Strains Of Ammonia-oxidizing Bacteria Research Articles

Chlorate as a comammox Nitrospira specific inhibitor reveals nitrification and N2O production activity in coastal wetland

Specific nitrification inhibitors (NIs) have been widely used to disentangle the contribution of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB) as well as nitrite oxidizers to the nitrification process in specific environments. However, if these previously reported NIs can also be used to evaluate the activity of the newly discovered complete ammonia oxidizers (comammox) Nitrospira, remains understudied. Here we evaluated various NIs for their impact and specificity regarding inhibition of comammox Nitrospira in batch cultures of pure and mixed strains of AOA, AOB and comammox. Using these cultures, we observed that chlorate could specifically inhibit the ammonia oxidation and nitrite oxidation activity of comammox Nitrospira, while it had no effect on the tested AOA and AOB strains. This inhibitory effect of chlorate on comammox Nitrospira was subsequently confirmed based on 13CO2-DNA-stable isotope probing (13C-DNA-SIP) analysis. Furthermore, by applying a set of specific NIs, the nitrification and nitrous oxide (N2O) production rates of comammox Nitrospira in coastal wetlands were estimated as 17.45 ng N g−1 h−1 (26.9% of the total rate) and 0.0083 μmol−1 L h−1 (28.5%). Altogether, we identified and applied an effective and specific inhibitor of comammox Nitrospira, which allowed quantifying comammox activity in wetlands of the Yangtze Estuary, shedding new light on the ecological roles of comammox bacteria in coastal wetland environments.

Screening methane-oxidizing bacteria from municipal solid waste landfills and simulating their effects on methane and ammonia reduction.

Municipal solid waste landfills are not only a crucial source of global greenhouse gas emissions; they also produce large amounts of ammonia (NH3), hydrogen sulfide, and other odorous gases that negatively affect the regional environment. Several types of methane-oxidizing bacteria (MOB) were proved to be effective in mitigating methane emission from landfills. Nevertheless, more MOB species and their technical parameters for best mitigating methane still need to be explored. In landfills, methane is simultaneously generated with ammonia, which may impede the CH4 bio-oxidizing process of MOB. However, very limited studies examined the enhancement of methane reduction by introducing ammonia-oxidizing bacteria (AOB) in landfills. In this study, two enriched MOB cultures were gained from a typical municipal solid waste landfill, and then were cultured with three strains of ammonia-oxidizing bacteria (AOB). The MOB enrichment culture used in this work includes Methylocaldum, Methylocystaceae, and Methyloversatilis, with a methane oxidation capacity of 43.6-65.0%, and the AOB includes Candida ethanolica, Bacillus cereus, and Alcaligenes faecalis. The effects on the emission reduction of both NH3 and CH4 were measured using self-made landfill-simulating equipment, as MOB, AOB, and a MOB-AOB mixture were added to the soil cover of the simulation equipment. The concentrations of CH4 and NH3 in the MOB-AOB mixture group decreased sharply, and the CH4 and NH3 concentration was 76.4% and 83.7% of the control group level. We also found that addition of AOB can help MOB oxidize CH4 and improve the emission reduction effect.

Comparison of Nitrogen Oxide Metabolism among Diverse Ammonia-Oxidizing Bacteria.

Ammonia-oxidizing bacteria (AOB) have well characterized genes that encode and express nitrite reductases (NIR) and nitric oxide reductases (NOR). However, the connection between presence or absence of these and other genes for nitrogen transformations with the physiological production of nitric oxide (NO) and nitrous oxide (N2O) has not been tested across AOB isolated from various trophic states, with diverse phylogeny, and with closed genomes. It is therefore unclear if genomic content for nitrogen oxide metabolism is predictive of net N2O production. Instantaneous microrespirometry experiments were utilized to measure NO and N2O emitted by AOB during active oxidation of ammonia (NH3) or hydroxylamine (NH2OH) and through a period of anoxia. This data was used in concert with genomic content and phylogeny to assess whether taxonomic factors were predictive of nitrogen oxide metabolism. Results showed that two oligotrophic AOB strains lacking annotated NOR-encoding genes released large quantities of NO and produced N2O abiologically at the onset of anoxia following NH3-oxidation. Furthermore, high concentrations of N2O were measured during active O2-dependent NH2OH oxidation by the two oligotrophic AOB in contrast to non-oligotrophic strains that only produced N2O at the onset of anoxia. Therefore, complete nitrifier denitrification did not occur in the two oligotrophic strains, but did occur in meso- and eutrophic strains, even in Nitrosomonas communis Nm2 that lacks an annotated NIR-encoding gene. Regardless of mechanism, all AOB strains produced measureable N2O under tested conditions. This work further confirms that AOB require NOR activity to enzymatically reduce NO to N2O in the nitrifier denitrification pathway, and also that abiotic reactions play an important role in N2O formation, in oligotrophic AOB lacking NOR activity.

Biotransformation of Two Pharmaceuticals by the Ammonia-Oxidizing Archaeon Nitrososphaera gargensis.

The biotransformation of some micropollutants has previously been observed to be positively associated with ammonia oxidation activities and the transcript abundance of the archaeal ammonia monooxygenase gene (amoA) in nitrifying activated sludge. Given the increasing interest in and potential importance of ammonia-oxidizing archaea (AOA), we investigated the capabilities of an AOA pure culture, Nitrososphaera gargensis, to biotransform ten micropollutants belonging to three structurally similar groups (i.e., phenylureas, tertiary amides, and tertiary amines). N. gargensis was able to biotransform two of the tertiary amines, mianserin (MIA) and ranitidine (RAN), exhibiting similar compound specificity as two ammonia-oxidizing bacteria (AOB) strains that were tested for comparison. The same MIA and RAN biotransformation reactions were carried out by both the AOA and AOB strains. The major transformation product (TP) of MIA, α-oxo MIA was likely formed via a two-step oxidation reaction. The first hydroxylation step is typically catalyzed by monooxygenases. Three RAN TP candidates were identified from nontarget analysis. Their tentative structures and possible biotransformation pathways were proposed. The biotransformation of MIA and RAN only occurred when ammonia oxidation was active, suggesting cometabolic transformations. Consistently, a comparative proteomic analysis revealed no significant differential expression of any protein-encoding gene in N. gargensis grown on ammonium with MIA or RAN compared with standard cultivation on ammonium only. Taken together, this study provides first important insights regarding the roles played by AOA in micropollutant biotransformation.

The production of nitric oxide by marine ammonia-oxidizing archaea and inhibition of archaeal ammonia oxidation by a nitric oxide scavenger.

Nitrification is a critical process for the balance of reduced and oxidized nitrogen pools in nature, linking mineralization to the nitrogen loss processes of denitrification and anammox. Recent studies indicate a significant contribution of ammonia-oxidizing archaea (AOA) to nitrification. However, quantification of the relative contributions of AOA and ammonia-oxidizing bacteria (AOB) to in situ ammonia oxidation remains challenging. We show here the production of nitric oxide (NO) by Nitrosopumilus maritimus SCM1. Activity of SCM1 was always associated with the release of NO with quasi-steady state concentrations between 0.05 and 0.08 μM. NO production and metabolic activity were inhibited by the nitrogen free radical scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). Comparison of marine and terrestrial AOB strains with SCM1 and the recently isolated marine AOA strain HCA1 demonstrated a differential sensitivity of AOB and AOA to PTIO and allylthiourea (ATU). Similar to the investigated AOA strains, bulk water column nitrification at coastal and open ocean sites with sub-micromolar ammonia/ammonium concentrations was inhibited by PTIO and insensitive to ATU. These experiments support predictions from kinetic, molecular and biogeochemical studies, indicating that marine nitrification at low ammonia/ammonium concentrations is largely driven by archaea and suggest an important role of NO in the archaeal metabolism.

Comparison of Denaturing Gradient Gel Electrophoresis and Microarray Technology for Detection of Betaproteobacteria Ammonia-Oxidizing Communities

Methods linking environmental function and microbial community composition are limited by phylogenetic resolution and throughput time. Denaturing gradient gel electrophoresis (DGGE) allows for resolution of ammonia-oxidizing bacteria strains separated by as little as one base pair (bp) in 16S rRNA gene sequence but is low throughput. In contrast, microarrays are high throughput. We have designed a microarray platform to identify the majority of environmental strains of betaproteobacterial ammonia oxidizers using signature oligonucleotide sequences. Seventy-seven target sequences or single-nucleotide polymorphisms (SNP) were derived from 183 betaproteobacterial ammonia oxidizer 16S rRNA gene sequences representing the genera Nitrosospira and Nitrosomonas of the β sub-class of Proteobacteria and the cluster groups of strains within each genera identified using DNA homology values. These sequences were obtained from the National Center for Biotechnology Information database. Templates isolated from pure cultures of betaproteobacterial ammonia oxidizers and soil samples incubated with and without dairy slurry were amplified using primers CTO189f and CTO654r. Amplicons were analyzed via DGGE and microarray hybridization. Mixtures of pure culture strains of betaproteobacterial ammonia oxidizers were used to simulate environmental samples containing a known number of sequences and template concentrations. Our DNA oligonucleotide microarray demonstrated highly specific detection of individual betaproteobacterial ammonia oxidizer strains amplified from pure cultures, model communities, and soil samples and provided resolution of community composition comparable to that of DGGE. High-throughput analyses of betaproteobacterial ammonia oxidizer communities will further our knowledge of their ecology and link process level functions to management practices such as dairy slurry applications and specific strains of betaproteobacterial ammonia-oxidizing bacteria.

The Characteristics of Ammonia-Oxidizing Bacteria Isolated from the Rhizosphere of Wetland Plants

A total of 8 pure strains of ammonia-oxidizing bacteria (AOB) were collected from the rhizosphere of wetland plants including Arundo donax, Herb of rice galingale and willow. By microscopic observation and color reaction of Griess reagent, two strains of YL-1 and YS-2 were selected as the dominant genus to conduct performance investigation. Bath experiment results demonstrated that YL-1 shows the similar properties with YS-2 on the degradation of ammonia under the conditions of different pH value, ammonia loading and oxygen concentration. Both the two strains all showed excellent adaptability to the conditions of strong alkaline (pH=8.5) and high free ammonia (FA) concentration (18.2 mg/L). In spite of it, the ammonia degradation performance of the two strains to the high ammonia loading was all inhibited by the low DO condition.

Characterization of bacterial structures in two-stage moving-bed biofilm reactor (MBBR) during nitrification of the landfill leachate

Differences in DNA banding patterns, obtained by ribosomal intergenic spacer analysis (RISA), and nitrification were followed in a moving-bed biofilm reactor (MBBR) receiving municipal landfill leachate. Complete nitrification (> 99%) to nitrate was obtained in the two-stage MBBR system with an ammonium load of 1.09 g N-NH(4)/m(2).d. Increasing the ammonium load to 2.03 g N-NH(4)/m(2).d or more caused a decline in process efficiency to 70-86%. Moreover, at the highest ammonium load (3.76 g N-NH(4)/m(2).d), nitrite was the predominant product of nitrification. Community succession was evident in both compartments in response to changes in ammonium load. Non-metric multidimensional scaling (NMDS) supported by similarity analysis (ANOSIM) showed that microbial biofilm communities differed between compartments. The microbial biofilm was composed mainly of ammonia-oxidizing bacteria (AOB), with Nitrosomonas europeae and N. eutropha being most abundant. These results suggest that high ammonium concentrations select for particular AOB strains.

Identification of Genus Nitrosovibrio, Ammonia-Oxidizing Bacteria, by Comparison of N-Terminal Amino Acid Sequences of Phosphoglycerate Kinase

Nitrosospira, Nitrosolobus , and Nitrosovibrio , three genera of ammonia-oxidizing bacteria (AOB), are morphologically distinct from each other ( Nitrosospira : spiral, Nitrosolobus : lobate, Nitrosovibrio : slender curved rods). However, these three genera cannot be classified using the phylogenetic tree based on 16S rDNA sequences. Phosphoglycerate kinase (PGK) was purified from 10 AOB strains as an index for identification, and its enzymatic properties were investigated. No distinction could be made between these three genera of AOB, because there was no notable difference in the molecular weight, K m , or other properties of their PGKs. However, the N-terminal amino acid residues of PGK of Pro(P)-1, Ile(I)-6 and Asp(D)-12 have sequences characteristic of genus Nitrosovibrio (three strains) in four AOB genera. Therefore, it is proved possible to use the differences in the sequence to identify individual strains of the genus Nitrosovibrio .

Identification of genus Nitrosovibrio, ammonia-oxidizing bacteria, by comparison of N-terminal amino acid sequences of phosphoglycerate kinase

Nitrosospira, Nitrosolobus, and Nitrosovibrio, three genera of ammonia-oxidizing bacteria (AOB), are morphologically distinct from each other ( Nitrosospira: spiral, Nitrosolobus: lobate, Nitrosovibrio: slender curved rods). However, these three genera cannot be classified using the phylogenetic tree based on 16S rDNA sequences. Phosphoglycerate kinase (PGK) was purified from 10 AOB strains as an index for identification, and its enzymatic properties were investigated. No distinction could be made between these three genera of AOB, because there was no notable difference in the molecular weight, K m, or other properties of their PGKs. However, the N-terminal amino acid residues of PGK of Pro(P)-1, Ile(I)-6 and Asp(D)-12 have sequences characteristic of genus Nitrosovibrio (three strains) in four AOB genera. Therefore, it is proved possible to use the differences in the sequence to identify individual strains of the genus Nitrosovibrio.

Susceptibility of ammonia-oxidizing bacteria to nitrification inhibitors.

Activity of nitrification inhibitors to several typical ammonia-oxidizing bacteria isolated recently, i. e. Nitrosococcus, Nitrosolobus, Nitrosomonas, Nitrosospira and Nitrosovibrio species was assayed using 2-amino-4-methyl-trichloromethyl-1,3,5-triazine (MAST), 2-amino-4-tribromomethyl-6-trichloromethyl-1,3,5-triazine (Br-MAST), 2-chloro-6-trichloromethylpyridine (nitrapyrin) and others, and compared to confirm the adequate control of ammonia-oxidizing bacteria by the inhibitors. The order of activity of the inhibitors to 13 species of ammonia-oxidizing bacteria examined was approximately summarized as Br-MAST > or = nitrapyrin > or = MAST > other inhibitors. Two Nitrosomonas strains, N. europaea ATCC25978 and N. sp. B2, were extremely susceptible to Br-MAST, exhibiting a pI50 > or = 6.40. These values are the position logarithms of the molar half-inhibition concentration. The 16S rRNA gene sequence similarity for the highly susceptible 4 strains of genus Nitrosomonas was 94% to 100% of Nitrosomonas europaea, although those of the less susceptible 3 strains of ammonia-oxidizing bacteria, Nitrosococcus oceanus C-107 ATCC19707, Nitrosolobus sp. PJA1 and Nitrosolobus multiformis ATCC25196, were 77.85, 91.53 and 90.29, respectively. However, no clear correlation has been found yet between pI50-values and percent similarity of 16S rRNA gene sequence among ammonia-oxidizing bacteria.

Identification of bacteria responsible for ammonia oxidation in freshwater aquaria.

Culture enrichments and culture-independent molecular methods were employed to identify and confirm the presence of novel ammonia-oxidizing bacteria (AOB) in nitrifying freshwater aquaria. Reactors were seeded with biomass from freshwater nitrifying systems and enriched for AOB under various conditions of ammonia concentration. Surveys of cloned rRNA genes from the enrichments revealed four major strains of AOB which were phylogenetically related to the Nitrosomonas marina cluster, the Nitrosospira cluster, or the Nitrosomonas europaea-Nitrosococcus mobilis cluster of the beta subdivision of the class Proteobacteria. Ammonia concentration in the reactors determined which AOB strain dominated in an enrichment. Oligonucleotide probes and PCR primer sets specific for the four AOB strains were developed and used to confirm the presence of the AOB strains in the enrichments. Enrichments of the AOB strains were added to newly established aquaria to determine their ability to accelerate the establishment of ammonia oxidation. Enrichments containing the Nitrosomonas marina-like AOB strain were most efficient at accelerating ammonia oxidation in newly established aquaria. Furthermore, if the Nitrosomonas marina-like AOB strain was present in the original enrichment, even one with other AOB, only the Nitrosomonas marina-like AOB strain was present in aquaria after nitrification was established. Nitrosomonas marina-like AOB were 2% or less of the cells detected by fluorescence in situ hybridization analysis in aquaria in which nitrification was well established.

The C.E.B.A.S.-Minimodule: behaviour of an artificial aquatic ecological system during spaceflight.

The C.E.B.A.S.-Minimodule, a closed aquatic ecosystem integrated into a middeck locker and consisting of a Zoological (animal tanks), a Botanical (plant bioreactor), a Microbial (bacteria filter) and an Electronic Component (data acquisition/control system) was flown on the STS-89 spaceshuttle mission in January 1998 for 9 days. Preflight the plant bioreactor was loaded with 53 g of Ceratophyllum demersum (coontail) and the animal tanks with 4 adult pregnant females of the fish, Xiphophorus helleri (sword-tails), 200 juveniles of the same species less than 1 week of age, 38 large and 30 juvenile Biomphalaria glabrata water snails. The filter compartment was filled with 200 g of lava grain inoculated with laboratory strains of ammonia-oxidizing bacteria. A ground reference was undertaken with the same biological setup with a delay of 4 d. After an adaptation period of 5 d the system was closed and integrated into the spaceshuttle one day before launch. Video recordings of the animals were automatically taken for 10 minutes in 2-hour periods; the tapes were changed daily by the astronauts. The chemical and physical data for the aquatic system were within the expected range and were closely comparable in comparison to the ground reference. After 9 d under space conditions, the plant biomass increased to 117 g. The plants were all found in very good condition. All 4 adult female fish were retrieved in a good physiological condition. The juvenile fishes had a survival rate of about 33%. Almost 97% of the snails had survived and produced more than 250 neonates and 40 spawning packs. All samples were distributed according to a defined schedule and satisfied all scientific needs of the involved 12 principal investigators. This was the first successful spaceflight of an artificial aquatic ecosystem containing vertebrates, invertebrates, higher plants and microorganisms self-sustained by its inhabitants only. C.E.B.A.S. in a modified form and biological setup is a promising candidate for the early space station utilization as a first midterm experiment.

Comparison of Deoxyribonucleic Acid Homologies of Six Strains of Ammonia-Oxidizing Bacteria

We assessed polynucleotide sequence homologies among representative strains from four genera of ammonia-oxidizing bacteria, including three morphological types of the genus Nitrosomonas, by deoxyribonucleic acid-deoxyribonucleic acid hybridization. Our results indicate that there is little homology among the four genera which we examined. Furthermore, the low degree of homology among the morphological types of Nitrosomonas suggests that each type should be considered a separate species.