Effect of adhesion to particles on the survival and activity of Nitrosomonas sp. and Nitrobacter sp.

The adhesion of Nitrosomonas sp. and Nitrobacter sp. cells isolated from fishpond sediment to different solid particles was studied. Nitrosomonas and Nitrobacter cells rapidly attached to particles of bentonite, calcium carbonate, amberlite, and fishpond sediment, however they did not adhere to phenyl-sepharose beads. The nitrifying activity of attached bacteria was greater than the activity of freely suspended cells or the activity of cells which have been detached from CaCO₃ particles. The enhancement in the nitrifying activity was rapid and was already observed within the first hour after attachment (which equals only 1/24 to 1/50 of the generation time of Nitrosomonas sp. or Nitrobacter sp. In addition, the survival of the attached bacteria under both anaerobic and under aerobic incubation was extended to weeks, compared to only a few days for the free cells. The presence of substrate (ammonia or nitrite) during the anaerobic incubation period was found not to affect the survival time of the bacteria. Finally, it was found that the attachment of Nitrosomonas and Nitrobacter cells to CaCO₃ particles affected the dispersal and sinking rate of these particles.     

Shifts between Nitrospira‐ and Nitrobacter‐like nitrite oxidizers underlie the response of soil potential nitrite oxidation to changes in tillage practices

Despite their role in soil functioning, the ecology of nitrite‐oxidizing bacteria, NOB, and their response to disturbances such as those generated by agricultural practices are scarcely known. Over the course of 17 months, we surveyed the potential nitrite oxidation, PNO, the abundance of the Nitrobacter‐ and Nitrospira‐like NOB (by quantitative PCR) and the community structure of the Nitrobacter‐like NOB (by PCR‐DGGE and cloning‐sequencing targeting the nxrA gene) in soils for four treatments: after establishment of tillage on a previously no‐tillage system, after cessation of tillage on a previously tillage system, and on control tillage and no‐tillage systems. Key soil variables (moisture, organic carbon content and gross mineralization – i.e. ammonification – measured by the ¹⁵N dilution technique) were also surveyed. PNO was always higher for the no‐tillage than tillage treatments. Establishment of tillage led to a strong and rapid decrease in PNO whereas cessation of tillage did not change PNO even after 17 months. PNO was strongly and positively correlated to the abundance of Nitrobacter‐like NOB and was also strongly related to gross mineralization, a proxy of N‐availability; in contrast, PNO was weakly and negatively correlated to the abundance of Nitrospira‐like NOB. Selection of a dominant population was observed under no‐tillage, and PNO was loosely correlated to the community structure of Nitrobacter‐like NOB. Our results demonstrate that Nitrobacter‐like NOB are the key functional players within the NOB community in soils with high N availability and high activity level, and that changes in PNO are due to shifts between Nitrospira‐like and Nitrobacter‐like NOB and to a weaker extent by shifts of populations within Nitrobacter‐like NOB.     

Isolation and characterization of a novel facultatively alkaliphilic Nitrobacter species, N. alkalicus sp. nov.

Five strains of lithotrophic, nitrite-oxidizing bacteria (AN1-AN5) were isolated from sediments of three soda lakes (Kunkur Steppe, Siberia; Crater Lake and Lake Nakuru, Kenya) and from a soda soil (Kunkur Steppe, Siberia) after enrichment at pH 10 with nitrite as sole electron source. Morphologically, the isolates resembled representatives of the genus Nitrobacter. However, they differed from recognized species of this genus by the presence of an additional S-layer in their cell wall and by their unique capacity to grow and oxidize nitrite under highly alkaline conditions. The influence of pH on growth of one of the strains (AN1) was investigated in detail by using nitrite-limited continuous cultivation. Under such conditions, strain AN1 was able to grow at a broad pH range from 6.5 to 10.2, with an optimum at 9.5. Cells grown at pH higher than 9 exhibited a clear shift in the optimal operation of the nitrite-oxidizing system towards the alkaline pH region with respect to both reaction rates and the affinity. Cells grown at neutral pH values behaved more like neutrophilic Nitrobacter species. These data demonstrated the remarkable potential of the new nitrite-oxidizing bacteria for adaptation to varying alkaline conditions. The 16S rRNA gene sequences of isolates AN1, AN2, and AN4 showed high similarity (≥ 99.8%) to each other, and to sequences of Nitrobacter strain R6 and of Nitrobacter winogradskyi. However, the DNA-DNA homology in hybridization studies was too low to consider these isolates as new strains. Therefore, the new isolates from the alkaline habitats are described as a new species of the genus Nitrobacter, N. alkalicus, on the basis of their substantial morphological, physiological, and genetic differences from the recognized neutrophilic representatives of this genus. Received: 3 April 1998 / Accepted: 2 July 1998     

An amperometric enzyme-linked immunosensor for Nitrobacter

A new amperometric enzyme-linked immunoassay for specific enumeration of Nitrobacter has been developed. This assay uses an electrode made of glassy carbon, on which the immunological reaction is carried out. The method is based on a competitive immunoassay principle, utilising monoclonal primary antibody and alkaline-phosphatase-labelled secondary antibody. The enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate generates an electroactive product which is amperometrically detected. The effects of different parameters on the performance of the sensor have been studied. Quantitative detection of Nitrobacter using the immunosensor has been compared to a previously developed enzyme-linked immunosorbent assay showing compatible results. In addition, the overall assay time can be shortened with this new sensor. A detection limit of approximately 3 × 10⁶ Nitrobacter cells/ml was obtained. Received: 27 May 1998 / Received revision: 28 August 1998 / Accepted: 28 August 1998     

Sequentially aerated membrane biofilm reactors for autotrophic nitrogen removal: microbial community composition and dynamics

Membrane‐aerated biofilm reactors performing autotrophic nitrogen removal can be successfully applied to treat concentrated nitrogen streams. However, their process performance is seriously hampered by the growth of nitrite oxidizing bacteria (NOB). In this work we document how sequential aeration can bring the rapid and long‐term suppression of NOB and the onset of the activity of anaerobic ammonium oxidizing bacteria (AnAOB). Real‐time quantitative polymerase chain reaction analyses confirmed that such shift in performance was mirrored by a change in population densities, with a very drastic reduction of the NOB Nitrospira and Nitrobacter and a 10‐fold increase in AnAOB numbers. The study of biofilm sections with relevant 16S rRNA fluorescent probes revealed strongly stratified biofilm structures fostering aerobic ammonium oxidizing bacteria (AOB) in biofilm areas close to the membrane surface (rich in oxygen) and AnAOB in regions neighbouring the liquid phase. Both communities were separated by a transition region potentially populated by denitrifying heterotrophic bacteria. AOB and AnAOB bacterial groups were more abundant and diverse than NOB, and dominated by the r‐strategists Nitrosomonas europaea and Ca. Brocadia anammoxidans, respectively. Taken together, the present work presents tools to better engineer, monitor and control the microbial communities that support robust, sustainable and efficient nitrogen removal.     

Surface attachment of nitrifying bacteria and their inhibition by potassium ethyl xanthate

Ion exchange resins and glass microscope slides were used to investigate factors affecting attachment of nitrifying bacteria to solid surfaces and the effect of attachment on inhibition ofNitrobacter by potassium ethyl xanthate. The ammonium oxidizerNitrosomonas attached preferentially to cation exchange resins while the nitrite oxidizerNitrobacter colonized anion exchange resins more extensively. Colonization was always associated with growth, and the site of substrate (NH₄ ⁺ or NO₂ ^–) adsorption was the major factor in attachment and colonization. The specific growth rate of cells colonizing either ion exchange resin beads or glass surfaces was greater than that of freely suspended cells, butNitrobacter populations colonizing glass surfaces were more sensitive to the inhibitor potassium ethyl xanthate. The findings indicate that surface growth alone does not protect soil nitrifying bacteria from inhibition by potassium ethyl xanthate and explain different patterns of inhibition for ammonium and nitrite oxidizers in the soil.     

Geospatial variation in co‐occurrence networks of nitrifying microbial guilds

Microbial communities transform nitrogen (N) compounds, thereby regulating the availability of N in soil. The N cycle is defined by interacting microbial functional groups, as inorganic N‐products formed in one process are the substrate in one or several other processes. The nitrification pathway is often a two‐step process in which bacterial or archaeal communities oxidize ammonia to nitrite, and bacterial communities further oxidize nitrite to nitrate. Little is known about the significance of interactions between ammonia‐oxidizing bacteria (AOB) and archaea (AOA) and nitrite‐oxidizing bacterial communities (NOB) in determining the spatial variation of overall nitrifier community structure. We hypothesize that nonrandom associations exist between different AO and NOB lineages that, along with edaphic factors, shape field‐scale spatial patterns of nitrifying communities. To address this, we sequenced and quantified the abundance of AOA, AOB, and Nitrospira and Nitrobacter NOB communities across a 44‐hectare site with agricultural fields. The abundance of Nitrobacter communities was significantly associated only with AOB abundance, while that of Nitrospira was correlated to AOA. Network analysis and geostatistical modelling revealed distinct modules of co‐occurring AO and NOB groups occupying disparate areas, with each module dominated by different lineages and associated with different edaphic factors. Local communities were characterized by a high proportion of module‐connecting versus module‐hub nodes, indicating that nitrifier assemblages in these soils are shaped by fluctuating conditions. Overall, our results demonstrate the utility of network analysis in accounting for potential biotic interactions that define the niche space of nitrifying communities at scales compatible to soil management.     

Free nitrous acid selectively inhibits and eliminates nitrite oxidizers from nitrifying sequencing batch reactor

In a complete nitrification sequencing batch reactor (CNSBR), where ammonium containing wastewater (200–1,000 mg N/L) is completely oxidized to nitrate up to 2.4 kg NH₄ ⁺–N/m³ d, both ammonia oxidizers and nitrite oxidizers were enriched in the sludge granules. Quantitative fluorescence in situ hybridization analyses of the sludge granules of the CNSBR showed that ammonia oxidizers and nitrite oxidizers occupied 31 and 4.2% of total bacteria, respectively. Most of the nitrite oxidizers were Nitrobacter species (95% of the nitrite oxidizers) and the remainder was Nitrospira species. The population of nitrite oxidizers was significantly higher than that of partial nitrification SBR (PNSBR) where most of the ammonium was oxidized to nitrite. The PNSBR had 37% (ammonia oxidizers) and 0.4% (nitrite oxidizers) of total bacteria. Comparative study with CNSBR and PNSBR revealed that free nitrous acid, rather than free ammonia, played a critical inhibition role to wash out nitrite oxidizers from the reactor. The concentrations of free ammonia and nitrite as well as free nitrous acid in the CNSBR selected Nitrobacter as the dominant nitrite oxidizers rather than Nitrospira.     

THE BIOCHEMISTRY OF NITRIFYING MICROORGANISMS

• 1 Biological nitrification is mediated primarily by two genera of bacteria, Nitrosomonas and its marine form Nitrosocystis, oxidizing ammonia to nitrite, and Nitrobacter, converting nitrite into nitrate. These are chemoautotrophic organisms since they usually derive their energy for growth by oxidizing these inorganic nitrogen compounds and their carbon from carbon dioxide, carbonates or bicarbonates.
• 2 The morphology and structure of these Gram-negative bacteria studied by electron microscopy show numerous intracellular membranes reminiscent of those in photosynthetic bacteria and blue-green algae. These structures may therefore be associated with the production of ATP.
• 3 The bacteria are difficult to grow in pure cultures in sufficient amounts for biochemical work since their generation time is around 10 hr. and the yields are only about one hundredth of those obtained with heterotrophic bacteria. Thus in continuous cultures great care must be taken to avoid ‘wash-out’ of the cells. Since Nitrosomonas and Nitrosocystis produce copious amounts of nitrous acid, which would eventually retard growth, pH stat units are used to titrate the cultures continuously with a solution of sodium carbonate, to hold the pH around 7–8.
• 4 The respiratory chain which is associated with cell membranes, contains flavin, quinones and many cytochromes linking to oxygen as a terminal acceptor. In Nitro-somonas-Nitrosocytis hydroxylamine is oxidized by the electron transfer chain and in Nitrobacter nitrous acid is utilized. The ammonia-oxidizing system, which in Nitrosomonas probably resides near the cell surface, does not appear to survive cell breakage. During the oxidation of hydroxylamine and nitrous acid by the respiratory chains, a phosphorylation occurs but the P/O ratios around 0–30 are low. There is little energy reserve material in the cells, possibly β-hydroxybutyrate and some metaphosphates and as soon as the oxidative processes are impaired the cells cease dividing.
• 5 Chemoautotrophic bacteria have a novel way of producing reduced nicotinamide adenine dinucleotide (NADH). This involves a reversal of electron flow from reduced cytochrome c to nicotinamide adenine dinucleotide (NAD) that is energy-dependent, thus requiring adenosine triphosphate.
• 6 Reductase enzymes, nitrate, nitrite and hydroxylamine reductases in Nitrobacter and nitrite and hydroxylamine reductases in Nitrosomonas, have been described. They appear to be readily extracted in soluble form and are probably assimilatory enzymes since ¹⁶N labelled nitrate, nitrite and hydroxylamine respectively in Nitrobacter and the last two in Nitrosomonas are readily incorporated into cell nitrogen. It has been suggested that a particulate nitrate reductase in Nitrobacter is coupled to the synthesis of adenosine triphosphate but adequate experimental evidence for this concept has not been produced.
• 7 Some recent observations with Nitrobacter suggest that it grows on acetate, deriving all its energy and carbon skeletons from this source but the mean generation time for the bacterium is unchanged. Under these conditions the carbon dioxide fixing enzymes of the pentose pathway are suppressed. This then is a case of facultative chemoautotrophy but there is no increase in the biosynthesis of the TCA enzymes. Whether this is a widespread phenomenon in other chemoautotrophic bacteria remains to be established. If this does prove to be the case it would aid their survival in a variety of habitats and extend their distribution in soils and seas.
• 8 The carbon dioxide fixing enzymes of the pentose pathway are found in the soluble parts of the cells. The major route is via the carboxydismutase system with only a small incorporation via the phosphoenolpyruvate carboxylase enzyme. Enzymes of the tricarboxylic acid cycle have low activities compared with those in heterotrophs and this overall slow metabolism, rather than the lack of a specific enzyme such as NADH oxidase, may well account for the slow growth of these bacteria. Although there is very active glutamic dehydrogenase in Nitrosomonas that utilizes ammonia, the enzyme has a very small activity in Nitrobacter. This poses a problem of the route of incorporation of nitrite nitrogen into cell nitrogen in the latter bacterium.
• 9 A few heterotrophic fungi have been described which oxidize ammonia to nitrate but their activity is small compared with that of the nitrifying bacteria.
• 10 It is concluded that the nitrifying bacteria which have many novel biochemical features not met with in other organisms merit further study.

     

Autoradiography and Immunofluorescence Combined for Autecological Study of Single Cell Activity with Nitrobacter as a Model System

Specific detection of a particular bacterium by immunofluorescence was combined with estimation of its metabolic activity by autoradiography. The nitrifying bacteria Nitrobacter agilis and N. winogradskyi were used as a model system. Nitrobacter were incubated with NaH¹⁴CO₃ and ¹⁴CO₂ prior to study. The same preparations made for autoradiograms were stained with fluorescent antibodies specific for the Nitrobacter species. Examination by epifluorescence and transmitted dark-field microscopy revealed Nitrobacter cells with and without associated silver grains. Direct detection and simultaneous evaluation of metabolic activity of Nitrobacter was demonstrated in pure cultures, in a simple mixed culture, and in a natural soil.     

Rapid start-up of nitrifying MBBRs at low temperatures: nitrification,biofilm response and microbiome analysis

The moving bed biofilm reactor (MBBR), operated as a post carbon removal system, requires long start-up times in comparison to carbon removal systems due to slow growing autotrophic organisms. This study investigates the use of carriers seeded in a carbon rich treatment system prior to inoculation in a nitrifying MBBR system to promote the rapid development of nitrifying biofilm in an MBBR system at temperatures between 6 and 8 °C. Results show that nitrification was initiated by the carbon removal carriers after 22 h of operation. High throughput 16S-rDNA sequencing indicates that the sloughing period was a result of heterotrophic organism detachment and the recovery and stabilization period included a growth of Nitrosomonas and Nitrospira as the dominant ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) in the biofilm. Peripheral microorganisms such as Myxococcales, a rapid EPS producer, appear to have contributed to the recovery and stabilization of the biofilm.

     

Isolation from soils ofNitrobacter and evidence for novel serotypes using immunofluorescence

To study the ecology of chemoautotrophic nitrifying bacteria (Nitrobacter), the immunofluorescence technique has been used. Fluorescent antibodies againstNitrobacter winogradskyi andNitrobacter agilis, the two known serotypes, have not labeled strains isolated from soils of the Lyon region (pH 8.1 and pH 4.7). The pure-culture isolates appeared to belong to the same genus, but to be serologically different from the reference strains. These results led us to question the diversity of strains ofNitrobacter in soils.     

Some observations on free ammonia inhibition to Nitrobacter in nitrifying biofilm reactor

Summary Free ammonia inhibition to Nitrobacter population in a nitrifying biofilm was investigated. It was found that when the tree ammonia concentration is greater than 0.1 mgN/l the oxidative activity of Nitrobacter was significantly inhibited, and resulting in a transient accumulation of nitrite ions. The results show that Nitrobacter population is capable of rapidly recovering its lost metabolic activity once the free ammonia concentration becomes less than 0.1 mgN/l, and a complete oxidation of nitrite to nitrate was achieved.     

Response of nitrifying bacterial communities to the increased thiocyanate concentration in pre-denitrification process

Changes in process performance and the nitrifying bacterial community associated with an increase of thiocyanate (SCN⁻) loading were investigated in a pre-denitrification process treating industrial wastewater. The increased SCN⁻ loading led to the concentration of total nitrogen (TN) in the final effluent, but increasing the internal recycling ratio as an operation parameter from 2 to 5 resulted in a 21% increase in TN removal efficiency. In the aerobic reactor, we found that the Nitrosomonas europaea lineage was the predominant ammonia oxidizing bacteria (AOB) and the percentages of the AOB population within the total bacteria increased from about 4.0% to 17% with increased SCN⁻ concentration. The increase of nitrite loading seemed to change the balance between Nitrospira and Nitrobacter, resulting in the high dominance of Nitrospira over Nitrobacter. Meanwhile, a Thiobacillus thioparus was suggested to be the main microorganism responsible for the SCN⁻ biodegradation observed in the system.     

Application of an amperometric immunosensor for the enumeration of Nitrobacter in activated sludge

A competitive immunosensor using a monoclonal antibody has been developed for the enumeration of Nitrobacter in activated sludge and other environmental samples. Its cross-reactivity was tested against a number of bacterial strains and isolates. All strains of the nitrite-oxidising genera Nitrobacter and Nitrococcus reacted strongly with the monoclonal antibody. The nitrite-oxidising Nitrospira moscoviensis, as well as the ammonia oxidising bacteria and the heterotrophic bacteria tested, did not show any affinity towards the antibody in the immunosensor. The numbers of Nitrobacter were analysed in sludge samples from several wastewater treatment plants in Sweden. Detectable amounts were found in all samples. This study shows the adequacy of using this immunosensor for the enumeration of Nitrobacter in natural environments. Received: 4 October 1999 / Received revision: 20 February 2000 / Accepted: 25 February 2000     

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.     

Dynamics of viable nitrifier community and nutrient availability in dry tropical forest habitat as affected by cultivation and soil texture

Seasonal dynamics of N-mineralization and the size of the viable community of nitrifying bacteria were studied for a forest site and an adjoining cropland site. The forest site was dominated by Boswellia serrata and Acacia catechu in the tree layer, and by Nyctanthes arbortristis and Zizyphus glaberrima in the shrub layer. Crop sequence on the cropland site was Oryza sativa/Lens culinaris. The soil type in both the sites was ultisol (USDA). The cropland soil had significantly higher bulk density, and clay content but lower organic C, total N and total P than forest soil. The soil moisture content, numbers of ammonia-and nitrite oxidizing bacteria and N-mineralization rates were highest in the wet season and lowest in the dry season, while the size of mineral N and P pools showed a reverse trend in both sites. The numbers of free-living cells of ammonia-and nitrite oxidizing bacteria were significantly related with each other as well as with the soil moisture content and N-mineralization rates. In N-mineralization, NO ₃ ^– was the dominating form in the forest site during rainy season, while in other seasons in this site and in all the seasons in the cropland site, NH ₄ ⁺ -N was predominant. The N-mineralization rate and the number of viable nitrifying cells were consistently higher for the forest soil compared to the clay-rich cropland soil. The combination of low soil organic matter and high clay content suppressed the number of free-living cells of nitrifying bacteria and N-mineralization rates in the cropland site.     

The close phylogenetic relationship of Nitrobacter and Rhodopseudomonas palustris

The phylogenetic position of Nitrobacter winogradskyi and two other nitrite-oxidizing bacteria was elucidated comparing oligonucleotides of the 16S ribosomal RNA. Nitrobacter winogradskyi and the Nitrobacter isolate Yukatan are genetically nearly identical; Nitrobacter isolate X14 is more distantly related. Phylogenetically, Nitrobacter is a member of a group of purple non-sulfur bacteria that is defined by various species of Rhodopseudomonas, Rhodomicrobium vannielii, Rhodospirillum rubum and their non-phototrophic relatives. Nitrobacter shares a high sequence similarity to Rhodopseudomonas palustris. These findings are in accord with several common taxonomic characteristics, and in addition support the conversion hypothesis for the origin of this group of chemolithotrophic bacteria.     

Stimulatory Effect of Xenobiotics on Oxidative Electron Transport of Chemolithotrophic Nitrifying Bacteria Used as Biosensing Element

Electron transport chain (ETCh) of ammonium (AOB) and nitrite oxidizing bacteria (NOB) participates in oxidation of ammonium to nitrate (nitrification). Operation of ETCh may be perturbed by a range of water-soluble xenobiotics. Therefore, consortia of nitrifying bacteria may be used as a biosensor to detect water contamination. A surprising feature of this system is an increase of oxygen consumption, detected in the presence of certain inhibitors of ETCh. Thus, to shed light on the mechanism of this effect (and other differences between inhibitors) we monitored separately respiration of the bacteria of the first (AOB - Nitrosomonas) and second (NOB -Nitrobacter) stages of nitrification. Furthermore, we measured plasma membrane potential and the level of reduction of NAD(P)H. We propose a novel model of ETCh in NOB to explain the role of reverse electron transport in the stimulation of oxygen consumption (previously attributed to hormesis).