Nitrifying Bacterial Populations Research Articles

High-yielding nitrate transporter cultivars also mitigate methane and nitrous oxide emissions in paddy.

Development of high yield rice varieties is critical to ensuring global food security. However, the emission of greenhouse gases (GHG) such as Methane (CH4) and Nitrous oxide (N2O) from paddy fields threatens environmental sustainability. In this study, we selected overexpressed high-affinity nitrate transporters (NRT2.3 along with their partner protein NAR2.1) cultivars, which are effective nitrogen use efficient transgenic lines pOsNAR2.1: OsNAR2.1 (Ox2) and p35S:OsNRT2.3b (O8). We used high (270 kg N/ha) and low (90 kg N/ha) nitrogen (N) fertilizers in paddy fields to evaluate morphophysiological traits, including GHG emission. We found that Ox2 and O8 reduced CH4 emissions by 40% and 60%, respectively, compared to their wild type (WT). During growth stages, there was no consistent N2O discharge pattern between WT and transgenics (Ox2, O8) in low and high N application. However, total cumulative N2O in a cropping season reduced in O8 and increased in Ox2 cultivars, compared to WT. Root aerenchyma formation reduced by 30-60% in transgenic lines. Methanogens like mcrA in low and high N were also reduced by up to 50% from rhizosphere of Ox2 and O8. However, the nitrifying bacterial population such as nosZ reduced in both transgenics significantly, but nirK and nirS did not show a consistent variation. The high yield of transgenic rice with limited aerenchyma mitigates the discharge of CH4 and N2O by reducing root exudates that provide substrates for GHG. Our results improve understanding for breeders to serve the purpose of sustainable development.

Bioaugmentation of nitrifying bacteria in up-flow biological aerated filter's microbial community for wastewater treatment and analysis of its microbial community

Bioaugmentation is a bioremediation approach involving the seeding of the competent microbial consortia to improve nitrogen removal from wastewater. The discharge of nitrogen-saturated wastewater into freshwater results in cultural eutrophication and huge outbreak of harmful algal bloom. The purpose of this study was to relate the population size of bioaugmented ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) with nitrification performance in an experimental up-flow biological aerated filter (UBAF) for domestic wastewater treatment. Fluorescent in situ hybridization (FISH) was used to enumerate the portions of AOB and NOB in UBAFs. The FISH method disclosed that nitrifying bacteria represented 31.3% of the total population. The AOB (16.6%) outcompeted NOB (14.6%) after 15 days of bioaugmentation. Additionally, AOB/NOB ratio (1.13) showed a higher nitrification process. The average effluent from experimental UBAF exhibited high nitrogen and nutrients removal, the averaged removal efficiencies for NH4+-N, NNO3-, COD, BOD5 and phosphate were 85.2%, nitrate accumulation (2.2 times), 67.3%, 75.6%, and 58.3%, respectively. Therefore, an increase in the number of nitrifying microorganisms confirms the efficacy of microbial seeding and correlates with the physical-chemical data on bioconversion of nitrogenous compounds. However, on day 35, there was a disparate distribution pattern of the nitrifying bacteria (AOB, NOB) in the experimental UBAF, which in turn declined the nitrification performance.The study demonstrated the importance of monitoring the shifts of the nitrifying bacterial population in UBAF. Moreover, the bioaugmentation of nitrifying bacteria in UBAF packed with cost-effective and eco-friendly ceramsite could be a promising alternative technique for the domestic wastewater treatment.

Dissolved oxygen concentrations affect the function but not the relative abundance of nitrifying bacterial populations in full-scale municipal wastewater treatment bioreactors during cold weather

This study aimed to understand the effect of different dissolved oxygen (DO) concentrations on the abundance and performance of nitrifying bacteria in full-scale wastewater treatment bioreactors, particularly during the winter when nitrifying bacterial activity is often negligible. Biomass samples were collected from three parallel full-scale bioreactors with low DO concentrations (<1.3 mg/ L) and from two full-scale bioreactors with higher DO concentrations (~4.0 and ~2.3 mg/ L). The relative abundance of nitrifying bacteria was determined by sequencing of PCR-amplified 16S rRNA gene fragments. In the three bioreactors with low DO concentrations, effluent ammonia concentrations sharply increased with a decline in temperature below approximately 17 °C, while the bioreactors with high DO concentrations showed stable nitrification regardless of temperature. Even with the decline in nitrification during the winter in the three low DO bioreactors, the relative abundance of ammonia oxidizing bacteria (mostly Nitrosomonas spp.) was curiously maintained. The relative abundance of nitrite oxidizing bacteria was similarly maintained, although there were substantial seasonal fluctuations in the relative abundance values of Nitrospira spp. versus Nitrotoga spp. This research suggests that nitrification activity can be controlled during the winter via DO to produce better effluent quality with high DO concentrations or to reduce aeration costs with a concomitant decline in nitrification activity.

Influence of chlorothalonil and carbendazim fungicides on the transformation processes of urea nitrogen and related microbial populations in soil.

To improve crop yielding, a large amount of fungicides is continuously applied during the agricultural management, while the effects of fungicides residues on microbial processing of N in soil need further study. In the present study, two broad spectrum fungicides, chlorothalonil and carbendazim, were applied at the rates of 5, 10, and 50 mg of active ingredient (A.I.) per kg of dry soil combined with urea with 200 mg of N per kg of dry soil under laboratory conditions. The results showed that chlorothalonil obviously retarded the hydrolysis of urea, whereas carbendazim accelerated it in 4 days after the treatments (P < 0.05). Chlorothalonil reduced denitrification, nitrification, and N2O production (P < 0.05), but not for carbendazim. Further analysis on N-associated microbial communities showed chlorothalonil reduced nitrosomonas populations at the rates of 10 and 50 mg of A.I. per kg and autotrophic nitrifying bacterial populations at three application rates (P < 0.05), but Carbendazim decreased nitrosomonas populations only at the rate of 50 mg of A.I. per kg and also autotrophic nitrifying bacterial populations at three rates and heterotrophic nitrifying bacterial populations at the rates of 10 and 50 mg of A.I. per kg. The reasons for this difference were ascribed to arrest urea hydrolysis and impediment of denitrification and nitrification processes by chlorothalonil. In conclusion, to improve crop yielding, chlorothalonil might be more beneficial to conserve soil N by improving soil N fertility, compared with carbendazim.

The N-fixing legume Periandra mediterranea constrains the invasion of an exotic grass (Melinis minutiflora P. Beauv) by altering soil N cycling

Melinis minutiflora is an invasive species that threatens the biodiversity of the endemic vegetation of the campo rupestre biome in Brazil, displacing the native vegetation and favouring fire spread. As M. minutiflora invasion has been associated with a high nitrogen (N) demand, we assessed changes in N cycle under four treatments: two treatments with contrasting invasion levels (above and below 50%) and two un-invaded control treatments with native vegetation, in the presence or absence of the leguminous species Periandra mediterranea. This latter species was considered to be the main N source in this site due to its ability to fix N2 in association with Bradyrhizobia species. Soil proteolytic activity was high in treatments with P. mediterranea and in those severely invaded, but not in the first steps of invasion. While ammonium was the N-chemical species dominant in plots with native species, including P.mediterranea, soil nitrate prevailed only in fully invaded plots due to the stimulation of the nitrifying bacterial (AOB) and archaeal (AOA) populations carrying the amoA gene. However, in the presence of P. mediterranea, either in the beginning of the invasion or in uninvaded plots, we observed an inhibition of the nitrifying microbial populations and nitrate formation, suggesting that this is a biotic resistance strategy elicited by P. mediterranea to compete with M. minutiflora. Therefore, the inhibition of proteolytic activity and the nitrification process were the strategies elicited by P.mediterranea to constrain M.munitiflora invasion.

Application of hierarchical oligonucleotide primer extension (HOPE) to assess relative abundances of ammonia- and nitrite-oxidizing bacteria

BackgroundEstablishing an optimal proportion of nitrifying microbial populations, including ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), complete nitrite oxidizers (comammox) and ammonia-oxidizing archaea (AOA), is important for ensuring the efficiency of nitrification in water treatment systems. Hierarchical oligonucleotide primer extension (HOPE), previously developed to rapidly quantify relative abundances of specific microbial groups of interest, was applied in this study to track the abundances of the important nitrifying bacterial populations.ResultsThe method was tested against biomass obtained from a laboratory-scale biofilm-based trickling reactor, and the findings were validated against those obtained by 16S rRNA gene-based amplicon sequencing. Our findings indicated a good correlation between the relative abundance of nitrifying bacterial populations obtained using both HOPE and amplicon sequencing. HOPE showed a significant increase in the relative abundance of AOB, specifically Nitrosomonas, with increasing ammonium content and shock loading (p < 0.001). In contrast, Nitrosospira remained stable in its relative abundance against the total community throughout the operational phases. There was a corresponding significant decrease in the relative abundance of NOB, specifically Nitrospira and those affiliated to comammox, during the shock loading. Based on the relative abundance of AOB and NOB (including commamox) obtained from HOPE, it was determined that the optimal ratio of AOB against NOB ranged from 0.2 to 2.5 during stable reactor performance.ConclusionsOverall, the HOPE method was developed and validated against 16S rRNA gene-based amplicon sequencing for the purpose of performing simultaneous monitoring of relative abundance of nitrifying populations. Quantitative measurements of these nitrifying populations obtained via HOPE would be indicative of reactor performance and nitrification functionality.

The effects of salinity on nitrification using halophilic nitrifiers in a Sequencing Batch Reactor treating hypersaline wastewater.

With annual increases in the generation and use of saline wastewater, the need to avoid environmental problems such as eutrophication is critical. A previous study identified ways to start up a halophilic sludge domesticated from estuarine sediments to remove nitrogen from wastewater with a salinity of 30 g/L. This investigation expands that work to explore the impact of salinity on nitrogen removal. This study demonstrated that the mixed halophilic consortia removed nitrogen from wastewater with a salinity of 30–85 g/L. A kinetic analysis showed that halophilic nitrifiers selected based on hypersalinity were characterized by low Ks, μmax and specific ammonium oxidization rates. This explains the decrease in ammonium removal efficiency in the high salinity operational phases. Salinity inhibited ammonia oxidizing bacteria (AOB) activity, as well as the number of dominant AOB, but did not significantly affect the AOB dominant species. Three most dominant AOB lineages in the halophilic sludge were Nitrosomonas marina, Nitrosomonas europaea, and Nitrosococcus mobilis. Nitrosomonas europaea and Nitrosococcus mobilis were mainly affected by salinity, while nitrite accumulation and ammonia loading played the key role in determining the abundance of Nitrosococcus mobilis and Nitrosococcus europaea. The study contributes insights about shifts in halophilic nitrifying bacterial populations.

Storage of Pacific oysters Crassostrea gigas in recirculating tank: Ammonia excretion and potential nitrification rates

In response to the closure of shellfish production sites due to increasing occurrence of toxic algal blooms, land-based recirculating aquaculture systems (RAS) could be used by producers to store a proportion of their stock temporarily. An RAS prototype was tested in this study to store Pacific oyster Crassostrea gigas over a range of temperatures. In such systems, the water temperature is a variable that could influence the bivalve excretion rate of ammonia, the concentration of which may become critical for the water quality. In this study, we first estimated the total ammonia nitrogen (TAN, N-NH4++N-NH3) excretion of the Pacific oyster and then investigated whether the bacterial population associated with the oysters could act as a natural biofilter by measuring the potential nitrification rate (PNR) of the shell. The TAN excretion rate varied significantly with temperature. The PNRs were significantly different depending on the shell surface considered with a PNR of 0.42±0.11 (SD)nmolNcm−2h−1 for the internal part, 0.96±0.15 (SD)nmolNcm−2h−1 for the external part of the shell. In addition, a 5-week experiment was conducted with a 180kg of oyster stock stored in a 1900L RAS to monitor TAN concentration and to establish a TAN budget at the level of an oyster population. The TAN concentration sharply declined in the RAS after 6 days and a stable concentration of 3.3±0.91 (SD)μmolNL−1 was reached till the end of the experiment. This measurement, as well as the estimation of a theoretical TAN budget based on the previous experiments, showed that the PNR and some other associated processes were sufficient to counteract the ammonia fluxes excreted by the oysters. This result underlines the significant role played by the nitrifying bacterial population colonizing the bivalve shell and suggests that such an oyster stock could be stored in a recirculating tank without the addition of a biofilter, which would represent a real economic advantage.

Physiological characteristics of predominant ammonia-oxidizing bacteria enriched from bioreactors with different influent supply regimes

Two acclimatized biomasses exposed to ammonium (NH4+) concentration of 600mg NL−1, one from a completely stirred tank reactor (CSTR), the other from a sequencing batch reactor (SBR), were assayed for nitritation performance, predominant nitrifying bacterial population and nitrous oxide (N2O) production. By virtue of fluctuating and constant NH4+ concentrations respectively, the SBR and CSTR wastewater supply regimes were hypothesized to support different predominant ammonia-oxidizing bacteria (AOB) exhibiting distinct biokinetic properties. Nitritation efficiency (NO2−-N/NO2+3−-N) was higher in the SBR (89%) than the CSTR (30%) likely due to free ammonia and dissolved oxygen concentration. Quantitative fluorescence in situ hybridization (FISH) analyses revealed that fast-growing (r-strategist) AOB of halophilic and halotolerant Nitrosomonas lineage were more highly enriched in the SBR (76±4.2%) than the CSTR (38±6.0%). The CSTR predominantly enriched slow-growing (K-strategist) AOB Nitrosospira spp. (42±1.9% versus 1.4±0.8% in the SBR). Biokinetic parameter estimation consolidated the FISH result: the maximum growth rate and half-saturation coefficients for NH4+ were higher in the SBR (μmax=0.92 day, KNH4+=28.9mg NL−1) relative to the CSTR (μmax=0.42 day, KNH4+=3.47mg NL−1), suggesting that the extent of nitritation may be controlled by choice of wastewater influent operational regime, which itself determines predominant AOB. N2O production was a maximum of 25 times higher (10.2mg N-N2Oh−1 at 0.5mg O2L−1) in CSTR-enriched biomass than in SBR-enriched biomass (0.41mg N-N2Oh−1 at 0.5mg O2L−1).

Nutrient removal performance and microbial community analysis of a combined ABR–MBR (CAMBR) process

In order to reduce the external nutrient loads in Lake Taihu, it was found that controlling nutrient input is the most effective way of reducing the risk of blooms. In this regard, nutrient removal by using a combined process consisting of an anaerobic baffled reactor and an aerobic membrane bioreactor for municipal wastewater treatment was investigated. The results demonstrated that hydraulic retention time, recycle ratio and dissolved oxygen had a negligible effect on the removal efficiencies of chemical oxygen demand and ammonia. Optimal operation conditions with a hydraulic retention time of 7.5h at a recycle ratio of 200% and dissolved oxygen of 3mg/L was shown to achieve higher quality effluent with chemical oxygen demand, ammonium, total nitrogen and total phosphorus of 26, 0.5, 10.5 and 0.4mg/L, respectively. Analysis of fluorescence in situ hybridization showed that the specialized microorganisms, including nitrifying bacteria (ammonia-oxidizing bacteria and nitrite-oxidizing bacteria), and phosphorus accumulating organisms, were enriched in this process. The profile of nitrite-oxidizing bacteria was similar to that of ammonia-oxidizing bacteria. The nitrifying bacterial population had a good correlation with nitrogen removal efficiency. A good correlation was also observed between phosphorus removal performance and the percentage of phosphorus accumulating organisms present in the sludge.

Fate and toxicity of melamine in activated sludge treatment systems after a long-term sludge adaptation

Melamine is a nitrogen-rich (67% nitrogen by mass) heterocyclic aromatic compound that could significantly increase effluent total nitrogen concentrations. In this study, we investigated the degradation of melamine and its impact on activated sludge operations by employing two common activated sludge processes, namely the Modified Ludzack-Ettinger (MLE) process and the continuous stirred tank reactor (CSTR) process. Melamine was dosed continuously from day 125 in both activated sludge treatment systems at an influent concentration of 3 mg/L for about 100 days. Even after such a long period of sludge adaptation, melamine appeared not to be easily biodegradable. The average melamine removal efficiencies in the CSTR and MLE systems were 14 ± 10% and 20 ± 15%, respectively. There was no significant difference in melamine removal between the two different activated sludge processes. The long-term input of melamine resulted in a decrease in the nitrifying bacterial activities (by 82 ± 8%) and population in both systems. Short-term microtiter assay results also showed that melamine reduced activated sludge growth by 80% when supplied at a concentration of 75.6 mg/L. These results suggest that sludge adaptation plays a minimal role in melamine degradation, as the enzymes responsible for hydrolytic deamination of melamine in activated sludge are not easily induced. The insignificant biodegradation of melamine is also attributed to bacterial growth inhibition under long-term dosing conditions with melamine, resulting in a significant decrease in effluent water quality.

Laboratory investigations of the effects of nitrification-induced acidification on Cr cycling in vadose zone material partially derived from ultramafic rocks

Sacramento Valley (California, USA) soils and sediments have high concentrations of Cr(III) because they are partially derived from ultramafic material. Some Cr(III) is oxidized to more toxic and mobile Cr(VI) by soil Mn oxides. Valley soils typically have neutral to alkaline pH at which Cr(III) is highly immobile. Much of the valley is under cultivation and is both fertilized and irrigated. A series of laboratory incubation experiments were conducted to assess how cultivation might impact Cr cycling in shallow vadose zone material from the valley. The first experiments employed low (7.1mmolN per kg soil) and high (35mmolNkg−1) concentrations of applied (NH4)2SO4. Initially, Cr(VI) concentrations were up to 45 and 60% greater than controls in low and high incubations, respectively. After microbially-mediated oxidation of all NH4+, Cr(VI) concentrations dropped below control values. Increased nitrifying bacterial populations (estimated by measurement of phospholipid fatty acids) may have increased the Cr(VI) reduction capacity of the vadose zone material resulting in the observed decreases in Cr(VI). Another series of incubations employed vadose zone material from a different location to which low (45meqkg−1) and high (128meqkg−1) amounts of NH4Cl, KCl, and CaCl2 were applied. All treatments, except high concentration KCl, resulted in mean soil Cr(VI) concentrations that were greater than the control. High concentrations of water-leachable Ba2+ (mean 38μmolkg−1) in this treatment may have limited Cr(VI) solubility. A final set of incubations were amended with low (7.1mmolNkg−1) and high (35mmolNkg−1) concentrations of commercial liquid ammonium polyphosphate (APP) fertilizer which contained high concentrations of Cr(III). Soil Cr(VI) in the low APP incubations increased to a concentration of 1.8μmolkg−1 (5× control) over 109days suggesting that Cr(III) added with the APP fertilizer was more reactive than naturally-occurring soil Cr(III).

Applied Method of Fluorescence In Situ Hybridization (FISH) with Luminescence Spectrometer for Estimating the Activity of Nitrifying Bacteria

The ability to detect and enumerate the specific bacteria is essential for determining their fate in various biological processes. Although fluorescent in situ hybridization (FISH) technique using rRNA-targeted oligonucleotide probes may be considered as the most rapid method for identification of a single bacterial cell, it could not efficiently count cells due to the limitations of light microscopy. In this study, to make accurate estimates of the active nitrifying bacterial populations from activated sludge samples, applied FISH method using Luminescence Spectrometer LS50B was used. Enumeration of nitrifying bacteria by FISH using Luminescence Spectrometer can help in the optimization of the performance of biological nitrogen removal processes.

Biomass characteristics of two types of submerged membrane bioreactors for nitrogen removal from wastewater

Biomass characteristics and microbial community diversity between a submerged membrane bioreactor with mixed liquor recirculation (MLE/MBR) and a membrane bioreactor with the addition of integrated fixed biofilm medium (IFMBR) were compared for organic carbon and nitrogen removal from wastewater. The two bench-scale MBRs were continuously operated in parallel at a hydraulic retention time (HRT) of 24 h and solids retention time (SRT) of 20 d. Both MBRs demonstrated good COD removal efficiencies (>97.7%) at incremental inflow organic loading rates. The total nitrogen removal efficiencies were 67% for MLE/MBR and 41% for IFMBR. The recirculation of mixed liquor from aerobic zone to anoxic zone in the MLE/MBR resulted in higher microbial activities of heterotrophic (46.96 mg O 2/gVSS h) and autotrophic bacteria (30.37 mg O 2/gVSS h) in the MLE/MBR compared to those from IFMBR. Terminal Restriction Fragment Length Polymorphism analysis indicated that the higher nitrifying activities were correlated with more diversity of nitrifying bacterial populations in the MLE/MBR. Membrane fouling due to bacterial growth was evident in both the reactors. Even though the trans-membrane pressure and flux profiles of MLE/MBR and IFMBR were different, the patterns of total membrane resistance changes had no considerable difference under the same operating conditions. The results suggest that metabolic selection via alternating anoxic/aerobic processes has the potential of having higher bacterial activities and improved nutrient removal in MBR systems.

Impact of pre-treatments on nitrifying bacterial community analysis from wastewater using fluorescent in situ hybridization and confocal scanning laser microscopy

Fluorescent in situ hybridization (FISH) and confocal scanning laser microscopy (CSLM) are the key techniques used to investigate bacterial community structure at wastewater treatment plants. An optimum nitrifying bacterial population is necessary for nitrification, which plays a significant ecological role in regulating the overall quality of water. Nitrifying bacteria mainly appear as dense aggregates within activated sludge flocs. The impacts of five different pre-treatment methods (physical, chemical, enzymatic and combinations) on floc dispersion from two different wastewater treatment plants were determined. The effect of pre-treatment on the enumeration of the nitrifying bacterial population was also investigated. This study on floc dispersion using CSLM images showed sonication was the superior method for all the samples tested, irrespective of the sludge type. For samples from industrial wastewater plants, an optimized sonication level of 8 W for 8 min could reduce the floc size to 10 microm, whereas for domestic wastewater samples, the floc size was reduced to 10 microm at 8 W for 5 min. The maximum number of nitrifying bacterial cells was observed at this optimized level for different samples. A decrease in the number of cells was observed beyond this optimized level for both the plants. The results presented here highlight the importance of optimizing pre-treatment methods for different types of wastewater for accurate bacterial community analysis using FISH-CSLM.

Long-term assessment of nitrification in a full-scale wastewater treatment plant

In this study, long term nitrification performances and microbial composition in a full-scale oxidation ditch type wastewater treatment plant (WWTP) were monitored and evaluated using different molecular methods; fluorescent in situ hybridization (FISH), slot-blot hybridization and quantitative real-time polymerase chain reaction (PCR) method. In situ and membrane hybridization results indicated that Nitrosomonas species were identified as the dominant ammonia oxidizing bacteria and Nitrospira related species were detected as the prevailing nitrite oxidizing bacteria in a full-scale wastewater treatment plant throughout 2 years of operation. Real-time PCR using the LightCyclerinstrument has been developed for the quantification of ammonia monooxygenase (amoA) and 16S rRNA genes. Results suggest that real-time PCR analysis, amoA/16S rRNA ratio, is an alternative method to understand nitrifying bacterial population and activity in wastewater treatment plant compared with the FISH and slot-blot hybridization assays. The autotrophic/heterotrophic bacterial ratio and their influence on reactor performances were investigated using real-time PCR amoA/16S rRNA gene copy ratios and the results showed that this ratio varied from 3.6% to 8.3% during operational period.

The variation of nitrifying bacterial population sizes in a sequencing batch reactor (SBR) treating low, mid, high concentrated synthetic wastewater

The purpose of this study was to correlate the population size of ammonia-oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) with nitrification performance under various operational conditions (chemical oxygen demand (COD) concentration, dissolved oxygen (DO), and hydraulic retention time (HRT)) and influent allylthiourea (ATU) shock. The AOB (genera Nitrosomonas and Nitrosospira ) and NOB (genera Nitrobacter and Nitrospira ) communities were analyzed using fluorescent in situ hybridization (FISH). Ammonia-oxidizing bacteria and NOB accounted for 6.2 ± 0.9% and 2.5 ± 0.3% in total biomass, respectively. The population sizes of AOB and NOB varied with different levels of COD, DO, and HRT. Nitrosomonas and Nitrospira were dominant under conditions favorable for nitrification, while Nitrosospira outcompeted Nitrosomonas under adverse conditions (low [NH4+], low DO, short HRT, and ATU shock), and Nitrobacter outcompeted Nitrospira at high substrate concentrations (COD and [NH4+]). Under ATU shock that inhibited the oxidation of NH4+ to NO2–, AOB population was substantially reduced with the stepwise increase of ATU dosage, and led to a corresponding decrease of NOB population. There was a discrepancy between nitrifying bacterial populations and their functions. Although AOB outnumbered NOB in all tests and became more dominant at low DO and short HRT, NH4+ oxidation, instead of NO2– oxidation, was the rate-limiting reaction for nitrification and susceptible to the adverse conditions. The study demonstrated the importance of elucidating the shifts of nitrifying bacterial population to optimize process design and operation at different influent characteristics, aeration intensity, retention time, and potential influent toxic shock.

Impact of Selected Pollutants in Synthetic Industrial Wastewater on Nitrifying Biofilms in Fixed Bed Biofilm Reactors‐Visualized with Fluorescence In‐Situ Hybridization

The impact of selected pollutants on the structure and activity of nitrifying bacterial populations within fixed bed biofilm reactors was examined. Clofibrinic acid and ibuprofen as pharmaceutically active compounds were added to a synthetic wastewater fed to the biofilm reactors and the concentrations of chloride and dissolved organic carbon were also varied. Clear impacts of increased salt and dissolved organic carbon concentrations on nitrification activity and nitrifying bacterial populations were observed whereas clofibrinic acid (up to 100 µg/l) and ibuprofen (10 µg/l) showed no impact on the nitrification activity. The exposure to clofibrinic acid also showed no significant influence on the nitrifying bacterial population, while the addition of ibuprofen resulted in a slight shift of the population to a lower abundance of the β‐proteobacteria.

Experimental and simulation analysis of community structure of nitrifying bacteria in a membrane-aerated biofilm

Until now, only few attempts have been made to assess biofilm models simulating microenvironments in a biofilm. As a first step, we compare the microenvironment observed in a membrane aerated biofilm (MAB) to that derived from a two-dimensional computational model with individual ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) embedded in a continuum EPS matrix. Gradients of oxygen were determined by means of microelectrodes. The change in nitrifying bacterial populations with the biofilm depth was quantified using fluorescence in situ hybridization (FISH) in combination with a confocal laser scanning microscopy (CLSM). Microelectrode measurements revealed that oxic and anoxic or anaerobic regions exist within the MAB. The oxygen profile predicted by the model showed good agreement with that obtained by microelectrode measurements. The oxic part of the biofilm was dominated by NSO190 probe-hybridized AOB, which formed relatively large clusters of cells directly on the membrane surface, and by the NOB belonging to genus Nitrobacter sp. On the other hand, NOB belonging to genus Nitrospira sp. were abundant at the oxic-anoxic interface. The model prediction regarding AOB and Nitrobacter sp. distribution was consistent with the experimental counterpart. Measurements of AOB cluster size distribution showed that colonies are slightly larger adjacent to the membrane than at the inner part of the biofilm. The sizes predicted by the current model are larger than those obtained in the experiment, leading to the arguments that some factors not contained in the model would affect the cluster size.

Identification of nitrifiers in a full-scale biological treatment system using fluorescent in situ hybridization

Diversity of nitrifying bacterial population was investigated in sludge samples taken from a full-scale biological wastewater treatment plant (WWTP) treating domestic wastewater by fluorescent in situ hybridization (FISH) during seasonal operation. Duplicate grab samples were collected in March 2003, June 2003, December 2003 and May 2004 from the aerobic tank of the treatment plant. FISH results were interpreted with system performance in terms of BOD5, TKN and NO3-N removals and also with operational parameters such as wastewater temperature and sludge age. BOD5 removal efficiencies were always greater than 90% whilst TKN removal in a range of 69–95% were achieved during the monitoring period. Although there were variations in operational conditions of the biological treatment system both Nitrosomonas and Nitrosospira genera from AOB and Nitrobacter genus from NOB were found to be present in all samples examined.