Comparison of the Morphology and Deoxyribonucleic Acid Composition of 27 Strains of Nitrifying Bacteria

The gross morphology, fine structure, and per cent guanine plus cytosine (GC) composition of deoxyribonucleic acid of 27 strains of nitrifying bacteria were compared. Based on morphological differences, the ammonia-oxidizing bacteria were separated into four genera. Nitrosomonas species and Nitrosocystis species formed one homogenous group, and Nitrosolobus species and Nitrosospira species formed a second homogenous group in respect to their deoxyribonucleic acid GC compositions. Similarly, the nitrite-oxidizing bacteria were separated into three genera based on their morphology. The members of two of these nitrite-oxidizing genera, Nitrobacter and Nitrococcus, had similar GC compositions, but Nitrospina gracilis had a significantly lower GC composition than the members of the other two genera.     

Stoichiometric and kinetic characterisation of Nitrosomonas sp. in mixed culture by decoupling the growth and energy generation processes

A novel method that relies on the decoupling of the energy production and biosynthesis processes was used to characterise the maintenance, cell lysis and growth processes of Nitrosomonas sp. A Nitrosomonas culture was enriched in a sequencing batch reactor (SBR) with ammonium as the sole energy source. Fluorescent in situ hybridization (FISH) showed that Nitrosomonas bound to the NEU probe constituted 82% of the bacterial population, while no other known ammonium or nitrite oxidizing bacteria were detected. Batch tests were carried out under conditions that both ammonium and CO2 were in excess, and in the absence of one of these two substrates. The oxygen uptake rate and nitrite production rate were measured during these batch tests. The results obtained from these batch tests, along with the SBR performance data, allowed the determination of the maintenance coefficient and the in situ cell lysis rate, as well as the maximum specific growth rate of the Nitrosomonas culture. It is shown that, during normal growth, the Nitrosomonas culture spends approximately 65% of the energy generated for maintenance. The maintenance coefficient was determined to be 0.14-0.16 mgN mgCOD(biomass)(-1)h(-1), and was shown to be independent of the specific growth rate. The in situ lysis rate and the maximum specific growth rate of the Nitrosomonas culture were determined to be 0.26 and 1.0 day(-1) (0.043 h(-1)), respectively, under aerobic conditions at 30 degrees C and pH 7.     

Autotrophic Nitrifying Bacteria in Acid Tea Soils from Bangladesh and Sri Lanka

Autotrophic ammonia-oxidizing bacteria were detected in some Bangladesh and Sri Lanka tea soils and in three other Bangladesh soils. Numbers ranged from 25 to 5500 organisms/g dry soil. Pure cultures were obtained from all the soils either by picking colonies from silica gel plates used for counts or by enrichment culture procedures. The isolates were identified as species of Nitrosolobus, Nitrosomonas and a new species of Nitrosospira .     

Ammonia- and nitrite-oxidizing bacterial communities in a pilot-scale chloraminated drinking water distribution system.

Nitrification in drinking water distribution systems is a common operational problem for many utilities that use chloramines for secondary disinfection. The diversity of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the distribution systems of a pilot-scale chloraminated drinking water treatment system was characterized using terminal restriction fragment length polymorphism (T-RFLP) analysis and 16S rRNA gene (ribosomal DNA [rDNA]) cloning and sequencing. For ammonia oxidizers, 16S rDNA-targeted T-RFLP indicated the presence of Nitrosomonas in each of the distribution systems, with a considerably smaller peak attributable to Nitrosospira-like AOB. Sequences of AOB amplification products aligned within the Nitrosomonas oligotropha cluster and were closely related to N. oligotropha and Nitrosomonas ureae. The nitrite-oxidizing communities were comprised primarily of Nitrospira, although Nitrobacter was detected in some samples. These results suggest a possible selection of AOB related to N. oligotropha and N. ureae in chloraminated systems and demonstrate the presence of NOB, indicating a biological mechanism for nitrite loss that contributes to a reduction in nitrite-associated chloramine decay.     

Changes in the community structure and activity of betaproteobacterial ammonia-oxidizing sediment bacteria along a freshwater-marine gradient

To determine whether the distribution of estuarine ammonia-oxidizing bacteria (AOB) was influenced by salinity, the community structure of betaproteobacterial ammonia oxidizers (AOB) was characterized along a salinity gradient in sediments of the Ythan estuary, on the east coast of Scotland, UK, by denaturant gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rRNA gene fragments. Ammonia-oxidizing bacteria communities at sampling sites with strongest marine influence were dominated by Nitrosospira cluster 1-like sequences and those with strongest freshwater influence were dominated by Nitrosomonas oligotropha-like sequences. Nitrosomonas sp. Nm143 was the prevailing sequence type in communities at intermediate brackish sites. Diversity indices of AOB communities were similar at marine- and freshwater-influenced sites and did not indicate lower species diversity at intermediate brackish sites. The presence of sequences highly similar to the halophilic Nitrosomonas marina and the freshwater strain Nitrosomonas oligotropha at identical sampling sites indicates that AOB communities in the estuary are adapted to a range of salinities, while individual strains may be active at different salinities. Ammonia-oxidizing bacteria communities that were dominated by Nitrosospira cluster 1 sequence types, for which no cultured representative exists, were subjected to stable isotope probing (SIP) with 13C-HCO3-, to label the nucleic acids of active autotrophic nitrifiers. Analysis of 13C-associated 16S rRNA gene fragments, following CsCl density centrifugation, by cloning and DGGE indicated sequences highly similar to the AOB Nitrosomonas sp. Nm143 and Nitrosomonas cryotolerans and to the nitrite oxidizer Nitrospira marina. No sequence with similarity to the Nitrosospira cluster 1 clade was recovered during SIP analysis. The potential role of Nitrosospira cluster 1 in autotrophic ammonia oxidation therefore remains uncertain.     

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.     

Desulfovibrionales-related bacteria in a paper mill environment as detected with molecular techniques and culture

The aim of the present study was to evaluate the suitability of a nested PCR-DGGE (denaturing gradient gel electrophoresis) method for the detection of Desulfovibrionales-related sulfate-reducing bacteria (SRB) from paper mill samples. The samples were also analyzed with culturing. SRB cause/enhance industrial problems, namely creation of foul-smelling gases (hydrogen sulfide) and biological corrosion, and so far there has not been a simple method to study these bacteria in paper mill laboratories. In our study, culturing was able to detect Desulfovibrionales-related bacteria from two different white waters, two different brokes, pulp, clay, and slime. Out of the isolated Desulfovibrionales, 23 enrichment cultures were further characterized with Desulfovibrionales-selective PCR-DGGE. An identical Desulfovibrio species sequence was found from paper machine I (broke I, slime, and pulp) and from paper machine II (broke II and white water II), suggesting an in-house contamination with the same strain. Desulfovibrionales-selective PCR-DGGE was also performed from DNA templates extracted directly from the paper mill samples. The DGGE profiles derived from the samples without prior enrichment were more diverse and the sequenced amplicons proved to belong to the Desulfovibrionales order. Moreover, molecular techniques were able to detect Desulfovibrionales-related bacteria from calcium carbonate samples whereas culture did not. Altogether, the nested PCR-DGGE method used in this study was suitable for the detection of Desulfovibrionales-related SRB directly from different paper mill samples and it could be used for the rapid identification of SRB-contaminated industrial sites and, when combined with sequencing, for tracing of the contamination routes.     

网柄细胞状黏菌纯化培养基的改进

网柄细胞状黏菌传统的分离培养是在添加细菌的培养基上进行的。选取了9种网柄细胞状黏菌作为研究对象,在不饲喂任何细菌的条件下于3种培养基上进行培养,通过对生长状态的比较,发现燕麦片琼脂培养基对于纯化网柄细胞状黏菌具有较好的效果。     

Seasonal variation in communities of ammonia-oxidizing bacteria based on polymerase chain reaction - denaturing gradient gel electrophoresis in a biofilm reactor for drinking water pretreatment

The diversity and variation of total and active ammonia-oxidizing bacteria in a full-scale aerated submerged biofilm reactor for drinking water pretreatment were characterized by clone libraries and denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA and its gene during a whole year. Sequences obtained from clone libraries affiliated with the Nitrosomonas oligotropha lineage and the Nitrosomonas communis lineage. An uncultured subgroup of Nitrosomonas communis lineage was also detected. Seasonal variations in both total and active ammonia-oxidizing bacteria communities were observed in the DGGE profiles, but an RNA-based analysis reflected more obvious dynamic changes in ammonia-oxidizer community than a DNA-based approach. Statistical study based on canonical correspondence analysis showed that a community shift of active ammonia oxidizers was significantly influenced by temperature and pH, but no significant correlation was found between environmental variables and total ammonia-oxidizer community shift.     

Effects of aeration cycles on nitrifying bacterial populations and nitrogen removal in intermittently aerated reactors

The effects of the lengths of aeration and nonaeration periods on nitrogen removal and the nitrifying bacterial community structure were assessed in intermittently aerated (IA) reactors treating digested swine wastewater. Five IA reactors were operated in parallel with different aeration-to-nonaeration time ratios (ANA). Populations of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were monitored using 16S rRNA slot blot hybridizations. AOB species diversity was assessed using amoA gene denaturant gradient gel electrophoresis. Nitrosomonas and Nitrosococcus mobilis were the dominant AOB and Nitrospira spp. were the dominant NOB in all reactors, although Nitrosospira and Nitrobacter were also detected at lower levels. Reactors operated with the shortest aeration time (30 min) showed the highest Nitrosospira rRNA levels, and reactors operated with the longest anoxic periods (3 and 4 h) showed the lowest levels of Nitrobacter, compared to the other reactors. Nitrosomonas sp. strain Nm107 was detected in all reactors, regardless of the reactor's performance. Close relatives of Nitrosomonas europaea, Nitrosomonas sp. strain ENI-11, and Nitrosospira multiformis were occasionally detected in all reactors. Biomass fractions of AOB and effluent ammonia concentrations were not significantly different among the reactors. NOB were more sensitive than AOB to long nonaeration periods, as nitrite accumulation and lower total NOB rRNA levels were observed for an ANA of 1 h:4 h. The reactor with the longest nonaeration time of 4 h performed partial nitrification, followed by denitrification via nitrite, whereas the other reactors removed nitrogen through traditional nitrification and denitrification via nitrate. Superior ammonia removal efficiencies were not associated with levels of specific AOB species or with higher AOB species diversity.     

Effect of free ammonia and free nitrous acid concentration on the anabolic and catabolic processes of an enriched Nitrosomonas culture

The effects of free ammonia (FA; NH(3)) and free nitrous acid (FNA; HNO(2)) concentrations on the metabolisms of an enriched ammonia oxidizing bacteria (AOB) culture were investigated using a method allowing the decoupling of growth and energy generation processes. A lab-scale sequencing batch reactor (SBR) was operated for the enrichment of an AOB culture. Fluorescent in-situ hybridization (FISH) analysis showed that 82% of the bacterial population in the SBR bound to the NEU probe specifically designed for Nitrosomonas europaea. Batch tests were carried out to measure the oxygen and ammonium consumption rates by the culture at various FA and FNA levels, in the presence or absence of inorganic carbon (CO(2), HCO(3) (-), and CO(3) (2-)). It was revealed that FA of up to 16.0 mgNH(3)-N . L(-1), which was the highest concentration used in this study, did not have any inhibitory effect on either the catabolic or anabolic processes of the Nitrosomonas culture. In contrast, FNA inhibited both the growth and energy production capabilities of the Nitrosomonas culture. The inhibition on growth initiated at approximately 0.10 mgHNO(2)-N . L(-1), and the data suggested that the biosynthesis was completely stopped at an FNA concentration of 0.40 mgHNO(2)-N . L(-1). The inhibition on energy generation initiated at a slightly lower level but the Nitrosomonas culture was still oxidizing ammonia at half of the maximum rate at an FNA concentration of 0.50-0.63 mgHNO(2)-N . L(-1). The affinity constant of the Nitrosomonas culture with respect to ammonia was determined to be 0.36 mgNH(3)-N . L(-1), independent of the presence or absence of inorganic carbon.     

Genome sequence of Nitrosomonas sp. strain AL212, an ammonia-oxidizing bacterium sensitive to high levels of ammonia

Nitrosomonas sp. strain AL212 is an obligate chemolithotrophic ammonia-oxidizing bacterium (AOB) that was originally isolated in 1997 by Yuichi Suwa and colleagues. This organism belongs to Nitrosomonas cluster 6A, which is characterized by sensitivity to high ammonia concentrations, higher substrate affinity (lower K(m)), and lower maximum growth rates than strains in Nitrosomonas cluster 7, which includes Nitrosomonas europaea and Nitrosomonas eutropha. Genome-informed studies of this ammonia-sensitive cohort of AOB are needed, as these bacteria are found in freshwater environments, drinking water supplies, wastewater treatment systems, and soils worldwide.     

Structure and function of denitrifying and nitrifying bacterial communities in relation to the plant species in a constructed wetland

The community structure and potential activities of nitrifying and denitrifying bacteria were studied in the rhizosphere of Typha latifolia and Phragmites australis present in a free water system constructed wetland (CW). Potential nitrate reduction and nitrification activities were shown to be significantly higher in the rhizosphere when compared with the nonvegetated sediment. Higher rates were generally obtained for P. australis . The community structure of denitrifying bacteria in the rhizosphere differed from that found at the bulk sediment, as revealed by PCR-denaturing gradient gel electrophoresis (DGGE) of the nitrous oxide reductase encoding gene nosZ . Results also show a greater nosZ genotype diversification and suggest a plant species effect in rhizosphere samples obtained during events of low hydraulic retention times. Ammonia-oxidizing communities were less complex on the basis of PCR-DGGE analysis of the 16S rRNA gene. Retrieved sequences were all related to Nitrosomonas marina and Nitrosomonas ureae , being both present in rhizosphere and bulk sediment regardless of environmental changes. The results demonstrate the effect of vegetation on the functioning and structure of bacterial communities involved in the removal of nitrogen in the treatment cells of a CW and point to the use of vegetation coverage to promote nitrification or denitrification in particular areas.     

Autecological Study of the Chemoautotroph Nitrobacter by Immunofluorescence

Fluorescent antibodies (FA) prepared for Nitrobacter agilis and N. winogradskyi were highly reactive in homologous staining. Low-level cross-reactions between the two species were removed by adsorption. All 15 pure-culture isolates of Nitrobacter tested reacted strongly with either N. agilis FA or N. winogradskyi FA. All pure-culture isolates from soils were determined to be N. winogradskyi; those from Mammoth Cave sediments and a cattle waste oxidation ditch were N. agilis. No cross-reaction was found in extensive tests that included five isolates of Nitrosomonas europaea and 668 heterotrophic aerobic and anaerobic bacteria isolated from soil, sewage, and cave sites. The FA preparations were used to detect Nitrobacter species in Mammoth Cave sediments, in a cattle waste oxidation ditch, and in surface waters and sediments of a river and to observe that N. winogradskyi can outgrow N. agilis in enrichment culture.     

Effects of nitrite on ammonia-oxidizing activity and gene regulation in three ammonia-oxidizing bacteria

Nitrite is the highly toxic end product of ammonia oxidation that accumulates in the absence of a nitrite-consuming process and is inhibitory to nitrifying and other bacteria. The effects of nitrite on ammonia oxidation rates and regulation of a common gene set were compared in three ammonia-oxidizing bacteria (AOB) to determine whether responses to this toxic metabolite were uniform. Mid-exponential-phase cells of Nitrosomonas europaea ATCC 19718, Nitrosospira multiformis ATCC 25196, and Nitrosomonas eutropha C-91 were incubated for 6 h in mineral medium supplemented with 0, 10, or 20 mM NaNO(2) . The rates of ammonia oxidation (nitrite production) decreased significantly only in NaNO(2) -supplemented incubations of N. eutropha; no significant effect on the rates was observed for N. europaea or N. multiformis. The levels of norB (nitric oxide reductases), cytL (cytochrome P460), and cytS (cytochrome c'-β) mRNA were unaffected by nitrite in all strains. The levels of nirK (nitrite reductase) mRNA increased only in N. europaea in response to nitrite (10 and 20 mM). Nitrite (20 mM) significantly reduced the mRNA levels of amoA (ammonia monooxygenase) in N. multiformis and norS (nitric oxide reductase) in the two Nitrosomonas spp. Differences in response to nitrite indicated nonuniform adaptive and regulatory strategies of AOB, even between closely related species.     

Investigation of total bacterial and ammonia-oxidizing bacterial community composition in a full-scale aerated submerged biofilm reactor for drinking water pretreatment in China

The community composition of total bacteria and ammonia-oxidizing bacteria in a full-scale aerated submerged biofilm reactor for drinking water pretreatment was characterized by analysis of 16S rRNA gene and the functional gene amoA, respectively. Sampling was performed in February and in July. 16S rRNA gene clone libraries revealed 13 bacterial divisions. At both sampling dates, the majority of clone sequences were related to the Alpha- and Betaproteobacteria. A minor proportion belonged to the following groups: Gammaproteobacteria, Deltaproteobacteria, Nitrospira, Firmicutes, Acidobacteria, Verrucomicrobia, Actinobacteria, Planctomycetes, Chloroflexi, Gemmatimonadetes and the Cytophaga-Flavobacterium-Bacteroides group. Some sequences related to bacteria owning high potential metabolic capacities were detected in both samples, such as Rhodobacter-like rRNA gene sequences. Surveys of cloned amoA genes from the two biofilm samples revealed ammonia-oxidizing bacterial sequences affiliated with the Nitrosomonas oligotropha lineage, Nitrosomonas communis lineage. An unknown Nitrosomonas group of amoA gene sequences was also detected.     

南美白对虾养殖底泥中氨氧化细菌与氨氧化古菌多态性分析

【目的】本研究皆在了解虾养殖底泥中氨氧化细菌与氨氧化古菌群落多态性。【方法】以功能基因为基础,构建氨氧化细菌(AOB)与氨氧化古菌(AOA)的氨单加氧酶α亚基基因(amoA)克隆文库。利用限制性片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)技术将克隆文库阳性克隆子进行归类分析分成若干个可操作分类单元(Operational Taxa Units,OTUs)。【结果】通过序列多态性分析,表明AOB amoA基因克隆文库中所有序列都属于变形杆菌门β亚纲(β-Proteobacteria)中的亚硝化单细胞菌属(Nitrosomonas)及Nitrosomonas-like,未发现亚硝化螺旋菌属(Nitrosospira)。AOA amoA基因克隆文库中只有一个OTU序列属于未分类的古菌(Unclassified-Archaea),其余序列都属于泉古菌门(Crenarchaeote)。AOA群落结构单一且存在一个绝对优势类群OTU3,其克隆子数目占克隆文库的57.45%。AOB和AOA amoA基因克隆文库分别包括13个OTUs和9个OTUs,其文库覆盖率分别为73.47%和90.43%。AOB amoA基因克隆文库的Shannon-Wiener指数、Evenness指数、Simpson指数、Richness指数均高于AOA。【结论】虾养殖塘底泥中存在氨氧化古菌的amoA基因,且多态性低于氨氧化细菌,表明氨氧化古菌在虾养殖塘底泥的氮循环中可能具有重要的作用。     

Amplification of the amoA gene from diverse species of ammonium-oxidizing bacteria and from an indigenous bacterial population from seawater.

Because the chemolithotrophic ammonium-oxidizing bacteria are an integral component of nitrogen biogeochemistry, a sensitive and accurate method to detect this ecologically important group of microorganisms is needed. The amoA gene of these organisms encodes the active site of ammonia monooxygenase, an enzyme unique to this group of nitrifying bacteria. We report here the use of the PCR technique to detect the amoA gene from pure cultures of chemolithotrophic ammonium-oxidizing bacteria, ammonium oxidizers introduced into filtered seawater, and the natural bacterial population of an unfiltered seawater sample. Oligonucleotide primers, based on the published amoA sequence from Nitrosomonas europaea, were used to amplify DNA from pure cultures of Nitrosomonas europaea, Nitrosomonas cryotolerans, and Nitrosococcus oceanus and from bacteria in seawater collected offshore near the Florida Keys. Partial sequencing of the amplification products verified that they were amoA. These primers, used in conjunction with a radiolabeled amoA gene probe from Nitrosomonas europaea, could detect Nitrosococcus oceanus inoculated into filter-sterilized seawater at 10(4) cells liter-1. Native marine bacteria containing amoA could also be detected at their naturally occurring titer in oligotrophic seawater. Amplification of the gene for ammonia monooxygenase may provide a method to estimate the distribution and relative abundance of chemolithotrophic ammonium-oxidizing bacteria in the environment.     

Moderately thermophilic nitrifying bacteria from a hot spring of the Baikal rift zone

Samples from three hot springs (Alla, Seya and Garga) located in the northeastern part of Baikal rift zone (Buryat Republic, Russia) were screened for the presence of thermophilic nitrifying bacteria. Enrichment cultures were obtained solely from the Garga spring characterized by slightly alkaline water (pH 7.9) and an outlet temperature of 75 degrees C. The enrichment cultures of the ammonia- and nitrite oxidizers grew at temperature ranges of 27-55 and 40-60 degrees C, respectively. The temperature optimum was approximately 50 degrees C for both groups and thus they can be designated as moderate thermophiles. Ammonia oxidizers were identified with classical and immunological techniques. Representatives of the genus Nitrosomonas and Nitrosospira-like bacteria with characteristic vibroid morphology were detected. The latter were characterized by an enlarged periplasmic space, which has not been previously observed in ammonia oxidizers. Electron microscopy, denaturing gradient gel electrophoresis analyses and partial 16S rRNA gene sequencing provided evidence that the nitrite oxidizers were members of the genus Nitrospira.     

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.