Nitrospira marina gen. nov. sp. nov.: a chemolithotrophic nitrite-oxidizing bacterium

A new chemolithotrophic nitrite-oxidizing bacterium, for which the name Nitrospira marina is proposed, was isolated from the Gulf of Maine. N. marina is a Gramnegative curved rod which may form spirals with 1 to 12 turns. Cells have a unique periplasmic space and lack intracytoplasmic membranes and carboxysomes. N. marina is an obligate chemolithotroph, but best growth is obtained in a mixotrophic medium. N. marina may be one of the most prevalent nitrite-oxidizing bacteria in some oceanic environments. Type strain is field with American Type Culture Collection (ATCC 43039).     

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 × 9 nm. SDS-immunoblotting analysis of crude extracts showed that the monoclonal antibodies Hyb 153–3, which recognize the β-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. Received: 22 August 1997 / Accepted: 1 November 1997     

A new obligately chemolithoautotrophic,nitrite-oxidizing bacterium,Nitrospira moscoviensis sp. nov. and its phylogenetic relationship

A gram-negative, non-motile, non-marine, nitrite-oxidizing bacterium was isolated from an enrichment culture initiated with a sample from a partially corroded area of an iron pipe of a heating system in Moscow, Russia. The cells were 0.9–2.2 μm × 0.2–0.4 μm in size. They were helical- to vibroid-shaped and often formed spirals with up to three turns 0.8–1.0 μm in width. The organism possessed an enlarged periplasmic space and lacked intracytoplasmic membranes and carboxysomes. The cells tended to excrete extracellular polymers, forming aggregates. The bacterium grew optimally at 39°C and pH 7.6– 8.0 in a mineral medium with nitrite as sole energy source and carbon dioxide as sole carbon source. The optimal nitrite concentration was 0.35 mM. Nitrite was oxidized to nitrate stoichiometrically. The doubling time was 12 h in a mineral medium with 7.5 mM nitrite. The cell yield was low; only 0.9 mg protein/l was formed during oxidation of 7.5 mM nitrite. Under anoxic conditions, hydrogen was used as electron donor with nitrate as electron acceptor. Organic matter (yeast extract, meat extract, peptone) supported neither mixotrophic nor heterotrophic growth. At concentrations as low as 0.75 g organic matter/l or higher, growth of nitrite-oxidizing cells was inhibited. The cells contained cytochromes of the b- and c-type. The G+C content of DNA was 56.9 ± 0.4 mol%. The chemolithoautotrophic nitrite-oxidizer differed from the terrestrial members of the genus Nitrobacter with regard to morphology and substrate range and equaled Nitrospira marina in both characteristics. The isolated bacterium is designated as a new species of the genus Nitrospira. Comparative analysis of 16S rRNA gene sequences revealed a moderate phylogenetic relationship to Nitrospira marina, leptospirilla, Thermodesulfovibrio yellowstonii, "Magnetobacterium bavaricum," and the isolate OPI-2. Initial evidence is given that these organisms represent a new phylum of the domain bacteria. Received: 17 February 1995 / Accepted: 18 April 1995     

Oxygen uptake activity by Nitrobacter spp. in the presence of metal ions and sulfooxyanions

Abstract Several metal ions inhibited the oxygen uptake activity of Nitrobacter agilis , but their effects on the kinetic parameters of nitrite oxidation were mixed. Growth of Nitrobacter winogradskyi was inhibited by persulfate (>0.1 mM), tetrathionate (>0.5 mM), and trithionate (>5 mM). Oxygen uptake activity was, however, relatively insensitive to persulfate and tetrathionate ions.     

Effect of nitrite concentration and pH on most probable number enumerations of non-growing Nitrobacter spp.

Abstract Enumerations of nitrite-oxidizing bacteria in soil samples by a Most Probable Number technique, often showed relatively high cell numbers at a low nitrite concentration compared with the numbers of ammonium-oxidizing bacteria. It was hypothesized that the high numbers enumerated at low nitrite concentration would represent non-growing or organotrophically growing cells of nitrite-oxidizing species. In this paper, the sensitivity of non-growing Nitrobacter species to high nitrite concentrations as well as to low pH was examined. Different Nitrobacter species were pre-cultured at 0.5 mM nitrite. Non-growing cells differing in age were enumerated at different nitrite concentrations and pH values. The incubation period lasted for 5 months at 20°C. However, during the incubation periods of the older non-growing cells, it appeared that a period of 5 months might have been too short for reaching constant numbers. Early stationary cells of all species that were studied appeared not to be affected by high nitrite concentrations or low pH. Eight- and 18-month-old non-growing cells of Nitrobacter hamburgensis were also insensitive to 5 mM nitrite. The numbers of 8- and 18-month-old resting cells of N. vulgaris were only repressed by a combination of 5 mM nitrite and a low pH. Eight-month-old non-growing cells of N. winogradskyi were sensitive to 5 mM irrespective of pH, but 18-month-old cells only to 5 mM nitrate at low pH. The numbers of 8- and 18-month-old resting cells of N. winogradskyi serotype agilis were repressed by low pH rather than high nitrite concentration. Hence, it was concluded that the large differences in numbers of nitrite-oxidizing bacteria obtained with low and high nitrite concentrations in the incubation medium, was not likely to be due to the presence of non-growing Nitrobacter species in soil samples, but rather to the existence of organotrophically growing Nitrobacter cells.     

Competition for limiting amounts of oxygen between Nitrosomonas europaea and Nitrobacter winogradskyi grown in mixed continuous cultures

Chemolithotrophic nitrifying bacteria are dependent on the presence of oxygen for the oxidation of ammonium via nitrite to nitrate. The success of nitrification in oxygen-limited environments such as waterlogged soils, will largely depend on the oxygen sequestering abilities of both ammonium- and nitrite-oxidizing bacteria. In this paper the oxygen consumption kinetics of Nitrosomonas europaea and Nitrobacter winogradskyi serotype agilis were determined with cells grown in mixed culture in chemostats at different growth rates and oxygen tensions.Reduction of oxygen tension in the culture repressed the oxidation of nitrite before the oxidation of ammonium was affected and hence nitrite accumulated. K _m values found were within the range of 1–15 and 22–166 M O₂ for the ammonium- and nitrite-oxidizing cells, respectively, always with the lowest values for the N. europaea cells. Reduction of the oxygen tension in the culture lowered the half saturation constant K _m for oxygen of both species. On the other hand, the maximal oxygen consumption rates were reduced at lower oxygen levels especially at 0 kPa. The specific affinity for oxygen indicated by the V _max/K _m ratio, was higher for cells of N. europaea than for N. winogradskyi under all conditions studied. Possible consequences of the observed differences in specific affinities for oxygen of ammonium-and nitrite-oxidizing bacteria are discussed with respect to the behaviour of these organisms in oxygen-limited environments.     

锡林河流域Nitrospira的生态位分化及环境驱动力

【背景】硝化螺菌属(Nitrospira)驱动的全程及半程硝化过程在全球氮循环中发挥关键作用,但关于锡林河流域Nitrospira的生态位分化和环境驱动力及其可能发挥的全程硝化功能尚不明晰。【目的】阐明锡林河流域不同生境Nitrospira的生态位分化及环境驱动力,探究可能的全程硝化功能。【方法】利用16S rRNA基因高通量测序及生物信息学分析研究不同生境沉积物及土壤Nitrospira类群的组成、丰度、生态位分化及其环境驱动力,并预测其全程硝化功能。【结果】共检测到9类Nitrospira,其中Nitrospira 1、2、4主要分布在旱生环境,与沙粒、水分含量、pH及氨氮间存在负相关关系,与硝态氮、可溶性盐、总有机碳、全氮、全磷、粉粒和粘粒含量间存在正相关关系;Nitrospira5、6、7、8、9主要分布在水生及湿生环境,与粉粒等存在负相关关系,与沙粒等存在正相关关系(Nitrospira 9与氨氮存在负相关除外);Nitrospira 3优先分布在河床中心水生环境,仅与沙粉粒含量存在正相关关系,与其他环境因子存在负相关关系。【结论】锡林河流域Nitrospira类群有明显的生态位分化和广泛的生境适应性。Nitrospira 1、2、4最适于营养相对丰富的旱生环境;Nitrospira 5、6、7、8、9最适于相对寡营养的水生及湿生环境;Nitrospira 3偏爱粉沙、自由水及低氨寡营养水生环境。粉粒含量、可溶性盐和氨氮是导致Nitrospira类群生态位分化及空间分异的最主要环境驱动力。推测Nitrospira 3很可能是驱动全程硝化的完全氨氧化菌(comammox),而Nitrospira 1、2、4、5、6、7、8、9是否为Comammox有待探究。     

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     

Temporal and spatial variation in the nitrite-oxidizing bacterial community of a grassland soil

Abstract The temporal and spatial distribution of the nitrite-oxidizing community of a non-fertilized, semi-natural grassland soil was studied to obtain more insight into the possible variation in nitrate production in this soil throughout the year. Data describing the size, potential nitrite-oxidizing activity and serotype composition of the nitrite-oxidizing community are reported. In addition, several abiotic soil parameters potentially related to the activity of this community were measured. Whereas numbers and potential activities largely varied with time and place, the specific affinity for nitrite oxidation, defined as the ratio V _max/ K _m, was relatively constant. The serotypes Nitrobacter agilis, N. winogradskyi and N. hamburgensis were all present in the top 5-cm soil in every 500-g sample examined, showing that these species co-exist in this soil.     

Growth and substrate consumption of Nitrobacter agilis cells immobilized in carrageenan: part 2. Model evaluation

A dynamic model that predicts substrate and biomass concentration profiles across gel beads and from that the overall substrate consumption rate by the gel beads containing growing cells was evaluated with immobilized Nitrobacter agilis cells in an airlift loop reactor with oxygen as the limiting substrate. The model predictions agreed well with the observed oxygen consumption rates at three different liquid phase oxygen concentrations. Image analysis showed that 90% of the immobilized cells after 42 days of cultivation was situated in the outer shells in a film of 140 mum, while the bead radius was about 1 mm. The maximum biomass concentration in the outmost film of 56 mum was 11 kg . m(-3) gel.     

Stoichiometric and kinetic characterisation of Nitrobacter in mixed culture by decoupling the growth and energy generation processes

The growth, maintenance and lysis processes of Nitrobacter were characterised. A Nitrobacter culture was enriched in a sequencing batch reactor (SBR). Fluorescent in situ hybridisation showed that Nitrobacter constituted 73% of the bacterial population. Batch tests were carried out to measure the oxygen uptake rate and/or nitrite consumption rate when both nitrite and CO2 were in excess, and in the absence of either of these two substrates. The results obtained, along with the SBR performance data, allowed the determination of the maintenance coefficient and in situ cell lysis rate of Nitrobacter. Nitrobacter spends a significant amount of energy for maintenance, which varies considerably with the specific growth rate. At maximum growth, Nitrobacter consume nitrite at a rate of 0.042 mgN/mgCOD(biomass) . h for maintenance purposes, which increases more than threefold to 0.143 mgN/mgCOD(biomass) . h in the absence of growth. In the SBR, where Nitrobacter grew at 40% of its maximum growth rate, a maintenance coefficient of 0.113 mgN/mgCOD . h was found, resulting in 42% of the total amount of nitrite being consumed for maintenance. The above three maintenance coefficient values obtained at different growth rates appear to support the maintenance model proposed in Pirt (1982). The in situ lysis rate of Nitrobacter was determined to be 0.07/day under aerobic conditions at 22 degrees C and pH 7.3. Further, the maximum specific growth rate of Nitrobacter was estimated to be 0.02/h (0.48/day). The affinity constant of Nitrobacter with respect to nitrite was determined to be 1.50 mgNO2(-)-N/L, independent of the presence or absence of CO2.     

Competition between Phytophthora cinnamomi and Trichoderma spp. in autoclaved soil.

Results from analyses of beta-glucosidase (EC 3.2.1.21) and phosphatase (EC 3.1.3.1;EC 3.1.3.2) activities indicated that presence of a Trichoderma isolate reduced development of Phytophthora cinnamomi. It was also observed that P. cinnamomi was more competitive in coinoculated cultures than in cultures where Trichoderma was added on day 3. Analysis of trehalase (EC 3.2.1.28) activity indicated that Trichoderma either utilized portions of the P. cinnamomi mycelium as substrate or the action of P. cinnamomi released additional nutrients not normally available to Trichoderma. Ther stronger Trichoderma isolate was T. harzianum.     

Nitrosomonas Nm143-like ammonia oxidizers and Nitrospira marina-like nitrite oxidizers dominate the nitrifier community in a marine aquaculture biofilm

Zero-discharge marine aquaculture systems are an environmentally friendly alternative to conventional aquaculture. In these systems, water is purified and recycled via microbial biofilters. Here, quantitative data on nitrifier community structure of a trickling filter biofilm associated with a recirculating marine aquaculture system are presented. Repeated rounds of the full-cycle rRNA approach were necessary to optimize DNA extraction and the probe set for FISH to obtain a reliable and comprehensive picture of the ammonia-oxidizing community. Analysis of the ammonia monooxygenase gene (amoA) confirmed the results. The most abundant ammonia-oxidizing bacteria (AOB) were members of the Nitrosomonas sp. Nm143-lineage (6.7% of the bacterial biovolume), followed by Nitrosomonas marina-like AOB (2.2% of the bacterial biovolume). Both were outnumbered by nitrite-oxidizing bacteria of the Nitrospira marina-lineage (15.7% of the bacterial biovolume). Although more than eight other nitrifying populations were detected, including Crenarchaeota closely related to the ammonia-oxidizer 'Nitrosopumilus maritimus', their collective abundance was below 1% of the total biofilm volume; their contribution to nitrification in the biofilter is therefore likely to be negligible.     

3种本地植物与入侵植物紫茎泽兰的竞争

为筛选替代控制紫茎泽兰的本地植物及探讨提高替代控制效率的方法,通过盆栽实验并利用相对产量(RY)和竞争攻击力系数(A)衡量了3种本地植物与紫茎泽兰的竞争关系,同时评估了活性炭(AC)、杀真菌剂(FC)和二者联合(AC+FC)对它们竞争的影响。结果表明：(1)不添加任何物质条件下：紫茎泽兰与南酸枣混种时,紫茎泽兰的生物量明显高于单种(P<0.05),其RY和A分别显著大于1和0(P<0.05);紫茎泽兰分别与假地豆和狗尾草混种时,紫茎泽兰的株高和生物量均明显低于单种(P<0.05),其RY和A均分别显著小于1和0(P<0.05)。说明紫茎泽兰的竞争力强于南酸枣而弱于假地豆和狗尾草,假地豆和狗尾草可以在一定区域作为替代控制紫茎泽兰的潜在目标植物。(2)与不添加任何物质相比,紫茎泽兰与南酸枣混种时,AC、FC及AC+FC处理增加了紫茎泽兰的根冠比,降低了其地上生物量比,也降低了南酸枣的生物量(P<0.05);紫茎泽兰与假地豆混种时,AC和AC+FC处理增加了假地豆的株高和生物量,FC处理增加了紫茎泽兰的株高(P<0.05);紫茎泽兰与狗尾草混种时,AC和A...     

Identification of bacteria in biofilm and bulk water samples from a nonchlorinated model drinking water distribution system: detection of a large nitrite-oxidizing population associated with Nitrospira spp

In a model drinking water distribution system characterized by a low assimilable organic carbon content (<10 microg/liter) and no disinfection, the bacterial community was identified by a phylogenetic analysis of rRNA genes amplified from directly extracted DNA and colonies formed on R2A plates. Biofilms of defined periods of age (14 days to 3 years) and bulk water samples were investigated. Culturable bacteria were associated with Proteobacteria and Bacteriodetes, whereas independently of cultivation, bacteria from 12 phyla were detected in this system. These included Acidobacteria, Nitrospirae, Planctomycetes, and Verrucomicrobia, some of which have never been identified in drinking water previously. A cluster analysis of the population profiles from the individual samples divided biofilms and bulk water samples into separate clusters (P = 0.027). Bacteria associated with Nitrospira moscoviensis were found in all samples and encompassed 39% of the sequenced clones in the bulk water and 25% of the biofilm community. The close association with Nitrospira suggested that a large part of the population had an autotrophic metabolism using nitrite as an electron donor. To test this hypothesis, nitrite was added to biofilm and bulk water samples, and the utilization was monitored during 15 days. A first-order decrease in nitrite concentration was observed for all samples with a rate corresponding to 0.5 x 10(5) to 2 x 10(5) nitrifying cells/ml in the bulk water and 3 x 10(5) cells/cm(2) on the pipe surface. The finding of an abundant nitrite-oxidizing microbial population suggests that nitrite is an important substrate in this system, potentially as a result of the low assimilable organic carbon concentration. This finding implies that microbial communities in water distribution systems may control against elevated nitrite concentrations but also contain large indigenous populations that are capable of assisting the depletion of disinfection agents like chloramines.     

Co-immobilized Nitrosomonas europaea and Nitrobacter agilis cells: validation of a dynamic model for simultaneous substrate conversion and growth in kappa-carrageenan gel beads

A dynamic model for two microbial species immobilized in a gel matrix is presented and validated with experiments. The model characterizes the nitrification of ammonia with Nitrosomonas europaea and Nitrobacter agilis co-immobilized in K-carrageenan gel beads. The model consists of kinetic equations for the microorganisms and mass transfer equations for the substrates and products inside and outside the gel beads. The model predicts reactor bulk concentrations together with the substrate consumption rate, product formation, and biomass growth inside the gel beads as a function of time. A 50-day experiment with immobilized cells in a 3.3-dm(3) air-lift loop reactor was carried out to validate the model. The parameter values for the model were obtained from literature and separate experiments. The experimentally determined reactor bulk concentrations and the biomass distribution of the two microorganisms in the gel beads were well predicted by the model. A sensitivity analysis of the model for the given initial values indicated the most relevant parameters to be the maximum specific growth rate of the microorganisms, the diffusion coefficient of oxygen, and the radius of the beads. The dynamic model provides a useful tool for further study and possible control of the nitrification process. (c) 1994 John Wiley & Sons, Inc.