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.     

Most Probable Numbers of chemolitho-autotrophic nitrite-oxidizing bacteria in well drained grassland soils: Stimulation by high nitrite concentrations

Abstract The results of Most Probable Number (MPN) enumerations of chemolitho-autotrophic nitrite oxidizers are very much dependent on the nitrite concentration applied in the incubation medium. In order to explain this dependency, the influence of pH, nitrite and resultant nitrous acid concentration of the incubation medium on the MPN-enumeration was investigated. It appeared that none of these factors were exclusively responsible for the result of the enumeration. In samples from a well drained grassland soil, highest number have been obtained with a combination of a low pH and a low nitrite concentration in the counting medium. The relation between the MPN-counting results and the nitrous acid concentration showed an optimum with the same soil samples. It was hypothesized that the relatively high numbers of nitrite-oxidizing cells determined in soil samples at a high nitrite concentration and pH 7.3 was due to the presence of dormant cells. However, this hypothesis could not be confirmed with enumerations of aged cell suspensions of different Nitrobacter species. In contrast to the field observations, these resting cells were always enumerated more efficiently at a low nitrite concentration. The importance of the use of more than one incubation medium for the enumeration of nitrite-oxidizing bacteria is emphasized.     

Enumeration of nitrite-oxidizing bacteria in grassland soils using a Most Probable Number technique: Effect of nitrite concentration and sampling procedure

Abstract Numbers of nitrite-oxidizing bacteria were determined in grassland soils using a Most Probable Number technique. Two concentrations of nitrite were used in the incubation medium, i.e. 0.05 and 5.0 mM. The results of the enumerations were highly dependent on the nitrite concentration as well as on the grassland soil sampled. In the one soil the highest numbers were counted with 5.0 mM, whereas in other soils highest numbers were obtained in media with 0.05 mM nitrite. The spatial distribution of nitrite-oxidizing bacteria was determined i two field plots. Variation between individual samples was low in one plot and high in the other. The implications of the observed differences for the sampling procedure in each sampling plot are discussed. In the waterlogged, oxygen-limited soils, numbers of nitrite-oxidizing bacteria were as high as in the drained soils. The chemolitho-autotrophic nature of these nitrifiers has been confirmed.     

The occurrence of chemolitho-autotrophic nitrifiers in water-saturated grassland soils

Relatively high most probable number (MPN) counts of chemolithotrophic nitrite oxidizers were present in water-saturated soils compared with MPNs and activity of ammonia oxidizers. These high numbers of nitrite oxidizers were confirmed by fluorescent antibody counts and potential activity measurements. Application of different nitrite concentrations in the MPN procedure discriminated within the community of nitrite oxidizers and revealed a large number of nitrite-sensitive nitrite oxidizers and a subcommunity of nitrite-insensitive nitrite oxidizers. The size of this subcommunity was small but corresponded with the low numbers of ammonium oxidizers. Numbers of nitrite-sensitive nitrite oxidizers outnumbered the ammonia oxidizing bacteria by 2–4 orders of magnitude in these soils. The possibility is discussed that the fraction of the nitrite-insensitive cells was active as aerobic nitrite oxidizers, whereas the nitrite-sensitive cells represented an inactive group of nitrite oxidizers growing as heterotrophs or as anaerobes reducing nitrite. In this situation, both MPN enumerations at a low nitrite concentration and activity measurements could give false information about the size of the in situ nitrite-oxidizing community.     

Numbers of nitrite-oxidizing bacteria in the root zone of grassland plants

Abstract The results of Most Probable Number determinations applying low and high concentrations of nitrite reveal the presence of at least two different communities of potential nitrite-oxidizing bacteria in a number of soil types. The effect of plant roots on these two communities was studied in pot experiments with soil from natural grassland in the presence or absence of either Festuca rubra or Plantago lanceolata . Both plant species are dominant on the grassland soil used in this study. Plant roots had a stimulating effect on the numbers of nitrite-oxidizing bacteria determined with 0.05 mM nitrite in the enumeration medium as well as on the potential nitrite-oxidizing activity. On the other hand, plants roots, especially in younger plants, repressed the numbers of nitrite-oxidizing bacteria enumerated with 5.0 mM nitrite in the counting medium. Pure culture studies with organotrophically grown Nitrobacter species clearly showed that this type of potential nitrite-oxidizing bacteria could not have been responsible for the relatively high Most Probable Numbers observed in the root zones when applying 0.05 mM nitrite in the enumeration medium.     

Nitrification of swine waste

Complete oxidation of ammonia nitrogen (approximately 1000 mg/L) to nitrite was observed in stabilized swine waste after 49 days in incubation at 400 rpm and 29 degrees C, only if 10% (v/v) activated sludge from a wastewater treatment unit and 1.5% (w/v) CaCO3, were added. Stabilized swine waste contains less than 0.09 most probable number (MPN) per millilitre of nitrosobacteria and 2.3 MPN/mL of nitrobacteria. In activated sludge, the concentrations of these bacteria were 2.4 MPN/mL for nitrosobacteria and 4.2 x 10(5) MPN/mL for nitrobacteria. In the swine waste where ammonia was oxidized to nitrite, the nitrosobacteria growth increased to 5.5 x 10(5) MPN/mL, while the nitrobacteria growth decreased to 2.3 MPN/mL. Inoculation of a freshly stabilized swine waste with 10% (v/v) of the active nitrifying waste and addition of 1.5% (w/v) CaCO3, accelerated the oxidation of ammonia nitrogen to nitrite; the reaction was completed after only 5 days of incubation. Increasing the incubation period to 10 days resulted in the complete oxidation of the accumulated nitrite to nitrate. In the stabilized swine waste, complete nitrification without accumulation of nitrite was obtained in only 5 days of incubation when the waste was inoculated with both enriched nitrifying populations (10(6)-10(7) MPN/mL).     

Nitrate Reduction to Nitrite, a Possible Source of Nitrite for Growth of Nitrite-Oxidizing Bacteria

Growth yields and other parameters characterizing the kinetics of growth of nitrite-oxidizing bacteria are presented. These parameters were measured during laboratory enrichments of soil samples with added nitrite. They were then used to reanalyze data for nitrite oxidizer growth in a previously reported field study (M. G. Volz, L. W. Belser, M. S. Ardakani, and A. D. McLaren, J. Environ. Qual. 4:179-182, 1975), where nitrate, but not nitrite or ammonium, was added. In that report, analysis of the field data indicated that in unsaturated soils, the reduction of nitrate to nitrite may be a significant source of nitrite for the growth of nitrite oxidizers. A yield of 1.23 × 10⁴ cells per μg of N was determined to be most appropriate for application to the field. It was determined that if nitrite came only from mineralized organic nitrogen via ammonium oxidation, 35 to 90% of the organic nitrogen would have had to have been mineralized to produce the growth observed. However, it is estimated that only about 2% of the organic nitrogen could have been mineralized during the growth period. Thus, it appears that another source of nitrite is required, the most likely being the reduction of nitrate to nitrite coupled to the oxidation of organic matter.     

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.     

Change in the community structure of ammonia-oxidizing bacteria in activated sludge during selective incubation for MPN determination

We investigated the changes in the community structure of ammonia-oxidizing bacteria (AOB) in activated sludge during incubation of the sludge in a medium selective for AOB. The number of AOB present in the activated sludge sample was enumerated by the most-probable-number (MPN) method. Both the activated sludge sample and the incubated samples for MPN determination were analyzed by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). Universal PCR-DGGE indicated that even after 40-d incubation in a medium selected for AOB, the MPN samples were predominantly composed of heterotrophic bacteria and not AOB. Denitrification by heterotrophic bacteria might lead to the underestimation of the MPN count of AOB. Not dominated in whole bacteria, one species of AOB was detected in both original activated sludge and samples after MPN incubation by PCR-DGGE targeting AOB. Furthermore, two new species of AOB were detected only after incubation. Therefore, the community structure of AOB in the MPN samples partially resembled that in the original activated sludge.     

A Most Probable Number method (MPN) for the estimation of cell numbers of heterotrophic nitrifying bacteria in soil

A Most Probable Number (MPN) method was developed allowing for the first time estimation of populations of bacteria capable of heterotrophic nitrification. The method was applied to an acidic soil of a coniferous forest exhibiting nitrate production. In this soil nitrate production was unlikely to be catalyzed by autotrophic nitrifiers, since autotrophic ammonia oxidizers never could be detected, and autotrophic nitrite oxidizers were usually not found in appreciable cell numbers. The developed MPN method is based on the demonstration of the presence/absence of nitrite/nitrate produced by heterotrophic nitrifying bacteria during growth in a complex medium (peptone-meat-extract softagar medium) containing low concentrations of agar (0.1%). Both the supply of the growing cultures in MPN test tubes with sufficient oxygen and the presence of low agar concentrations in the medium were found to be favourable for sustainable nitrite/nitrate production. The results demonstrate that in the acidic forest soil the microbial population capable of heterotrophic nitrifcation represents a significant part of the total aerobic heterotrophic population. By applying the developed MPN method, several bacterial strains of different genera not previously described to perform heterotrophic nitrification have been isolated from the soil and have been identified by bacterio-diagnostic tests.     

Nitrifying populations and the destruction of nitrogen dioxide in soil

The nitrite formed from nitrogen dioxide (NO₂) was oxidized more readily in soil that had been treated previously with the gas than in soil not so pre-exposed. The reaction was inhibited by 1.0 but not by 0.01 mM chlorate. The population of nitrite-oxidizing autotrophs estimated by the most-probable-number procedure was too small and often grew too late to account for oxidation of the nitrite generated from NO₂. The reaction also proceeded in soil heated to 42° to 45°C or treated with 0.16 mM chlorate, although the countable autotrophs did not increase during the transformation or grew only late in the active period of nitrite oxidation. The data suggest that unknown populations are responsible for metabolism of the nitrite produced from NO₂ entering soil.     

海水亚硝酸氧化细菌初始富集过程中硝酸盐的定性检测

本文对海水亚硝酸氧化细菌初始富集过程中硝酸盐的定性检测方法及其适用范围进行了研究。结果表明, 在NaNO2起始浓度为100mg/L的亚硝酸氧化细菌初始富集培养系统中,1mL培养液中残余的NaNO2,可先用 1．0mol/L盐酸溶液20μL和50g/L氨基磺酸铵溶液10-20μL将其去除,然后再用二苯胺试剂对培养液中经亚硝酸 氯化细菌转化来的NaNO3进行定性检测,可检测出的NaNO3浓度下限在20mg/L左右。在NaNO2起始浓度不同的 富集培养系统中,去除NaNO2所需盐酸溶液、氨基磺酸铵溶液的量可根据其起始浓度按比例相应增减,但NaNO2的 起始浓度不宜超过200mg/L。该方法亦适用于淡水亚硝酸氧化细菌初始富集培养过程中硝酸盐的定性检测。       

Marine Ammonia- and Nitrite-Oxidizing Bacteria: Serological Diversity Determined by Immunofluorescence in Culture and in the Environment

Immunofluorescence assays for marine ammonium- and nitrite-oxidizing bacteria were used to assess the diversity of nitrifying bacteria isolated from marine environments. The antisera show relatively broad specificity, in that each reacts with several strains of the same physiological type as the strain to which the antiserum was prepared. The antisera do not, however, react with any strains of differing physiological type. Seventy percent of the 30 unidentified ammonium-oxidizing isolates tested reacted with one or both of the antisera produced to marine ammonium-oxidizing strains, and 8 of the 9 unidentified nitrite-oxidizing strains tested reacted with 1 or more of the 3 nitrite oxidizer antisera used. Ammonium- and nitrite-oxidizing bacteria were enumerated in samples taken in a depth profile (to 750 m) in the Southern California Bight by immunofluorescence assays for two ammonium oxidizers and two nitrite oxidizers. Average abundances of the two types of nitrifiers were 3.5 × 10⁵ and 2.8 × 10⁵ cells liter⁻¹, respectively. Nitrifiers constitute 0.1 to 0.8% of the total bacterial population in these samples.     

Characterization of the Microbial Community in a Partial Nitrifying Sequencing Batch Biofilm Reactor

A lab-scale partial nitrifying sequencing batch biofilm reactor was a successful start-up. Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial community dynamics in three periods together with inocula sludge at ambient temperature. The DGGE profiles of bacteria and Shannon–Wiener index (H′) results showed that high free ammonia (FA) concentration referred to lower diversity in the bioreactor system. Cluster analysis indicated that microorganism in period III was similar with inocula sludge and was different from that in periods I and II. Similar results also appeared in ammonia-oxidizing bacteria (AOB) community structure and nitrite-oxidizing bacteria (NOB) community structure, and at least four AOB species and two NOB species were present in period III, respectively. Phylogenetic analysis of amoA gene sequences showed that Nitrosomonas eutropha cluster was predominant in all the three periods. With lower ammonium loads, three new operational taxonomic units formed and consisted Nitrosomonas sp. Cluster. This article demonstrated that microbial community, AOB, and NOB diversity were related with FA concentration closely at ambient temperature.     

Autotrophic growth of nitrifying community in an agricultural soil

The two-step nitrification process is an integral part of the global nitrogen cycle, and it is accomplished by distinctly different nitrifiers. By combining DNA-based stable isotope probing (SIP) and high-throughput pyrosequencing, we present the molecular evidence for autotrophic growth of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) in agricultural soil upon ammonium fertilization. Time-course incubation of SIP microcosms indicated that the amoA genes of AOB was increasingly labeled by ¹³CO₂ after incubation for 3, 7 and 28 days during active nitrification, whereas labeling of the AOA amoA gene was detected to a much lesser extent only after a 28-day incubation. Phylogenetic analysis of the ¹³C-labeled amoA and 16S rRNA genes revealed that the Nitrosospira cluster 3-like sequences dominate the active AOB community and that active AOA is affiliated with the moderately thermophilic Nitrososphaera gargensis from a hot spring. The higher relative frequency of Nitrospira-like NOB in the ¹³C-labeled DNA suggests that it may be more actively involved in nitrite oxidation than Nitrobacter-like NOB. Furthermore, the acetylene inhibition technique showed that ¹³CO₂ assimilation by AOB, AOA and NOB occurs only when ammonia oxidation is not blocked, which provides strong hints for the chemolithoautotrophy of nitrifying community in complex soil environments. These results show that the microbial community of AOB and NOB dominates the nitrification process in the agricultural soil tested.     

Methane oxidation and the competition for oxygen in the rice rhizosphere

A mechanistic approach is presented to describe oxidation of the greenhouse gas methane in the rice rhizosphere of flooded paddies by obligate methanotrophic bacteria. In flooded rice paddies these methanotrophs compete for available O(2) with other types of bacteria. Soil incubation studies and most-probable-number (MPN) counts of oxygen consumers show that microbial oxygen consumption rates were dominated by heterotrophic and methanotrophic respiration. MPN counts of methanotrophs showed large spatial and temporal variability. The most abundant methanotrophs (a Methylocystis sp.) and heterotrophs (a Pseudomonas sp. and a Rhodococcus sp.) were isolated and characterized. Growth dynamics of these bacteria under carbon and oxygen limitations are presented. Theoretical calculations based on measured growth dynamics show that methanotrophs were only able to outcompete heterotrophs at low oxygen concentrations (frequently < 5 microM). The oxygen concentration at which methanotrophs won the competition from heterotrophs did not depend on methane concentration, but it was highly affected by organic carbon concentrations in the paddy soil. Methane oxidation was severely inhibited at high acetate concentrations. This is in accordance with competition experiments between Pseudomonas spp. and Methylocystis spp. carried out at different oxygen and carbon concentrations. Likely, methane oxidation mainly occurs at microaerophilic and low-acetate conditions and thus not directly at the root surface. Acetate and oxygen concentrations in the rice rhizosphere are in the critical range for methane oxidation, and a high variability in methane oxidation rates is thus expected.     

Microbial niche differentiation explains nitrite oxidation in marine oxygen minimum zones

Nitrite is a pivotal component of the marine nitrogen cycle. The fate of nitrite determines the loss or retention of fixed nitrogen, an essential nutrient for all organisms. Loss occurs via anaerobic nitrite reduction to gases during denitrification and anammox, while retention occurs via nitrite oxidation to nitrate. Nitrite oxidation is usually represented in biogeochemical models by one kinetic parameter and one oxygen threshold, below which nitrite oxidation is set to zero. Here we find that the responses of nitrite oxidation to nitrite and oxygen concentrations vary along a redox gradient in a Pacific Ocean oxygen minimum zone, indicating niche differentiation of nitrite-oxidizing assemblages. Notably, we observe the full inhibition of nitrite oxidation by oxygen addition and nitrite oxidation coupled with nitrogen loss in the absence of oxygen consumption in samples collected from anoxic waters. Nitrite-oxidizing bacteria, including novel clades with high relative abundance in anoxic depths, were also detected in the same samples. Mechanisms corresponding to niche differentiation of nitrite-oxidizing bacteria across the redox gradient are considered. Implementing these mechanisms in biogeochemical models has a significant effect on the estimated fixed nitrogen budget.Subject terms: Biogeochemistry, Water microbiology, Microbial ecology     

Nitrogen oxide air pollutants interfere with the measurement of nitric oxide using 2,3-diaminonaphthalene: Reduction of background interference

Nitrite, a stable metabolite of nitric oxide (NO), is measured fluorometrically as an indicator of NO production using 2,3-diaminonaphthalene. In cultured cells, it has been believed that a longer period of incubation improves the detection sensitivity because of the accumulation of nitrite formed from NO in culture media. However, here we show that nitrite formed from nitrogen oxide air pollutants accumulates continuously in culture media during the incubation and interferes with the measurement of NO as nitrite. Thus, a proper period of incubation is important to allow maximum nitrite signals from NO with minimum background nitrite from the air.