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.

Treatment of petrochemical wastewater by microaerobic hydrolysis and anoxic/oxic processes and analysis of bacterial diversity.

Microaerobic hydrolysis-acidification (MHA)-anoxic-oxic (A/O) processes were developed to treat actual petrochemical wastewater. The results showed that the overall COD removal efficiency was 72-79% at HRT=20h, and MHA accounted for 33-42% of COD removal, exhibiting good efficiency of acidogenic fermentation. Ammonium removal was more than 94%. The main pollutants in the influent were identified to be benzene, ketone, alcohols, amine, nitrile and phenols by GC-MS, and the majority of pollutants could be removed by MHA-A/O treatment. Proteobacteria was the most dominant bacteria in the system, accounting for more than 55% of the reads. The predominant genera in MHA, anoxic and oxic reactors were Anaerolineaceae and Sulfuritalea, Lactococcus and Blastocatella, and Saprospiraceae uncultured and Nitrosomonadaceae, respectively. This treatment system exhibited good performance in degrading the complex compounds in the petrochemical wastewater.

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.

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.

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.

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.

Effects of substratum on the diversity and stability of ammonia-oxidizing communities in a constructed wetland used for wastewater treatment.

AIM: To study the relationship between the nature of the substratum and the diversity and stability of the ammonia-oxidizing microbial community in a constructed wetland for the treatment of wastewaters. METHODS AND RESULTS: Samples have been taken the year around from sections of the wetland filled with different substrata. When present, the root zones of the helophyte Phragmites australis were also sampled. The diversity of the ammonia-oxidizing community was established by a coupled PCR-DGGE method based on the 16s rRNA gene. Averaged over the seasons, no large differences in community composition were observed between the different substrata, although the section with zeolite always showed the highest frequencies of bands belonging to ammonia-oxidizing bacteria of the beta-subclass of the Proteobacteria. Only sequences related to the Nitrosospira lineage were detected. Averaged again over the seasons, the section with zeolite was also most constant with respect to the potential ammonia-oxidizing activity. CONCLUSIONS: Although the ammonia-oxidizing communities did not differ significantly between the different sections of the constructed wetland, the characteristics of zeolite were most appropriate to accommodate a stable and active community of ammonia-oxidizing bacteria. The presence of the helophyte had no effect on the diversity and stability of the ammonia-oxidizing community. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been shown that substrata used in constructed wetlands made no distinction between ammonia-oxidizing strains in relation to attachment. However, zeolite had the best performance with respect to activity over the seasons.

Diversity of ammonia oxidising bacteria in a vertical flow constructed wetland.

Vertical flow constructed wetlands (VFCWs) with intermittent loading are very suitable for nitrification. Ammonia oxidising bacteria (AOB) are the limiting step of nitration. Therefore the AOB community of a full-scale VFCW, receiving municipal wastewater, was investigated within this study. The diversity of the functional gene encoding the alpha-subunit of the ammonia monooxygenase (amoA), present only in AOB, was assessed by denaturing gradient gel electrophoresis (DGGE). Only very few amoA sequence types dominated the wetland filter substrate; nevertheless a stable nitrification performance could be observed. During the cold season the nitrification was slightly reduced, but it has been shown that the same AOB could be identified. No spatial AOB pattern could be observed within the filter body of the VFCW. The most prominent bands were excised from DGGE gels and sequenced. Sequence analyses revealed two dominant AOB lineages: Nitrosomonas europaea/"Nitrosococcus mobilis" and Nitrosospira. Species of the Nitrosomonas lineage are commonly found in conventional wastewater treatment plants (WWTPs). In contrast, members of the Nitrosospira lineage are rarely present in WWTPs. Our observations indicate that the AOB community in this VFCW is similar to that found in horizontal flow constructed wetlands, but differs from common WWTPs regarding the presence of Nitrosospira.

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.

Identification of nitrifiers in a full-scale biological treatment system using fluorescent in situ hybridization.

Diversity of nitrifying bacterial population was investigated in sludge samples taken from a full-scale biological wastewater treatment plant (WWTP) treating domestic wastewater by fluorescent in situ hybridization (FISH) during seasonal operation. Duplicate grab samples were collected in March 2003, June 2003, December 2003 and May 2004 from the aerobic tank of the treatment plant. FISH results were interpreted with system performance in terms of BOD5, TKN and NO3-N removals and also with operational parameters such as wastewater temperature and sludge age. BOD5 removal efficiencies were always greater than 90% whilst TKN removal in a range of 69-95% were achieved during the monitoring period. Although there were variations in operational conditions of the biological treatment system both Nitrosomonas and Nitrosospira genera from AOB and Nitrobacter genus from NOB were found to be present in all samples examined.

Diversity study of nitrifying bacteria in full-scale municipal wastewater treatment plants.

We hypothesize that activated-sludge processes having stable and complete nitrification have significant and similar diversity and functional redundancy among its ammonia- and nitrite-oxidizing bacteria, despite differences in temperature, solids retention time (SRT), and other operating conditions. To evaluate this hypothesis, we examined the diversity of nitrifying bacterial communities in all seven water-reclamation plants (WRPs) operated by Metropolitan Water Reclamation District of Greater Chicago (MWRDGC). These plants vary in types of influent waste stream, plant size, water temperature, and SRT. We used terminal restriction fragment length polymorphism (T-RFLP) targeting the 16S rRNA gene and group-specific ammonia-monooxygenase functional gene (amoA) to investigate these hard-to-culture nitrifying bacteria in the full-scale WRPs. We demonstrate that nitrifying bacteria carrying out the same metabolism coexist in all WRPs studied. We found ammonia-oxidizing bacteria (AOB) belonging to the Nitrosomonas europaea/eutropha, Nitrosomonas oligotropha, Nitrosomonas communis, and Nitrosospira lineages in all plants. We also observed coexisting Nitrobacter and Nitrospira genera for nitrite-oxidizing bacteria (NOB). Among the factors that varied among the WRPs, only the seasonal temperature variation seemed to change the nitrifying community, especially the balance between Nitrosospira and Nitrosomonas, although both coexisted in winter and summer samples. The coexistence of various nitrifiers in all WRPs is evidence of functional redundancy, a feature that may help maintain the stability of the system for nitrification.

Phylogeny of nitrite reductase (nirK) and nitric oxide reductase (norB) genes from Nitrosospira species isolated from soil.

Ammonia-oxidizing bacteria are believed to be an important source of the climatically important trace gas nitrous oxide (N(2)O). The genes for nitrite reductase (nirK) and nitric oxide reductase (norB), putatively responsible for nitrous oxide production, have been identified in several ammonia-oxidizing bacteria, but not in Nitrosospira strains that may dominate ammonia-oxidizing communities in soil. In this study, sequences from nirK and norB genes were detected in several cultured Nitrosospira species and the diversity and phylogeny of these genes were compared with those in other ammoniaoxidizing bacteria and in classical denitrifiers. The nirK and norB gene sequences obtained from Nitrosospira spp. were diverse and appeared to be less conserved than 16S rRNA genes and functional ammonia monooxygenase (amoA) genes. The nirK and norB genes from some Nitrosospira spp. were not phylogenetically distinct from those of denitrifiers, and phylogenetic analysis suggests that the nirK and norB genes in ammonia-oxidizing bacteria have been subject to lateral transfer.

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.

Emergence of competitive dominant ammonia-oxidizing bacterial populations in a full-scale industrial wastewater treatment plant.

Ammonia-oxidizing bacterial populations in an industrial wastewater treatment plant were investigated with amoA and 16S rRNA gene real-time PCR assays. Nitrosomonas nitrosa initially dominated, but over time RI-27-type ammonia oxidizers, also within the Nitrosomonas communis lineage, increased from below detection to codominance. This shift occurred even though nitrification remained constant.

Removal of carbonaceous and nitrogenous pollutants from a synthetic wastewater using a membrane-coupled bioreactor.

Two modified Ludzack-Ettinger (MLE)-type membrane-coupled bioreactors (MBRs) were investigated in this study for the purpose of removing both nitrogenous and carbonaceous pollutants from a synthetic wastewater. During the first MBR experiment, removal efficiencies were high (>90%) for chemical oxygen demand (COD) and ammonia, but total nitrogenous pollutant removal efficiency was poor (approximately 25%). Bacterial community analysis of ammonia oxidizing bacteria (AOB) by a nested PCR-DGGE approach detected two Nitrosomonas-like populations and one Nitrosospira-like population. During the initial portion of the second MBR experiment, COD and ammonia removal efficiencies were similar to the first MBR experiment until the COD of the influent wastewater was increased to provide additional electron donors to support denitrification. Total nitrogen removal efficiencies eventually exceeded 90%, with a hydraulic residence time (HRT) of 24 h and a recirculation ratio of 8. When the HRT of the MBR experiment was decreased to 12 h, however, ammonia removal efficiency was adversely affected. A subsequent increase in the HRT to 18 h helped improve removal efficiencies for both ammonia (>85%) and total nitrogenous compounds (approximately 70%). Our research demonstrates that MBRs can be effectively designed to remove both carbonaceous and nitrogenous pollutants. The ability of the microbial community to switch between anoxic (denitrifying) and oxic (nitrifying) conditions, however, represents a critical process constraint for the application of MLE-type MBR systems, such that little benefit is gained compared to conventional designs.

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.