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.

Insights into the effect of mixed engineered nanoparticles on activated sludge performance.

In this study, the effects, fate and transport of ENPs in wastewater treatment plants (WWTP) were investigated using three parallel pilot WWTPs operated under identical conditions. The WWTPs were spiked with (i) an ENP mixture consisting of silver oxide, titanium dioxide and zinc oxide, and (ii) bulk metal salts. The third plant served as control (unspiked). ENP effects were evaluated for (i) bulk contaminant removal, (ii) activated sludge (AS) process performance, (iii) microbial community structure and dynamics and (iv) microbial inhibition. ENPs showed a strong affinity for biosolids and induced a specific oxygen uptake rate two times higher than the control. The heterotrophic biomass retained its ability to nitrify and degrade organic matter. However, non-recovery of ammonia- and nitrite-oxidizing bacteria such as Nitrosomonas, Nitrobacter or Nitrospira in the ENP spiked reactors suggests selective inhibitory effects. The results further suggest that ENPs and metal salts have antimicrobial properties which can reduce synthesis of extracellular polymeric substances and therefore floc formation. Scanning electron microscopy evidenced selective damage to some microbes, whereas lipid fingerprinting and 454 pyrosequencing indicated a temporal shift in the microbial community structure and diversity. Acidovorax, Rhodoferax, Comamonas and Methanosarcina were identified as nano-tolerant species. Competitive growth advantage of the nano-tolerant species influenced the removal processes and unlike other xenobiotic compounds, ENPs can hasten the natural selection of microbial species in AS.

Performance evaluation and bacterial characterization of membrane bioreactors.

A bench-scale conventional membrane bioreactor (C-MBR), a moving bed membrane bioreactor (MB-MBR) and an anoxic/oxic membrane bioreactor (A/O-MBR), operating under similar feed, environmental and operating conditions, were each evaluated for their treatment performance and bacterial diversity. MBRs were compared for the removal of organics (COD) and nutrients (N and P) while pure culture techniques were employed for bacterial isolation and an API 20E kit was used to identify the isolates. Pseudomonas aeruginosa, selected as a representative of denitrifying microorganisms, was isolated only from the A/O-MBR using Citrimide Agar. Using PCR, the nitrifying bacteria Nitrosomonas europaea was detected only in the MB-MBR. On the other hand, Nitrobacter winogradskyi was detected in all three reactors. Addition of media and maintenance of a lesser DO resulted in the highest TN removal in the A/O-MBR as compared to the C-MBR and the MB-MBR, whereas better nitrification was observed in the MB-MBR than in the C-MBR.

Crude oil-degradation and plasmid profile of nitrifying bacteria isolated from oil-impacted mangrove sediment in the Niger Delta of Nigeria.

The crude oil degradability and plasmid profile of autotrophic nitrifying bacteria, Nitrosomonas and Nitrobacter species, isolated from mangrove sediment in the Niger Delta of Nigeria were studied. The effects of temperature, pH and optical density on the utilization of different carbon sources by the bacteria were also investigated. Results showed that nitrifying bacteria could utilize kerosene, diesel oil, jet fuel and engine oil as carbon sources. None utilized hexane and xylene but moderate growth was observed in benzene, phenol and toluene. However, their ability to utilized crude oil varied both in rates of utilization and in growth profiles. Mixed culture of the isolates degrades 52 % of crude oil introduced into the medium followed by Nitrosomonas sp. with 40 % degradation. The least was Nitrobacter sp. with 20 % degradation. The ability of the autotrophs to degrade crude oil was found to be plasmid-mediated through curing experiment and electrophoresis. The size of the plasmid involved was estimated to be 23 kb. The high crude oil utilization of the mixed culture implies that nitrifying bacteria isolated from contaminated ecosystem are excellent crude oil degraders and can be harnessed for bioremediation purposes.

Cultivation, growth physiology, and chemotaxonomy of nitrite-oxidizing bacteria.

Lithoautotrophic nitrite-oxidizing bacteria (NOB) are known as fastidious microorganisms, which are hard to maintain and not many groups are trained to keep them in culture. They convert nitrite stoichiometrically to nitrate and growth is slow due to the poor energy balance. NOB are comprised of five genera, which are scattered among the phylogenetic tree. Because NOB proliferate in a broad range of environmental conditions (terrestrial, marine, acidic) and have diverse lifestyles (lithoautotrophic, mixotrophic, and heterotrophic), variation in media composition is necessary to match their individual growth requirements in the laboratory. From Nitrobacter and Nitrococcus relatively high cell amounts can be achieved by consumption of high nitrite concentrations, whereas accumulation of cells belonging to Nitrospira, Nitrospina, or the new candidate genus Nitrotoga needs prolonged feeding procedures. Isolation is possible for planktonic cells by dilution series or plating techniques, but gets complicated for strains with a tendency to develop microcolonies like Nitrospira. Physiological experiments including determination of the temperature or pH-optimum can be conducted with active laboratory cultures of NOB, but the attainment of reference values like cell protein content or cell numbers might be hard to realize due to the formation of flocs and the low cell density. Monitoring of laboratory enrichments is necessary especially if several species or genera coexist within the same culture and due to population shifts over time. Chemotaxonomy is a valuable method to identify and quantify NOB in biofilms and pure cultures alike, since fatty acid profiles reflect their phylogenetic heterogeneity. This chapter focusses on methods to enrich, isolate, and characterize NOB by various cultivation-based techniques.

Seasonal variations of nitrifying community in trickling filter-solids contact (TF/SC) activated sludge systems.

Two full-scale trickling filter/solids contact (TF/SC) basin plants, each successfully performing nitrification, were sampled throughout various seasons over a period of one year. Concentrations of ammonia, nitrate and nitrite were measured at various sampling locations along the treatment train. DNA was also extracted from mixed liquor in the solids contact basins. These DNA samples were subjected to terminal restriction fragment length polymorphism (TRFLP) in order to profile the ammonia oxidizing bacteria and nitrite oxidizing bacteria communities. In both plants, there was a prevalence of Nitrosomonas europaea among the ammonia oxidizing bacteria (AOBs). However, during the summer months, there was increased diversity of Nitrosomonas species. Likewise, Nitrospira spp. was the dominant nitrite oxidizing bacteria (NOBs) in both plants regardless of season. Yet there was an increased presence of Nitrobacter among the NOBs in the summer months. These results add an important understanding of the ecology and dynamics in nitrifying population in full-scale TF/SC wastewater treatment plants.

Greenhouse gas microbiology in wet and dry straw crust covering pig slurry.

Liquid manure (slurry) storages are sources of gases such as ammonia (NH(3)) and methane (CH(4)). Danish slurry storages are required to be covered to reduce NH(3) emissions and often a floating crust of straw is applied. This study investigated whether physical properties of the crust or crust microbiology had an effect on the emission of the potent greenhouse gases CH(4) and nitrous oxide (N(2)O) when crust moisture was manipulated ("dry", "moderate", and "wet"). The dry crust had the deepest oxygen penetration (45 mm as compared to 20 mm in the wet treatment) as measured with microsensors, the highest amounts of nitrogen oxides (NO(2)(-) and NO(3)(-)) (up to 36 mumol g(-1) wet weight) and the highest emissions of N(2)O and CH(4). Fluorescent in situ hybridization and gene-specific polymerase chain reaction (PCR) were used to detect occurrence of bacterial groups. Ammonia-oxidizing bacteria (AOB) were abundant in all three crust types, whereas nitrite-oxidizing bacteria (NOB) were undetectable and methane-oxidizing bacteria (MOB) were only sparsely present in the wet treatment. A change to anoxia did not affect the CH(4) emission indicating the virtual absence of aerobic methane oxidation in the investigated 2-mo old crusts. However, an increase in N(2)O emission was observed in all crusted treatments exposed to anoxia, and this was probably a result of denitrification based on NO(x)(-) that had accumulated in the crust during oxic conditions. To reduce overall greenhouse gas emissions, floating crust should be managed to optimize conditions for methanotrophs.

Nitrifying community structures and nitrification performance of full-scale municipal and swine wastewater treatment plants.

This study evaluated nitrification performance and microbial ecology of nitrifying sludge in two full-scale wastewater treatment plants (WWTPs) including a municipal WWTP treating 20mgNL(-1) of ammonium and a swine WWTP treating 220mgNL(-1) of ammonium. These two plants differed in both wastewater characteristics and operating parameters, such as influent COD, TKN, ammonium, hydraulic retention time, and solids retention time, even though both plants achieve >85% nitrification efficiency. By employing molecular techniques, including terminal restriction fragment length polymorphism, cloning-sequencing and phylogenetic analyses targeting the 16S ribosomal RNA and group specific ammonia-monooxygenase functional gene (amoA), microbial community structures of nitrifying sludge and their significance to nitrification performance were evaluated. The results reveal that for the municipal WWTP Nitrosomonas marina-like AOB (ammonia-oxidizing bacteria) and Nitrospira-like NOB (nitrite-oxidizing bacteria) were the ubiquitously dominant nitrifiers, while Nitrosomonas europaea-, Nitrosomonas oligotropha-, and Nitrosospira-like AOB and Nitrobacter- and Nitrospira-like NOB were the major nitrifying populations found in the swine WWTP. The observed dissimilar nitrifying populations prevailing in these two plants may be related to niche differentiation concerning ammonium concentrations, system operation, and salinity. Moreover, our results suggest that the swine nitrifying sludge, involving relatively diverse AOB and NOB populations that perform the same task but with distinct growth and survival characters, may allow communities to maintain nitrifying capabilities when conditions change such as sudden increases in ammonium concentrations as examined with nitrification kinetic batch tests.

Investigation of the microbial community structure and activity as indicators of compost stability and composting process evolution.

In a bid to identify suitable microbial indicators of compost stability, the process evolution during windrow composting of poultry manure (PM), green waste (GW) and biowaste was studied. Treatments were monitored with regard to abiotic factors, respiration activity (determined using the SOUR test) and functional microflora. The composting process went through typical changes in temperature, moisture content and microbial properties, despite the inherent feedstock differences. Nitrobacter and pathogen indicators varied as a monotonous function of processing time. Some microbial groups have shown a potential to serve as fingerprints of the different process stages, but still they should be examined in context with respirometric tests and abiotic parameters. Respiration activity reflected well the process stage, verifying the value of respirometric tests to access compost stability. SOUR values below 1 mg O(2)/g VS/h were achieved for the PM and the GW compost.

Molecular characterization of microbial populations in groundwater sources and sand filters for drinking water production.

In full-scale drinking water production from groundwater, subsurface aeration is an effective means of enhancing the often troublesome process of nitrification. Until now the exact mechanism, however, has been unknown. By studying the microbial population we can improve the understanding of this process. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments of bacteria, archaea and ammonia-oxidizing bacteria was used to characterize the microbial populations in raw groundwater and trickling filters of an active nitrifying surface aerated system and an inactive non-surface aerated system. Only in the active filter were nitrifying microorganisms found above the detection limit of the method. In ammonia oxidation in this groundwater filter both bacteria and archaea played a role, while members belonging to the genus Nitrospira were the only nitrite-oxidizing species found. The subsurface aerated groundwater did not contain any of the nitrifying organisms active in the filter above the detection limit, but did contain Gallionella species that might play a major role in iron oxidation in the filter.

Sustainable nitrogen removal from wastewater with the hybrid membrane biofilm process (HMBP): bench-scale studies.

The hybrid membrane biofilm process (HMBP) is a novel approach to achieving total nitrogen removal from wastewater. It incorporates air-supplying, hollow-fiber membranes into a conventional activated sludge tank, where bulk aeration is suppressed. A nitrifying biofilm grows on the membranes and exports nitrite and nitrate to the anoxic bulk liquid, where suspended heterotrophic bacteria denitrify using influent BOD as an electron donor. Bench scale tests were used to assess the performance of the HMBP for a variety of nitrogen and BOD loading rates. For a nitrogen loading of 1.6 gN m(-2), the nitrification flux remained at approximately 1.0 gN m(-2) d(-1) for BOD loadings ranging from 4 to 17 gBOD m(-2) d(-1) day(-1). Full denitrification was achieved when sufficient BOD was available in the influent. Microsensor measurements indicated nitrite was the dominant form of oxidized nitrogen produced by the biofilm, showing that shortcut nitrogen removal was taking place. Fluorescence in-situ hybridization (FISH) tests on the biofilm revealed a unique stratification, with three distinct regions: AOB and NOB near the membrane, strictly AOB at intermediate depths, and AOB and heterotrophs at the outer edge of the biofilm.

Application of high rate nitrifying trickling filters for potable water treatment.

The interference of ammonia with chlorination is a prevalent problem encountered by water treatment plants located throughout South East Asia. The efficacy of high rate, plastic-packed trickling filters as a pre-treatment process to remove low concentrations of ammonia from polluted surface water was investigated. This paper presents the findings from a series of pilot experiments, which were designed to investigate the effect of specific conditions-namely low ammonia feed concentrations (0.5-5.0 mg NH(4)-NL(-1)), variations in hydraulic surface load (72.5-145 m(3)m(-2)d(-1)) and high suspended solid loads (51+/-25 mgL(-1))-on filter nitrifying capacity. The distribution of nitrification activity throughout a trickling filter bed was also characterised. Results confirmed that high hydraulic rate trickling filters were able to operate successfully, under ammonia-N concentrations some 10- to 50-fold lower and at hydraulic loading rates 30-100 times greater than those of conventional wastewater applications. Mass transport limitations posed by low ammonia-N concentrations on overall filter performance were insignificant, where apparent nitrification rates (0.4-1.6 g NH(4)-Nm(-2)d(-1)), equivalent to that of wastewater filters were recorded. High inert suspended solid loadings had no adverse effect on nitrification. Results imply that implementation of high rate trickling filters at the front-end of a water treatment train would reduce the ammonia-related chlorine demand, thereby offering significant cost savings.

[Characteristic and prospects of heterotrophic nitrification].

Nitrification is a key step in the global nitrogen cycle. Classically, nitrifying bacteria are chemoautotroph. Recently, bacteria that have the ability of heterotrophic nitrification have been intensively studied as potential microorganisms that may be used to overcomeproblems inherent in the conventional method. This review gives a broad overview of the current status of heterotrophic nitrification including the heterotrophic species which nitrify actively, optimal condition for nitrification, heterotrophic nitrification pathway, enzymatic system and isolation methods. Also, the promising prospects of heterotrophic nitrification are especially introduced.

Distribution of Nitrosomonas europaea and Nitrobacter winogradskyi in an autotrophic nitrifying biofilm reactor as depicted by molecular analyses and mathematical modelling.

The autotrophic two-species biofilm from the packed bed reactor of a life-support system, containing Nitrosomonas europaea ATCC 19718 and Nitrobacter winogradskyi ATCC 25391, was analysed after 4.8 years of continuous operation performing complete nitrification. Real-time quantitative polymerase chain reaction (Q-PCR) was used to quantify N. europaea and N. winogradskyi along the vertical axis of the reactor, revealing a spatial segregation of N. europaea and N. winogradskyi. The main parameters influencing the spatial segregation of both nitrifiers along the bed were assessed through a multi-species one-dimensional biofilm model generated with AQUASIM software. The factor that contributed the most to this distribution profile was a small deviation from the flow pattern of a perfectly mixed tank towards plug-flow. The results indicate that the model can estimate the impact of specific biofilm parameters and predict the nitrification efficiency and population dynamics of a multispecies biofilm.

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.

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.

[Isolation and characterization of heterotrophic nitrifying bacteria].

Method of isolating the heterotrophic nitrifiers and the characterization of the heterotrophic nitrification were studied. Two heterotrophic nitrifiers were newly isolated from a membrane bioreactor (MBR) in which the TN removal efficiency was 80.1%. The batch test results indicate that Bacillus sp. LY and Brevibacillus sp. LY could utilize the organic carbon as the source of assimilation when they grew on glucose and ammonium chloride medium companying the formation of oxidized-nitrogen. After 24 days incubation, the removal efficiencies of the COD by Bacillus sp. LY and Brevibacillus sp. LY were 71.7% and 52.6%, respectively. The removal efficiencies of ammonium nitrogen by the two isolates were 78.2% and 51.2% and the TN removal efficiencies by the two isolates were 61.2% and 35.6%, respectively.

Influence of inorganic nitrogen management regime on the diversity of nitrite-oxidizing bacteria in agricultural grassland soils.

To assess links between the diversity of nitrite-oxidizing bacteria (NOB) in agricultural grassland soils and inorganic N fertilizer management, NOB communities in fertilized and unfertilized soils were characterized by analysis of clone libraries and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Previously uncharacterized Nitrospira-like sequences were isolated from both long-term-fertilized and unfertilized soils, but DGGE migration patterns indicated the presence of additional sequence types in the fertilized soils. Detailed phylogenetic analysis of Nitrospira-like sequences suggests the existence of one newly described evolutionary group and of subclusters within previously described sublineages, potentially representing different ecotypes; the new group may represent a lineage of noncharacterized Nitrospira species. Clone libraries of Nitrobacter-like sequences generated from soils under different long-term N management regimes were dominated by sequences with high similarity to the rhizoplane isolate Nitrobacter sp. strain PJN1. However, the diversity of Nitrobacter communities did not differ significantly between the two soil types. This is the first cultivation-independent study of nitrite-oxidizing bacteria in soil demonstrating that nitrogen management practices influence the diversity of this bacterial functional group.