Ammonia-oxidizing Bacteria Cells Research Articles

Sealing solid agar in serum bottles for rapid isolation and long-term preservation of chemoautotrophic ammonia-oxidizing bacteria

Ammonia-oxidizing bacteria (AOB) are ubiquitous on the earth and have broad applications in bioremediation. However, the number of their species with standing in nomenclature and deposited in Microbial Culture Collections still remains low. Moreover, only a few novel species have been reported over the last decades. In this study, we sealed agar in serum bottles to develop a kind of solid agar plate with the oxygen concentration in the headspace maintained at low levels. By using these plates, eight AOB isolates including two novel species were obtained. When AOB cells were grown on the sealed solid agar plates, the time to form visible colonies was largely reduced and the maximum diameter of colonies reached 2 mm, which makes the process of AOB isolation rapid and efficient. Based on five AOB isolates, the headspace oxygen concentration had a significant influence on AOB growth either on solid plate or in liquid culture. Especially, when grown under 21 % O2, the number of colonies formed on solid agar plates was very low and sometimes no visible colony formed. Besides the application on AOB isolation, the sealed solid agar plate was also effective for the enumeration and preservation of AOB cells. When preserved under room temperature for more than ten months, the AOB colonies on the plate could still be recovered. This method provides a feasible way to isolate more novel AOB species from the environment and deposit more species in Microbial Culture Collections.

Oxygen diffusivity and reaction rate in spherical gel entrapping ammonia-oxidizing bacteria

Nitrifying biomass immobilized in a gel has been applied in wastewater treatment, as immobilization results in a high bacterial cell density in the reactor and facilitates easy solid–liquid separation in the settling tank. Herein, the diffusivity, reaction rate, and concentration profile of oxygen in a spherical gel entrapping ammonia-oxidizing bacteria (AOB) were investigated. Calcium alginate gel and 2,2-bis[4-(methacryloxy polyethoxy)phenyl] propane (BPE) gel were used as models to prepare millimeter-sized spherical gels. A novel method involving controlled oxygen transfer into/out of the spherical gel with alternative flow of air and N2 gas, measurement of oxygen concentration at the center of the spherical gel using an oxygen microsensor, and analysis using the Fickian diffusion equation was developed to determine oxygen diffusivity in the gel. Ammonia oxidation in the AOB-entrapping gel was also monitored by measuring the oxygen concentration at the center of the gel using an oxygen microsensor. The oxygen concentration profile in the gel was estimated based on the oxygen diffusivity and Michaelis–Menten model parameters determined for suspended AOB cells. The kinetic rate-determining step in the AOB-entrapping gel was identified using the effectiveness factor. The results would be useful for the development of aerobic bacteria-entrapping gels and the design of wastewater treatment processes using these gels.

Effect of free ammonia inhibition on process recovery of partial nitritation in a membrane bioreactor

Long-term inhibitory effect of free ammonia (FA) on the catabolic and anabolic activities of an enriched Nitrosomonas culture was investigated. Ammonia oxidizing bacteria (AOB) was inhibited at FA concentration of 34.34 ± 6.85 mg NH3-N/L while threshold inhibitory FA concentration was likely 23.64 ± 4.51 mg NH3-N/L. Below the latter, cell-specific ammonia oxidation rate (AOR), cell number and extracellular polymeric substances (EPS) increased. The calculated yield showed AOB had tended to increase cell mass under FA inhibition. Nitrite concentration increased by 58.6% 12 days after FA decreased to 8.89 ± 0.95 mg NH3-N/L. However, AOB cells became more sensitive to free nitrous acid (FNA) after 70 days of FA inhibition. Long-term FA inhibition changed AOB cell morphology and this could have increased AOB vulnerability to FNA. This study indicated FA inhibited catabolic and anabolic activities, and that long-term FA inhibition had adverse impact on process recoverability and subsequent failure threshold.

Fabrication of Bacteria Environment Cubes with Dry Lift-Off Fabrication Process for Enhanced Nitrification.

We have developed a 3D dry lift-off process to localize multiple types of nitrifying bacteria in polyethylene glycol diacrylate (PEGDA) cubes for enhanced nitrification, a two-step biological process that converts ammonium to nitrite and then to nitrate. Ammonia-oxidizing bacteria (AOB) is responsible for converting ammonia into nitrite, and nitrite-oxidizing bacteria (NOB) is responsible for converting nitrite to nitrate. Successful nitrification is often challenging to accomplish, in part because AOB and NOB are slow growers and highly susceptible to many organic and inorganic chemicals in wastewater. Most importantly, the transportation of chemicals among scattered bacteria is extremely inefficient and can be problematic. For example, nitrite, produced from ammonia oxidation, is toxic to AOB and can lead to the failure of nitrification. To address these challenges, we closely localize AOB and NOB in PEGDA cubes as microenvironment modules to promote synergetic interactions. The AOB is first localized in the vicinity of the surface of the PEGDA cubes that enable AOB to efficiently uptake ammonia from a liquid medium and convert it into nitrite. The produced nitrite is then efficiently transported to the NOB localized at the center of the PEGDA particle and converted into non-toxic nitrate. Additionally, the nanoscale PEGDA fibrous structures offer a protective environment for these strains, defending them from sudden toxic chemical shocks and immobilize in cubes. This engineered microenvironment cube significantly enhances nitrification and improves the overall ammonia removal rate per single AOB cell. This approach—encapsulation of multiple strains at close range in cube in order to control their interactions—not only offers a new strategy for enhancing nitrification, but also can be adapted to improve the production of fermentation products and biofuel, because microbial processes require synergetic reactions among multiple species.

익산 왕궁지역 논 토양에서의 질산화 세균과 질산화 고세균의 미생물학적 작용

Spatial and temporal changes in nitrification activities and distribution of microbial population of ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) in paddy soils were investigated. Soil samples were collected in March and October 2015 from rice paddy with and without the presence of confined animal feeding operations. Incubation experiments and quantitative polymerase chain reaction showed that AOA's contribution to nitrification kinetics was much higher in locations where organic nitrogen in animal waste is expected to significantly contribute to overall nitrogen budget, and temporal variations in nitrification kinetics were much smaller for AOA than AOB. These differences were interpreted to indicate that different microbial responses of two microbial populations to the types and concentrations of nitrogen substrates were the main determining factors of nitrification processes in the paddy soils. The copy numbers of ammonium monooxygenase gene showed that AOA colonized the paddy soils in higher numbers than AOB with stable distribution while AOB showed variation especially in March. Although small in numbers, AOB population turned out to exert more influence on nitrification potential than AOA, which was attributed to higher fluctuation in AOB cell numbers and nitrification reaction rate per cells.

Optimized inhibition assays reveal different inhibitory responses of hydroxylamine oxidoreductases from beta- and gamma-proteobacterial ammonium-oxidizing bacteria

Ammonia-oxidizing bacteria (AOB), ubiquitous chemoautotrophic bacteria, convert ammonia (NH3) to nitrite (NO2−) via hydroxylamine as energy source. Excessive growth of AOB, enhanced by applying large amounts of ammonium-fertilizer to the farmland, leads to nitrogen leaching and nitrous oxide gas emission. To suppress these unfavorable phenomena, nitrification inhibitors, AOB specific bactericides, are widely used in fertilized farmland. However, new nitrification inhibitors are desired because of toxicity and weak-effects of currently used inhibitors. Toward development of novel nitrification inhibitors that target hydroxylamine oxidoreductase (HAO), a key enzyme of nitrification in AOB, we established inhibitor evaluation systems that include simplified HAO purification procedure and high-throughput HAO activity assays for the purified enzymes and for the live AOB cells. The new assay systems allowed us to observe distinct inhibitory responses of HAOs from beta-proteobacterial AOB (βAOB) Nitrosomonas europaea (NeHAO) and gamma-proteobacterial AOB (γAOB) Nitrosococcus oceani (NoHAO) against phenylhydrazine, a well-known suicide inhibitor for NeHAO. Consistently, the live cells of N. europaea, Nitrosomonas sp. JPCCT2 and Nitrosospira multiformis of βAOB displayed higher responses to phenylhydrazine than those of γAOB N. oceani. Our homology modeling studies suggest that different inhibitory responses of βAOB and γAOB are originated from different local environments around the substrate-binding sites of HAOs in these two classes of bacteria due to substitutions of two residues. The results reported herein strongly recommend inhibitor screenings against both NeHAO of βAOB and NoHAO of γAOB to develop HAO-targeting nitrification inhibitors with wide anti-AOB spectra.

Fertility practices and rhizosphere effects alter ammonia oxidizer community structure and potential nitrification activity in pepper production soils

Increasing nitrogen use efficiency is critical to the productivity and long-term sustainability of vegetable production systems in the US Midwest. Understanding the impact of alternative fertility sources on the ecology of the soil nitrogen cycle has potential to allow for management that will increase crop N uptake and reduce loss. We determined how repeated applications of urea, composted chicken litter, hairy vetch green manure with alfalfa meal, and an unfertilized control affected the structure and potential nitrification activity (PNA) of ammonia oxidizers in bulk and rhizosphere soil when N needs were expected to be high. PNA was greater in animal and green manure treatments relative to urea and the unfertilized control in bulk soil, and greater in the rhizosphere relative to bulk soil regardless of fertility treatment. A strong correlation was observed between PNA and ammonia oxidizing bacteria (AOB) abundance, suggesting that AOB rather than ammonia oxidizing archaea (AOA) controlled nitrification in this system. However, AOB abundance did not differ between bulk and rhizosphere soil, and PNA was lower in the urea treatment despite greater AOB abundance indicating that other factors could have affected PNA. For example, greater availability of labile carbon (C) could have stimulated PNA through various mechanisms, and lower pH and/or specific nitrification potential per AOB cell could have reduced PNA in the urea treatment. AOB community structure was more diverse in all fertility treatments relative to the unfertilized control in bulk soil, and community structure differed between bulk and rhizosphere soil indicating niche differentiation. However, differences in AOB community structure and PNA were only observed in rhizosphere relative to bulk soil indicating that the rhizosphere had a greater effect on nitrification dynamics than fertility practices. These findings indicate that organic fertility amendments stimulate PNA, but they could also increase N loss and should be investigated further. The rhizosphere appears to play a greater role in nitrification dynamics than fertility practices, and more detailed investigations at this key plant-soil interface are warranted.

Monitoring the abundance and the activity of ammonia-oxidizing bacteria in a full-scale nitrifying activated sludge reactor.

Cell-specific ammonia oxidation rate (AOR) has been suggested to be an indicator of the performance of nitrification reactors and to be used as an operational parameter previously. However, published AOR values change by orders of magnitude and studies investigating full-scale nitrification reactors are limited. Therefore, this study aimed at quantifying ammonia-oxidizing bacteria (AOB) and estimating their in situ cell-specific AOR in a full-scale activated sludge reactor treating combined domestic and industrial wastewaters. Results showed that cell-specific AOR changed between 5.30 and 9.89 fmol cell(-1) h(-1), although no significant variation in AOB cell numbers were obtained (1.54E + 08 ± 0.22 cell/ml). However, ammonia-removal efficiency varied largely (52-79 %) and was proportional to the cell-specific AOR in the reactor. This suggested that the cell-specific AOR might be the factor affecting the biological ammonia-removal efficiency of nitrification reactors independent of the AOB number. Further investigation is needed to establish an empirical relationship to use cell-specific AOR as a parameter to operate full-scale nitrification systems more effectively.

A kinetic study of nitrification inhibition in water distribution systems using low levels of chlorite

Water utilities use operational strategies such as increasing pH, chlorine-ammonia ratio, and/or chloramine residual, distribution main flushing, and periodic break-point chlorination to control nitrification of chloraminated water in distribution systems. Although these methods are proven to be useful in controlling nitrification, the results are utility dependent and sometimes not effective in controlling the loss of chloramines. In various pilot studies, the direct application of chlorite at 0.1 to 0.8 mg/L is shown to be an effective alternative to control and prevent nitrification. Chlorite inactivates ammonia-oxidizing bacteria (AOB), the root cause of nitrification. In this study, we developed a kinetic model for nitrification inhibition through the addition of chlorite and the observation of residual chloramines and AOB. The important water quality variables examined were: chlorite concentration ranging from 0.02 to 0.4 mg/L, pH ranging from 7 to 9, ammonia concentrations ranging from 0.5 to 2.0 mg/L-N, and temperature ranging from 15 to 35 °C. Instead of measuring the viability of the AOB cells, the production of nitrite and the consumption of ammonia are used as surrogates for cell activity. This information was used to develop a kinetic model (modified Intrinsic Quenching model) that was able to fit the experimental data.

Three-Dimensional Stratification of Bacterial Biofilm Populations in a Moving Bed Biofilm Reactor for Nitritation-Anammox

Moving bed biofilm reactors (MBBRs) are increasingly used for nitrogen removal with nitritation-anaerobic ammonium oxidation (anammox) processes in wastewater treatment. Carriers provide protected surfaces where ammonia oxidizing bacteria (AOB) and anammox bacteria form complex biofilms. However, the knowledge about the organization of microbial communities in MBBR biofilms is sparse. We used new cryosectioning and imaging methods for fluorescence in situ hybridization (FISH) to study the structure of biofilms retrieved from carriers in a nitritation-anammox MBBR. The dimensions of the carrier compartments and the biofilm cryosections after FISH showed good correlation, indicating little disturbance of biofilm samples by the treatment. FISH showed that Nitrosomonas europaea/eutropha-related cells dominated the AOB and Candidatus Brocadia fulgida-related cells dominated the anammox guild. New carriers were initially colonized by AOB, followed by anammox bacteria proliferating in the deeper biofilm layers, probably in anaerobic microhabitats created by AOB activity. Mature biofilms showed a pronounced three-dimensional stratification where AOB dominated closer to the biofilm-water interface, whereas anammox were dominant deeper into the carrier space and towards the walls. Our results suggest that current mathematical models may be oversimplifying these three-dimensional systems and unless the multidimensionality of these systems is considered, models may result in suboptimal design of MBBR carriers.

Abundance and distribution of ammonia-oxidizing archaea in Tibetan and Yunnan plateau agricultural soils of China

As low oxygen and high ultraviolet (UV) exposure might significantly affect the microbial existence in plateau, it could lead to a specialized microbial community. To determine the abundance and distribution of ammonia-oxidizing archaea (AOA) in agricultural soil of plateau, seven soil samples were collected respectively from farmlands in Tibet and Yunnan cultivating the wheat, highland-barley, and colza, which are located at altitudes of 3200–3800 m above sea level. Quantitative PCR (q-PCR) and clone library targeting on amoA gene were used to quantify the abundances of AOA and ammonia-oxidizing bacteria (AOB), and characterize the community structures of AOA in the samples. The number of AOA cells (9.34 × 107−2.32 × 108 g−1 soil) was 3.86–21.84 times greater than that of AOB cells (6.91 × 106−1.24 × 108 g−1 soil) in most of the samples, except a soil sample cultivating highland-barley with an AOA/AOB ratio of 0.90. Based Kendall’s correlation coefficient, no remarkable correlation between AOA abundance and the environmental factor was observed. Additionally, the diversities of AOA community were affected by total nitrogen and organic matter concentration in soils, suggesting that AOA was probably sensitive to several environmental factors, and could adjust its community structure to adapt to the environmental variation while maintaining its abundance.

Spatial distribution of ammonia-oxidizing bacteria in the biofilm and suspended growth biomass of the full- and partial-bed biological aerated filters

The extent of comparable nitrogen removal in the full- and partial-bed biological aerated reactors needs further microbiological evidence, specifically the existence of ammonia-oxidizing bacteria (AOB). The nitrogen removal process in such systems is typically initiated by chemoliautotrophic ammonia-oxidizing bacteria converting ammonia to nitrite and traces of oxidized nitrogen gases. The formation of a dense biofilm as a result of higher turbulence would account for the higher number of AOB cells enumerated in the biofilm samples from the partial-bed reactor (4·3 × 105 ± 1·9 × 105 No. of AOB cells/mL sample) as compared with those from the full-bed reactor (1·5 × 105 ± 8·0 × 104 No. of AOB cells/mL sample).

Effects of Long Exposure to Low Temperatures on Nitrifying Biofilm and Biomass in Wastewater Treatment

Attached growth biological treatment systems are a promising solution to ammonia removal in cold-temperature climates. Environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy in combination with fluorescent in situ hybridization (FISH) was used to investigate the effects of 4 months of exposure to 4 degrees C on nitrifying biofilm and biomass. These molecular and microscopic methods were modified to minimize loss of mass and distortion of in situ perspectives. Environmental scanning electron microscopy revealed that nitrifying biofilm did not exhibit significant changes in volume with exposure to 4 degrees C. Confocal laser scanning microscopy in combination with FISH showed that the number of ammonia-oxidizing bacteria (AOB) cells present in the biofilm was statistically consistent during exposure to 4 degrees C. The RNA content of AOB cells remained sufficient for FISH enumeration. The number of nitrite-oxidizing bacteria cells remained steady during exposure to 4 degrees C; however, the RNA content of the cells appeared to decrease with exposure to 4 degrees C, thereby preventing their enumeration using FISH.

Nitrification of high-strength ammonium landfill leachate with microbial community analysis using fluorescence in situ hybridization (FISH)

Nitrification of mature sanitary landfill leachate with high-strength of N-NH(4) + (1080-2350 mg L(-1)) was performed in a 10 L continuous nitrification activated sludge reactor. The nitrification system was acclimatized with synthetic leachate during feed batch operation to avoid substrate inhibition before being fed with actual mature leachate. Successful nitrification was achieved with an approximately complete ammonium removal (99%) and 96% of N-NH(4) + conversion to N-NO(-) (3) . The maximum volumetric and specific nitrification rates obtained were 2.56 kg N-NH(4) (+) m(-3) day(-1) and 0.23 g N-NH(4) ( +) g(-1) volatile suspended solid (VSS) day(-1), respectively, at hydraulic retention time (HRT) of 12.7 h and solid retention time of 50 days. Incomplete nitrification was encountered when operating at a higher nitrogen loading rate of 3.14 kg N-NH(4) (+) m(-3) day(-1). The substrate overloading and nitrifiers competition with heterotrophs were believed to trigger the incomplete nitrification. Fluorescence in situ hybridization (FISH) results supported the syntrophic association between the ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria. FISH results also revealed the heterotrophs as the dominant and disintegration of some AOB cell aggregates into single cells which further supported the incomplete nitrification phenomenon.

Spatial distribution of ammonia-oxidizing bacteria in the biofilm and suspended growth biomass of the full- and partial-bed biological aerated filtersA paper submitted to the Journal of Environmental Engineering and Science.

The extent of comparable nitrogen removal in the full- and partial-bed biological aerated reactors needs further microbiological evidence, specifically the existence of ammonia-oxidizing bacteria (AOB). The nitrogen removal process in such systems is typically initiated by chemoliautotrophic ammonia-oxidizing bacteria converting ammonia to nitrite and traces of oxidized nitrogen gases. The formation of a dense biofilm as a result of higher turbulence would account for the higher number of AOB cells enumerated in the biofilm samples from the partial-bed reactor (4.3 × 105 ± 1.9 × 105No. of AOB cells/mL sample) as compared with those from the full-bed reactor (1.5 × 105 ± 8.0 × 104No. of AOB cells/mL sample).

Population dynamics of ammonia-oxidizing bacteria in an aerated submerged biofilm reactor for micropolluted raw water pretreatment

Population dynamics of ammonia-oxidizing bacteria (AOB) in a full-scale aerated submerged biofilm reactor for micropolluted raw water pretreatment was investigated using molecular techniques for a period of 1 year. The ammonia monooxygenase (amoA) gene fragments were amplified from DNA and RNA extracts of biofilm samples. Denaturing gradient gel electrophoresis (DGGE) profile based on the amoA messenger RNA approach exhibited a more variable pattern of temporal dynamics of AOB communities than the DNA-derived approach during the study. Phylogenetic analysis of excised DGGE bands revealed three AOB groups affiliated with the Nitrosomonas oligotropha lineage, Nitrosomonas communis lineage, and an unknown Nitrosomonas group. The population size of betaproteobacterial AOB, quantified with 16S ribosomal RNA gene real-time polymerase chain reaction assay, ranged from 6.63 x 10(5) to 2.67 x 10(9) cells per gram of dry biofilm and corresponded to 0.23-1.8% of the total bacterial fraction. Quantitative results of amoA gene of the three specific AOB groups revealed changes in competitive dominance between AOB of the N. oligotropha lineage and N. communis lineage. Water temperature is shown to have major influence on AOB population size in the reactor by the statistic analysis, and a positive correlation between AOB cell numbers and ammonia removal efficiency is suggested (r = 0.628, P < 0.05).

Population size and specific nitrification potential of soil ammonia-oxidizing bacteria under long-term fertilizer management

Population size of soil ammonia-oxidizing bacteria (AOB) was quantified by real-time PCR in a long-term (16 years) field experiment under different fertilizer managements. AOB population sizes in mineral nitrogen-fertilized soils and organic manure-fertilized soil were 10.3 and 3.1 times, respectively, that of the control, while phosphorus and potassium fertilization had no significant effect. On the other hand, the AOB specific nitrification potential (soil nitrification potential per AOB cell) was significantly higher ( P < 0.05) in organic manure-fertilized soil than in mineral-fertilized soils and the control, indicating that AOB was likely more metabolically active in organic manure-fertilized soils than in mineral nitrogen-fertilized soils after long-term application.

Use of real-time PCR to examine the relationship between ammonia oxidizing bacterial populations and nitrogen removal efficiency in a small decentralized treatment system ‘Johkasou’

The aim of this study was to examine the relationship between ammonia oxidizing bacterial populations and biological nitrogen removal in a small on-site domestic wastewater treatment system "Johkasou". The population dynamics of ammonia oxidizing bacteria (AOB) in six full-scale advanced Johkasous was surveyed using real-time PCR assay over a period of one year. These Johkasous were selected to compare the AOB populations in different treatment performance. When the effluent NH4-N concentration was higher than 2 mg L(-1), it was difficult to meet the effluent standard of advanced Johkasous (T-N 10 mg L(-1)). In contrast, the nitrogen removal efficiency was hardly affected by nitrite oxidation and denitrification in these systems. In other words, ammonia oxidation was a rate-limiting step. Furthermore, we focused on the relationship between NH4-N loading per AOB cell and nitrogen removal. Real time PCR monitoring results demonstrated that it is important to regulate NH4-N loading per AOB cell below 210 pg cell(-1) day(-1) to meet the effluent standard of advanced Johkasou. It is considered that NH4-N loading per AOB cell is a useful parameter for determining suitable nitrogen loading and small decentralized system design.

Quantification of ammonia oxidizing bacteria in soil using microcolony technique combined with fluorescence in situ hybridization (MCFU–FISH)

A new technique was developed for quantification of viable ammonia oxidizing bacteria (AOB) extracted from soil. Extracted bacteria were grown to microcolony-forming units (MCFU) on membrane filters and selectively stained with 16S rDNA probes using fluorescence in situ hybridization (FISH). The MCFU–FISH technique was used to enumerate a Nitrosomonas europaea population after inoculation to soil and the indigenous AOB populations in native and N-enriched soils. A low extraction efficiency (0.3–3%) of AOB in the native soil indicated predominance of a sessile AOB cell type. In contrast, N enrichments increased extractability of AOB from soil particles. Comparing all soils, a close correlation (R2=0.98) existed between extractable AOB numbers (MCFU–FISH counts) and potential ammonia oxidation activities covering more than three orders of magnitude, which indicated that MCFU–FISH counts reflected the active AOB extracted from the soil. By comparison, the correlation between AOB number estimated by the most-probable number (MPN) method and potential ammonia oxidation was considerably lower (R2=0.56), suggesting that MCFU–FISH counts better reflected the number of active AOB extracted from soil. Hence, the MCFU–FISH technique seems suitable for enumeration of viable and active AOB populations in soils, as long as the extractable population of microcolony-forming AOB is above a current minimum threshold for detection of 103–104 MCFU g−1 dry wt. soil.

Quantification of ammonia oxidizing bacteria in soil using microcolony technique combined with fluorescence in situ hybridization (MCFU–FISH)

A new technique was developed for quantification of viable ammonia oxidizing bacteria (AOB) extracted from soil. Extracted bacteria were grown to microcolony-forming units (MCFU) on membrane filters and selectively stained with 16S rDNA probes using fluorescence in situ hybridization (FISH). The MCFU–FISH technique was used to enumerate a Nitrosomonas europaea population after inoculation to soil and the indigenous AOB populations in native and N-enriched soils. A low extraction efficiency (0.3–3%) of AOB in the native soil indicated predominance of a sessile AOB cell type. In contrast, N enrichments increased extractability of AOB from soil particles. Comparing all soils, a close correlation ( R 2=0.98) existed between extractable AOB numbers (MCFU–FISH counts) and potential ammonia oxidation activities covering more than three orders of magnitude, which indicated that MCFU–FISH counts reflected the active AOB extracted from the soil. By comparison, the correlation between AOB number estimated by the most-probable number (MPN) method and potential ammonia oxidation was considerably lower ( R 2=0.56), suggesting that MCFU–FISH counts better reflected the number of active AOB extracted from soil. Hence, the MCFU–FISH technique seems suitable for enumeration of viable and active AOB populations in soils, as long as the extractable population of microcolony-forming AOB is above a current minimum threshold for detection of 10 3–10 4 MCFU g −1 dry wt. soil.