Nitrobacter Hamburgensis Research Articles

Microplastics drive nitrification by enriching functional microorganisms in aquaculture pond waters

The plastisphere refers to biofilm formation on the microplastic (MP) surface, but its subsequent functions, especially driving the nitrogen biogeochemical cycle, are rarely studied. Here, MPs were incubated in the pelagic water and benthic water-sediment interface of an aquaculture pond, and the two corresponding microcosms amended with incubated plastisphere were simulated. The results showed decreased ammonia concentrations and increased nitrification rates in microcosms with either pelagic or benthic plastispheres. To uncover the possible mechanisms, the community structure and function of the plastisphere were investigated. As clarified by 16S rRNA, the community diversity of the pelagic plastisphere was significantly higher than that of the corresponding hydrosphere. Plastisphere communities, especially those incubated in pelagic water, were separated from the hydrosphere. Moreover, the abundance of Proteobacteria increased while the abundance of Cyanobacteria decreased in both plastispheres. Metagenome further revealed that the abundance of amoA and annotated Nitrososphaeraceae_archaeon and hao and affiliated Nitrosomonas_europaea, which contributed to ammonia oxidation to nitrite, was higher in the benthic plastisphere. Comparing the pelagic plastisphere with the corresponding hydrosphere, however, the abundance of nxrA and annotated Nitrobacter hamburgensis and nxrB and the affiliated Nitrospira moscoviensis, which are involved in nitrite oxidation, was more abundant in the plastisphere. These findings suggest that the plastisphere might selectively enrich functional microorganisms and genes in a habitat-dependent manner to promote nitrification in aquaculture ponds.

Analysis of the Bacterial Biocenosis of Activated Sludge Treated with Leachate from Municipal Landfills.

The influx of toxic pollutants into wastewater treatment plants can negatively affect the quality of the activated sludge (AS). One source is landfill leachate. The identification of microorganisms present in AS is very important, e.g., while improving wastewater treatment technology. Therefore, the aim of the study was to investigate the effect of raw leachate and after purification of Phragmites australis and Ceratophyllum demersum on the composition of the AS bacterial biocenosis. In addition, AS status was assessed by LIVE/DEAD BacLight ™ fluorescent staining. The obtained results showed that the leachate did not significantly affect the cell membranes of AS bacteria, and even a slight improvement was noted. The research carried out using the next-generation sequencing method shows that the origin of the samples (active and closed storage) and the method of processing do not significantly affect the composition of the AS bacterial biocenosis at higher taxonomic levels. However, at the species level, the appearance of bacteria not previously present in AS was observed, namely: Flavobacterium luticocti, Candidimonas nitroreducens and Nitrobacter hamburgensis. The obtained results suggest that the leachate may be a source of microorganisms positively influencing the condition of AS bacteria.

Temperature affects the oxidation kinetics of co-cultured Nitrobacter winogradskyi and Nitrobacter hamburgensis in nitrite-oxidizing bioreactor

Nitrobacter is an important chemolithoautotrophic nitrite-oxidizing bacteria and has exhibited potential for the oxidation of nitrite to nitrate in natural and engineered ecosystems. Microbial kinetics of Nitrobacter have been widely reported and are used to model the nitrite oxidation process and optimize the design parameters of the biological processes for wastewater treatment. However, limited information is available about the nitrite oxidation kinetics of the two coexisting Nitrobacter stains, namely the Nitrobacter winogradskyi and the Nitrobacter hamburgensis. The current work mainly focuses on the influence of temperature on the kinetic characteristics of the coexistence of N. winogradskyi and hamburgensis, which were enriched in a lab-scale sequencing batch reactor (SBR) through 419 operating cycles. The results for the kinetic properties of nitrite oxidation of the enrichments within the temperature range of 15–35 °C showed that the maximum specific rate (rmax), half-saturation coefficients (KS), growth-rate constants (μmax), and temperature coefficient (θ) lied within the ranges of 22.91–93.58 mM/g/d, 0.13–2.47 mM, 0.026–0.105 1/d, and 1.038–1.137, respectively. The maximum activity and nitrite affinities of tested microorganisms were sensitive to temperature. Furthermore, the relationships between temperature and normalized kinetic parameters (rmax, μmax, and θ) were developed as: 0.069T − 0.076 (15–30 °C), 0.0057T − 0.062 (15–30 °C), and 5.7T40.9+T+T210.5 (15–35 °C), respectively. Moreover, the capabilities of N. winogradskyi and N. hamburgensis to oxidize nitrite were lost at the temperature of 35 °C and were non-recoverable at temperature of 25 °C. Overall, the findings of the present work can provide available data for future study about the mathematic models of nitrification kinetics and ecological niche differentiation of Nitrobacter species.

Response of activity and community composition of nitrite-oxidizing bacteria to partial substitution of chemical fertilizer by organic fertilizer.

Nitrite oxidation as the second step of nitrification can become the determining step in disturbed soil systems. As a beneficial fertilization practice to maintain high crop yield and soil fertility, partial substitution of chemical fertilizer (CF) by organic fertilizer (OF) may exert a notable disturbance to soil systems. However, how nitrite oxidation responds to different proportions of CF to OF is still unclear. We sampled soils from a 4-year field experiment subject to a gradient of increasing proportions of OF to CF application. Activity, size, and structure of Nitrospira-like and Nitrobacter-like nitrite-oxidizing bacteria (NOB) community were measured. The results revealed that with increasing proportion of OF to CF application, potential nitrite oxidation activity (PNO) showed a marked decreasing trend. PNO was significantly correlated with the abundance of Nitrobacter-like but not Nitrospira-like NOB. The abundance of Nitrobacter-like was significantly influenced by soil organic matter, organic nitrogen (N), and available N. In addition, PNO was also affected by the structure of Nitrobacter-like NOB. The relative abundance of Nitrobacter hamburgensis, alkalicus, winogradskyi, and vulgaris responded differently to the proportions of OF to CF application. Organic N, organic matter, and available N were the main factor shaping their community structure. Overall, Nitrobacter-like NOB is more sensitive and plays a more important role than Nitrospira-like NOB in responding to different proportions of OF to CF application.

Biological inhibition of soil nitrification by forest tree species affects Nitrobacter populations.

Some temperate tree species are associated with very low soil nitrification rates, with important implications for forest N dynamics, presumably due to their potential for biological nitrification inhibition (BNI). However, evidence for BNI in forest ecosystems is scarce so far and the nitrifier groups controlled by BNI-tree species have not been identified. Here, we evaluated how some tree species can control soil nitrification by providing direct evidence of BNI and identifying the nitrifier group(s) affected. First, by comparing 28 year-old monocultures of several tree species, we showed that nitrification rates correlated strongly with the abundance of the nitrite oxidizers Nitrobacter (50- to 1000-fold changes between tree monocultures) and only weakly with the abundance of ammonia oxidizing archaea (AOA). Second, using reciprocal transplantation of soil cores between low and high nitrification stands, we demonstrated that nitrification changed 16 months after transplantation and was correlated with changes in the abundance of Nitrobacter, not AOA. Third, extracts of litter or soil collected from the low nitrification stands of Picea abies and Abies nordmanniana inhibited the growth of Nitrobacter hamburgensis X14. Our results provide for the first time direct evidence of BNI by tree species directly affecting the abundance of Nitrobacter.

Contrasting the microbiomes from forest rhizosphere and deeper bulk soil from an Amazon rainforest reserve

Pristine forest ecosystems provide a unique perspective for the study of plant-associated microbiota since they host a great microbial diversity. Although the Amazon forest is one of the hotspots of biodiversity around the world, few metagenomic studies described its microbial community diversity thus far. Understanding the environmental factors that can cause shifts in microbial profiles is key to improving soil health and biogeochemical cycles. Here we report a taxonomic and functional characterization of the microbiome from the rhizosphere of Brosimum guianense (Snakewood), a native tree, and bulk soil samples from a pristine Brazilian Amazon forest reserve (Cuniã), for the first time by the shotgun approach. We identified several fungi and bacteria taxon significantly enriched in forest rhizosphere compared to bulk soil samples. For archaea, the trend was the opposite, with many archaeal phylum and families being considerably more enriched in bulk soil compared to forest rhizosphere. Several fungal and bacterial decomposers like Postia placenta and Catenulispora acidiphila which help maintain healthy forest ecosystems were found enriched in our samples. Other bacterial species involved in nitrogen (Nitrobacter hamburgensis and Rhodopseudomonas palustris) and carbon cycling (Oligotropha carboxidovorans) were overrepresented in our samples indicating the importance of these metabolic pathways for the Amazon rainforest reserve soil health. Hierarchical clustering based on taxonomic similar microbial profiles grouped the forest rhizosphere samples in a distinct clade separated from bulk soil samples. Principal coordinate analysis of our samples with publicly available metagenomes from the Amazon region showed grouping into specific rhizosphere and bulk soil clusters, further indicating distinct microbial community profiles. In this work, we reported significant shifts in microbial community structure between forest rhizosphere and bulk soil samples from an Amazon forest reserve that are probably caused by more than one environmental factors such as rhizosphere and soil depth.

Microbial Community Structure and Distribution of Nitrite-Oxidizing Bacteria in CANON System Operated at Ambient Temperature

The existence of nitrite-oxidizing bacteria (NOB) is regarded as one of the main problems in completely autotrophic nitrogen removal over nitrite (CANON) process. In this study, the microbial community structure and distribution of NOB in a lab-scale CANON reactors were investigated at different dissolved oxygen (DO) levels at ambient temperature. Results indicated a distinct community shift of NOB when DO increased from 0.5 mg/L to 1.0 mg/L. Phylogenetic results indicated the predominant NOB were Nitrobacter hamburgensis, Nitrobacter alkalicus and Nitrobacter winogradskyi in the system, all of which coexisted with aerobic ammonium-oxidizing bacteria (AerAOB) and anaerobic ammonium-oxidizing bacteria (AnAOB) without a distinguishable niche on the biofilm.

Nitrospira are more sensitive than Nitrobacter to land management in acid, fertilized soils of a rapeseed-rice rotation field trial

Nitrite oxidation is recognized as an essential process of biogeochemical nitrogen cycling in agricultural ecosystems. How nitrite-oxidizing bacteria (NOB) respond to land managements (the effect from the long-term straw incorporation and environmental variability caused by the shift from the upland stage to the paddy stage) in a rapeseed-rice rotation field remains unclear. We found the nitrite oxidation (NO) in soils increased from the upland stage to the paddy stage. An inhibitory effect of the long-term straw incorporation on NO was detectable in the upland stage. The abundance of Nitrospira was always greater than Nitrobacter, and it was affected by the rice-growing and straw incorporation while Nitrobacter was not. NO correlated positively with the abundance of Nitrospira and with soluble sulfate (SO42−), soil moisture, pH and NH4+. The high-throughput sequencing analysis of the nitrite oxidoreductase nxrA and nxrB genes for Nitrobacter- and Nitrospira-like NOB was performed respectively. The dominating (relative abundance>1%) operational taxonomic units (OTUs) from Nitrobacter were closely related to Nitrobacter hamburgensis, whereas those from Nitrospira were affiliated with or related to lineage II, lineage V and several unknown groups. Heatmap analysis showed that a few dominant Nitrobacter OTUs were affected by the straw treatment or the rice-growing, and half of the dominant Nitrospira ones were explained by at least one of the variables. Multi-response permutation procedure (MRPP) and redundancy analyses showed that the Nitrospira-like NOB community changes were significantly shaped by the land managements and the soil chemical properties, including pH, moisture and NH4+, whereas that of the Nitrobacter-like NOB community was not. These results suggested that Nitrospira are more sensitive than Nitrobacter to land management in acid and fertilized soils of a rapeseed-rice rotation field trial.

Do warming-induced changes in quantity and stoichiometry of root exudation promote soil N transformations via stimulation of soil nitrifiers, denitrifiers and ammonifiers?

Despite the recognized importance of root exudation to soil biogeochemical processes and soil function in forest ecosystems, few studies have investigated the impact of quantity and stoichiometry of root exudation on soil nitrogen (N) transformations. In addition, the potential mechanism of how climate warming influences soil N cycling via root-soil interactions remains unclear. We performed an experiment in which in situ root exudates were collected from Picea asperata seedling to examine the response of root carbon (C) & N exudation rates and the C: N ratio stoichiometry along with the soil N transformation rates to warming using infrared heaters in the Eastern Tibetan Plateau. The results showed that warming enhanced root C exudation rates (ug C g−1 root biomass h−1), with higher C: N ratio stoichiometry simultaneously. Experimental warming also significantly increased the soil net mineralization rates, net nitrification rates, and denitrification rates. Then, we conducted an in vitro experiment in which three bacterial strains (Bacillus subtilis, Nitrobacter hamburgensis, and Brachymonas denitrificans) representing ammonifiers, nitrifiers, and denitrifiers, respectively, were cultured with exudates as the culture media. The results indicated that root exudates collected in warmed plots significantly stimulated the growth of the three soil N transformation bacteria, compared with the treatments in control plots. Collectively, our results, to some degree, evidence a linkage that climate warming-induced changes in C fluxes and C: N ratio stoichiometry of root exudation stimulated the growth and reproduction of soil N transformation bacteria, consequently accelerating the soil organic matter decomposition and soil N transformations.

Management of Microbial Communities through Transient Disturbances Enhances the Functional Resilience of Nitrifying Gas-Biofilters to Future Disturbances.

Microbial communities have a key role for the performance of engineered ecosystems such as waste gas biofilters. Maintaining constant performance despite fluctuating environmental conditions is of prime interest, but it is highly challenging because the mechanisms that drive the response of microbial communities to disturbances still have to be disentangled. Here we demonstrate that the bioprocess performance and stability can be improved and reinforced in the face of disturbances, through a rationally predefined strategy of microbial resource management (MRM). This strategy was experimentally validated in replicated pilot-scale nitrifying gas-biofilters, for the two steps of nitrification. The associated biological mechanisms were unraveled through analysis of functions, abundances and community compositions for the major actors of nitrification in these biofilters, that is, ammonia-oxidizing bacteria (AOB) and Nitrobacter-like nitrite-oxidizers (NOB). Our MRM strategy, based on the application of successive, transient perturbations of increasing intensity, enabled to steer the nitrifier community in a favorable way through the selection of more resistant AOB and NOB sharing functional gene sequences close to those of, respectively, Nitrosomonas eutropha and Nitrobacter hamburgensis that are well adapted to high N load. The induced community shifts resulted in significant enhancement of nitrification resilience capacity following the intense perturbation.

Cropping systems modulate the rate and magnitude of soil microbial autotrophic CO2 fixation in soil.

The effect of different cropping systems on CO2 fixation by soil microorganisms was studied by comparing soils from three exemplary cropping systems after 10 years of agricultural practice. Studied cropping systems included: continuous cropping of paddy rice (rice-rice), rotation of paddy rice and rapeseed (rice-rapeseed), and rotated cropping of rapeseed and corn (rapeseed-corn). Soils from different cropping systems were incubated with continuous 14C-CO2 labeling for 110 days. The CO2-fixing bacterial communities were investigated by analyzing the cbbL gene encoding ribulose-1,5-bisphosphate carboxylase oxygenase (RubisCO). Abundance, diversity and activity of cbbL-carrying bacteria were analyzed by quantitative PCR, cbbL clone libraries and enzyme assays. After 110 days incubation, substantial amounts of 14C-CO2 were incorporated into soil organic carbon (14C-SOC) and microbial biomass carbon (14C-MBC). Rice-rice rotated soil showed stronger incorporation rates when looking at 14C-SOC and 14C-MBC contents. These differences in incorporation rates were also reflected by determined RubisCO activities. 14C-MBC, cbbL gene abundances and RubisCO activity were found to correlate significantly with 14C-SOC, indicating cbbL-carrying bacteria to be key players for CO2 fixation in these soils. The analysis of clone libraries revealed distinct cbbL-carrying bacterial communities for the individual soils analyzed. Most of the identified operational taxonomic units (OTU) were related to Nitrobacter hamburgensis, Methylibium petroleiphilum, Rhodoblastus acidophilus, Bradyrhizobium, Cupriavidus metallidurans, Rubrivivax, Burkholderia, Stappia, and Thiobacillus thiophilus. OTUs related to Rubrivivax gelatinosus were specific for rice-rice soil. OTUs linked to Methylibium petroleiphilum were exclusively found in rice-rapeseed soil. Observed differences could be linked to differences in soil parameters such as SOC. We conclude that the long-term application of cropping systems alters underlying soil parameters, which in turn selects for distinct autotrophic communities.

A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria

Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography–Mass Spectrometry (LC–MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in the E. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase (upp) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential. This pipeline could facilitate the functional genomics of the strains that are difficult to transform.

D-Lactate metabolism and the obligate requirement for CO2 during growth on nitrite by the facultative lithoautotroph Nitrobacter hamburgensis

Nitrobacter hamburgensis X14 is a facultative lithoautotroph that conserves energy from the oxidation of nitrite (NO(-)2) and fixes carbon dioxide (CO(2)) as its sole source of carbon. The availability of the N. hamburgensis X14 genome sequence initiated a re-examination of its mixotrophic and organotrophic potential, as genes encoding three flavin-dependent oxidases were identified that may function to oxidize lactate, providing energy and carbon for growth. The response of N. hamburgensis to D- and L-lactate in the presence (mixotrophy) and absence (organotrophy) of NO(-)2 was examined. L-lactate did not support organotrophic growth or stimulate mixotrophic growth. In contrast, D-lactate enhanced the growth rate and yield of N. hamburgensis in the presence of NO(-)2, and served as the sole carbon and energy source for growth in the absence of NO(-)2 with ammonium as the sole nitrogen source. Lithoautotrophically grown cells immediately consumed D-lactate, suggesting that a lactate metabolic pathway is constitutively expressed. Nevertheless, a physiological adaptation to lactate occurred, as D-lactate-grown cells consumed and assimilated lactate at a faster rate than NO(-)2-grown cells, and the D-lactate-dependent O(2) uptake rate was significantly greater in cells grown either organotrophically or mixotrophically compared with cells grown lithoautotrophically. Although D-lactate was assimilated and metabolized to CO(2) in the presence or absence of NO(-)2, exposure to atmospheric CO(2) or the addition of 0.75 mM sodium carbonate was required for mixotrophic growth and for optimum organotrophic growth on D-lactate.

First exploration of Nitrobacter diversity in soils by a PCR cloning-sequencing approach targeting functional gene nxrA

Nitrite oxidoreductase (NXR) is the key enzyme responsible for the oxidation of NO(2)(-) to NO(3)(-) in nitrite-oxidizing bacteria. For the first time a molecular approach for targeting the nxrA gene was developed, encoding the catalytic subunit of the NXR, to study diversity of Nitrobacter-like organisms based on the phylogeny of nxrA gene sequences in soils. NxrA sequences of the Nitrobacter strains analysed (Nitrobacter hamburgensis, Nitrobacter vulgaris, Nitrobacter winogradskyi, Nitrobacter alkalicus) by PCR, cloning and sequencing revealed the occurrence of multiple copies of nxrA genes in these strains. The copy number and similarity varied among strains. The diversity of Nitrobacter-like nxrA sequences was explored in three soils (a French permanent pasture soil, a French fallow soil, and an African savannah soil) using a cloning and sequencing approach. Most nxrA sequences found in these soils (84%) differed from nxrA sequences obtained from Nitrobacter strains. Moreover, the phylogenetic distribution and richness of nxrA-like sequences was extremely variable depending on soil type. This nxrA tool extends the panel of functional genes available for studying bacteria involved in the nitrogen cycle.

Characterization and performance of constructed nitrifying biofilms during nitrogen bioremediation of a wastewater effluent

Constructed ammonium oxidizing biofilms (CAOB) and constructed nitrite oxidizing biofilms (CNOB) were characterized during the bioremediation of a wastewater effluent. The maximum ammonium removal rate and removal efficiency in CAOB was 322 mg N-NH4+ m(-3) d(-1) and 96%, respectively, while in CNOB a maximum removal rate of 255 mg N-NH4+ m(-3) d(-1) and a removal efficiency of 76% was achieved. Both constructed biofilms on low-density polyester Dacron support achieved removal efficiencies higher than that of the concentrations normally present in reactors without constructed biofilms (P < 0.05). Nitrifying bacteria from the constructed biofilms cultures were typed by sequencing 16S rRNA genes that had been amplified by PCR from genomic DNA. Analysis of enrichment biofilms has therefore provided evidence of high removal of ammonium and the presence of Nitrosomonas eutropha, N. halophila and N. europaea in CAOB, while in CNOB Nitrobacter hamburgensis, N. winogradskyi and N. alkalicus were identified according to 16S rRNA gene sequences comparison. The biofilm reactors were nitrifying over the whole experimental period (15 days), showing a definite advantage of constructed biofilms for enhancing a high biomass concentration as evidenced by environmental electron microscopic analysis (ESEM). Our research demonstrates that low-density polyester Dacron can be effectively used for the construction of nitrifying biofilms obtaining high removal efficiencies of nitrogen in a relatively short time from municipal effluents from wastewater treatment plants. CAOB and CNOB are potentially promissory for the treatment of industrial wastewaters that otherwise requires very large and expensive reactors for efficient bioremediation of effluents.

Use of polyclonal antibodies to detect and quantify the NOR protein of nitrite oxidizers in complex environments

In the approaches or models which aim to understand and/or predict how the functioning of ecosystems may be affected by perturbations or disturbances, little attention is generally given to microorganisms. Even when they are taken into account as indicators, variables which are poorly informative about the changes in the microbial functioning (microbial biomass or diversity or total number of microorganisms) are often used. To be able to estimate, in complex environments, the quantity of enzymes involved in key ecosystem processes may constitute a useful complementary tool. Here, we describe an immunological method for detecting and quantifying, in complex environments, the nitrite oxidoreductase (NOR), responsible for the oxidation of nitrite to nitrate. The α-catalytic subunit of the enzyme was purified from Nitrobacter hamburgensis and used for the production of polyclonal antibodies. These antibodies were used to detect and quantify the NOR by a chemifluorescence technique on Western blots after separation of total proteins from pure cultures and soil samples. They recognized the α-NOR of all the Nitrobacter species described to date, but no reaction was observed with members of other nitrite-oxidizing genera. The detection threshold and reproducibility of the proposed method were evaluated. The feasibility of its use to quantify NOR in a soil was tested.

Competition between two nitrite-oxidizing bacterial populations: a model for studying the impact of wastewater treatment plant discharge on nitrification in sediment

Abstract Nitrobacter, a ubiquitous nitrite oxidizer in natural and anthropized environments, is commonly studied as the model genus performing the second stage of nitrification. In rivers, wastewater treatment plant discharges may affect the nitrite-oxidizing activity and the responsible genera that are largely associated with sediment. We used a laboratory batch culture approach with Nitrobacter wynogradskyi ssp. agilis strain AG and Nitrobacter hamburgensis strain X(14) to characterize the possible stress effect of wastewater effluent on these populations and to study the possible competition between an effluent strain (X(14)) and a sediment strain (AG) over a 42-day incubation time. Immunofluorescence enumerations of each strain showed that they both survived and settled in the sediment, indicating that there was no significant stress effect due to chemical changes caused by the effluent. The development of the strains' density and activity was directly correlated with the available nitrite concentration. Nevertheless, the potential specific activity was not constant along the so-called mixotrophic (non-limiting nitrite concentration) and heterotrophic (nitrite depletion) conditions. This illustrates the inducibility of the nitrite oxidoreductase and indicates the metabolic versatility of the strains. In our experimental conditions, the preferentially autotrophic AG strain appeared more competitive than the preferentially mixo- or heterotrophic X(14) strain, including in heterotrophic environment.

Competition between two nitrite-oxidizing bacterial populations: a model for studying the impact of wastewater treatment plant discharge on nitrification in sediment

Nitrobacter, a ubiquitous nitrite oxidizer in natural and anthropized environments, is commonly studied as the model genus performing the second stage of nitrification. In rivers, wastewater treatment plant discharges may affect the nitrite-oxidizing activity and the responsible genera that are largely associated with sediment. We used a laboratory batch culture approach with Nitrobacter wynogradskyi ssp. agilis strain AG and Nitrobacter hamburgensis strain X 14 to characterize the possible stress effect of wastewater effluent on these populations and to study the possible competition between an effluent strain (X 14) and a sediment strain (AG) over a 42-day incubation time. Immunofluorescence enumerations of each strain showed that they both survived and settled in the sediment, indicating that there was no significant stress effect due to chemical changes caused by the effluent. The development of the strains’ density and activity was directly correlated with the available nitrite concentration. Nevertheless, the potential specific activity was not constant along the so-called mixotrophic (non-limiting nitrite concentration) and heterotrophic (nitrite depletion) conditions. This illustrates the inducibility of the nitrite oxidoreductase and indicates the metabolic versatility of the strains. In our experimental conditions, the preferentially autotrophic AG strain appeared more competitive than the preferentially mixo- or heterotrophic X 14 strain, including in heterotrophic environment.

The aquatic budding bacterium Blastobacter denitrificans is a nitrogen-fixing symbiont of Aeschynomene indica.

Blastobacter spp. are freshwater bacteria that form rosette structures by cellular attachment to a common base. Comparative analyses of ribosomal 16S rRNA gene and internally transcribed spacer region sequences indicated that B. denitrificans is a member of the alpha-subdivision of Proteobacteria. Among the alpha-Proteobacteria, B. denitrificans was related to a cluster of genera, including Rhodopseudomonas palustris, Afipia felis, Nitrobacter hamburgensis, and Bradyrhizobium spp. Although the precise phylogenetic relationships among these genera could not be established with a high degree of confidence, the sequences of B. denitrificans and several bradyrhizobial isolates from nodules of Aeschynomene indica were almost identical. Bradyrhizobia are bacteria that form nitrogen-fixing symbioses with legumes, including soybeans (Glycine max) and members of the genus Aeschynomene. From symbiotic infectiveness tests we demonstrated that the type strain for B. denitrificans, IFAM 1005, was capable of forming an effective nitrogen-fixing symbiosis with A. indica. Not only do these results reveal a previously unknown ecological adaptation of a relatively obscure aquatic bacterium, but they also demonstrate how evidence gathered from molecular systematic analyses can sometimes provide clues for predicting ecological behavior.

Isolation and immunocytochemical location of the nitrite-oxidizing system in nitrospira moscoviensis

A membrane-associated nitrite-oxidizing system of Nitrospira moscoviensis was isolated from heat-treated membranes. The four major proteins of the enzyme fraction had apparent molecular masses of 130, 62, 46, and 29 kDa, respectively. The nitrite-oxidizing activity was dependent on the presence of molybdenum. In contrast to the nitrite oxidoreductase of Nitrobacter hamburgensis X14, the activity of the nitrite-oxidizing system of Ns. moscoviensis increased when solubilized by heat treatment. Electron microscopy of the purified enzyme revealed uniform particles with a size of approximately 7 x 9 nm. SDS-immunoblotting analysis of crude extracts showed that the monoclonal antibodies Hyb 153-3, which recognize the beta-subunit of the nitrite oxidoreductase from Nitrobacter, reacted with a protein of 50 kDa in Ns. moscoviensis. This protein corresponded to the protein of 46 kDa of the purified enzyme and contained a b-type cytochrome. Using electron microscopic immunocytochemistry and the monoclonal antibodies Hyb 153-3, the nitrite-oxidizing system of Ns. moscoviensis was shown to be located in the periplasmic space. Here a periodic arrangement of membrane-associated particles was found on the outside of the cytoplasmic membrane in the form of a hexagonal pattern. It is supposed that these particles represent the nitrite-oxidizing system in Nitrospira.