreased with the increasing application of N fertilizer. However, AOA community composition remained unchanged according to the denaturing gradient gel electrophoresis analyses.

Molecular biological characterization and biostimulation of ammonia-oxidizing bacteria in brackishwater aquaculture.

In the present study, molecular methods based on sequencing of clone libraries have been used to provide sequence and the phylogenetic information of ammonia oxidizing bacteria (AOB). Ammonia monooxygenase (amoA) gene, which catalyzed the oxidation of ammonia to hydroxyl amine in the initial rate-determining step of nitrification was targeted for detection and characterization of AOB using gene-specific primers. The amoA genes obtained through the clone library construction are closely affiliated with Nitrosomonas sp. and other uncultured beta proteobacteria. The levels of nucleotide similarity and amino acid similarity ranged from 85-99% and 83-88%, respectively. The level of conservation of the amino acid sequences is 73%. The use of a matrix prepared from abundantly available lignocellulosic agrowaste-bagasse has successfully been demonstrated for biostimulation of AOB in aquaculture environment by supplementing nutritional requirement facilitating the biofilm mode of growth of the autotrophic consortia. Present study is useful in predictability and reliability of the treatment of ammonia in brackishwater aquaculture.

Nitrospira community composition in nitrifying reactors operated with two different dissolved oxygen levels.

Nitrospira is a dominant member of nitrite-oxidizing bacteria (NOB) in nitrifying bioreactors as well as in natural habitats. In this study, Nitrospira NOB were investigated in the two nitrifying reactors operated with high and low dissolved oxygen (DO) concentrations for a period of 300 days. Phylogenetic and terminal restriction fragment length polymorphism analyses based on 16S rRNA gene sequences revealed that the Nitrospira community compositions of the two reactors during the early period related to group 1 and half of the Nitrospira community composition shifted to group 2 in the high-DO reactor after day 179, although there was no significant change in the low-DO reactor. These results suggested that DO was an important factor affecting Nitrospira community compositions in the nitrifying reactors.

Use of quantitative real-time PCR to monitor population dynamics of ammonia-oxidizing bacteria in batch process.

A quantitative real-time PCR (QPCR) assay with the TaqMan system was used to quantify 16S rRNA genes of beta-proteobacterial ammonia-oxidizing bacteria (AOB) in a batch nitrification bioreactor. Five different sets of primers, together with a TaqMan probe, were used to quantify the 16S rRNA genes of beta-proteobacterial AOB belonging to the Nitrosomonas europaea, Nitrosococcus mobilis, Nitrosomonas nitrosa, and Nitrosomonas cryotolerans clusters, and the genus Nitrosospira. We also used PCR followed by denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing of their 16S rRNA genes to identify the AOB species. Seed sludge from an industrial wastewater treatment process controlling high-strength nitrogen wastewater (500 mg/L NH4+-N) was used as the inoculum for subsequent batch experiment. The Nitrosomonas nitrosa cluster was the predominant AOB (2.3x10(5) copies/mL) in the start-up period of the batch experiment. However, from the exponential growth period, the Nitrosomonas europaea cluster was the most abundant AOB, and its 16S rRNA gene copy number increased to 8.9x10(6) copies/mL. The competitive dominance between the two AOB clusters is consistent with observed differences in ammonia tolerance and substrate affinity. Analysis of the DGGE results indicated the presence of Nitrosomonas europaea ATCC19718 and Nitrosomonas nitrosa Nm90, consistent with the QPCR results.

Simultaneous effect of temperature, cyanide and ammonia-oxidizing bacteria concentrations on ammonia oxidation.

For biological nitrification, a set of experiments were carried out to approximate the response of lag period along with ammonia oxidation rate with respect to different concentrations of cyanide (CN-) and ammonia-oxidizing bacteria (AOB), and temperature variation in laboratory-scale batch reactors. The effects of simultaneous changes in these three factors on ammonia oxidation were quantitatively estimated and modeled using response surface analysis. The lag period and the ammonia oxidation rate responded differently to changes in the three factors. The lag period and the ammonia oxidation rate were significantly affected by the CN- and AOB concentrations, while temperature changes only affected the ammonia oxidation rate. The increase of AOB concentration and temperature alleviated the inhibition effect of cyanide on ammonia oxidation. The statistical method used in this study can be extended to estimate the quantitative effects of other environmental factors that can change simultaneously.

Complete genome sequence of Nitrosospira multiformis, an ammonia-oxidizing bacterium from the soil environment.

The complete genome of the ammonia-oxidizing bacterium Nitrosospira multiformis (ATCC 25196(T)) consists of a circular chromosome and three small plasmids totaling 3,234,309 bp and encoding 2,827 putative proteins. Of the 2,827 putative proteins, 2,026 proteins have predicted functions and 801 are without conserved functional domains, yet 747 of these have similarity to other predicted proteins in databases. Gene homologs from Nitrosomonas europaea and Nitrosomonas eutropha were the best match for 42% of the predicted genes in N. multiformis. The N. multiformis genome contains three nearly identical copies of amo and hao gene clusters as large repeats. The features of N. multiformis that distinguish it from N. europaea include the presence of gene clusters encoding urease and hydrogenase, a ribulose-bisphosphate carboxylase/oxygenase-encoding operon of distinctive structure and phylogeny, and a relatively small complement of genes related to Fe acquisition. Systems for synthesis of a pyoverdine-like siderophore and for acyl-homoserine lactone were unique to N. multiformis among the sequenced genomes of ammonia-oxidizing bacteria. Gene clusters encoding proteins associated with outer membrane and cell envelope functions, including transporters, porins, exopolysaccharide synthesis, capsule formation, and protein sorting/export, were abundant. Numerous sensory transduction and response regulator gene systems directed toward sensing of the extracellular environment are described. Gene clusters for glycogen, polyphosphate, and cyanophycin storage and utilization were identified, providing mechanisms for meeting energy requirements under substrate-limited conditions. The genome of N. multiformis encodes the core pathways for chemolithoautotrophy along with adaptations for surface growth and survival in soil environments.

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.