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Comparative analysis of nitrifying bacteria associated with freshwater and marine aquaria.

Three nucleic acid probes, two for autotrophic ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria and one for alpha subdivision nitrite-oxidizing bacteria, were developed and used to study nitrifying bacterial phylotypes associated with various freshwater and seawater aquarium biofilters. Nitrosomonas europaea and related species were detected in all nitrifying seawater systems and accounted for as much as 20% of the total eubacterial rRNA. In contrast, nitrifying bacteria belonging to the beta-proteobacterial subdivision were detected in only two samples from freshwater aquaria showing vigorous nitrification rates. rRNA originating from nitrite-oxidizing alpha subdivision proteobacteria was not detected in samples from either aquarium environment. The data obtained indicate that chemolithotrophic ammonia oxidation in the freshwater aquaria was not due to beta-proteobacterial phylotypes related to members of the genus Nitrosomonas and their close relatives, the organisms usually implicated in freshwater nitrification. It is likely that nitrification in natural environments is even more complex than nitrification in these simple systems and is less well characterized with regard to the microorganisms responsible.

Ammonia↗

Recovery of microbially mediated processes in soil augmented with a pentachlorophenol-mineralizing bacterium.

Specific physiological groups can be used to evaluate the recovery of soil microbial communities following disturbance. In this study, soil was contaminated with pentachlorophenol (PCP) to assess the resiliency of microorganisms responsible for carbon and nitrogen cycling. Methane fluxes were monitored in soil microcosms to evaluate the effects of contamination and augmentation on microbial populations involved in carbon cycling. The addition of a PCP-mineralizing bacterium, Sphingomonas chlorophenolica strain RA2, enhanced the recovery of methane-oxidizing capacity relative to uninoculated treatments when soil was contaminated with low concentrations of PCP (10 and 50 microg/g soil, ppm). At the highest level of PCP contamination tested (300 ppm), there was no recovery of methane-oxidizing capacity whether or not the PCP-mineralizing bacterium was added to soil. The nitrogen-cycling capacity of contaminated and augmented soil was tested by measuring nitrification potentials. The addition of PCP resulted in a concentration-dependent reduction in nitrification rates. After three months of incubation, all augmented soils had nitrification rates equivalent to the uncontaminated control, and the uninoculated soils contaminated with 100 and 300 ppm PCP still were impaired in their nitrification potentials. These results indicate that biological removal of a contaminant has the potential to restore microbially mediated processes to levels observed prior to contamination.

Ammonia↗

Nitrite formation and nitrous oxide emissions as affected by reclaimed effluent application.

The effect of irrigation with reclaimed effluent (RE) (after secondary treatment) on the mechanisms and rates of nitrite formation, N2O emissions, and N mineralization is not well known. Grumosol (Chromoxerert) soil was incubated for 10 to 14 d with fresh water (FW) and RE treated with 15NO3- and 15NH4+ to provide a better insight on N transformations in RE-irrigated soil. Nitrite levels in RE-irrigated soil were one order of magnitude higher than in FW- irrigated soil and ranged between 15 to 30 mg N kg(-1) soil. Higher levels of NO2- were observed at a moisture content of 60% than at 70% and 40% w/w. Nitrite levels were also higher when RE was applied to a relatively dry Grumosol (20% w/w) than at subsequent applications of RE to soil at 40% w/w. Isotopic labeling indicated that the majority of NO2 was formed via nitrification. The amount of N2O emitted from RE-treated Grumosol was double the amount emitted from FW treatments at 60% w/w. Nitrification was responsible for about 42% of the emissions. The N20 emission from the RE-treated bulk soil (passing a 9.5-mm sieve) was more than double the amount formed in large aggregates (4.76-9.5 mm in diameter). No dinitrogen was detected under the experimental conditions. Results indicate that irrigation with secondary RE stimulates nitrification, which may enhance NO3 leaching losses. This could possibly be a consequence of long-term exposure of the nitrifier population to RE irrigation. Average gross nitrification rate estimates were 11.3 and 15.8 mg N kg(-1) soil d(-1) for FW- and RE-irrigated bulk soils, respectively. Average gross mineralization rate estimates were about 3 mg N kg(-1) soil d(-1) for the two water types.

Agriculture↗

Innovations in wastewater treatment: the moving bed biofilm process.

This paper describes the moving bed biofilm reactor (MBBR) and presents applications of wastewater treatment processes in which this reactor is used. The MBBR processes have been extensively used for BOD/COD-removal, as well as for nitrification and denitrification in municipal and industrial wastewater treatment. This paper focuses on the municipal applications. The most frequent process combinations are presented and discussed. Basic design data obtained through research, as well as data from practical operation of various plants, are presented. It is demonstrated that the MBBR may be used in an extremely compact high-rate process (<1 h total HRT) for secondary treatment. Most European plants require P-removal and performance data from plants combining MBBR and chemical precipitation is presented. Likewise, data from plants in Italy and Switzerland that are implementing nitrification in addition to secondary treatment are presented. The results from three Norwegian plants that are using the so-called combined denitrification MBBR process are discussed. Nitrification rates as high as 1.2 g NH4-N/m2 d at complete nitrification were demonstrated in practical operation at low temperatures (11 degrees C), while denitrification rates were as high as 3.5g NO3-Nequiv./m2.d. Depending on the extent of pretreatment, the total HRT of the MBBR for N-removal will be in the range of 3 to 5 h.

Biofilms↗

Dynamics of the nitrogen transformation in a shallow stream and possible interventions.

This paper presents the assessment of the efficiency of the main biological nitrogen transformation processes in a shallow well-oxygenated river and conditions under which they are active and stabilise. The process dynamics was studied with the help of mathematical modelling of 2 years on-line data series measured in a reach of the Toess River, Switzerland. The algal nitrogen uptake was very stable and unaffected by most but frequent flood events. Daylight photosynthetic nitrogen uptake stabilised at 6 mgN mstreambed(-2) h(-1) (15 degrees C), dark uptake on storage products at rates of 0.5-2.5 mgN mstreambed(-2) h(-1). Nitrogen uptake by heterotrophic bacteria in the hyporheic zone was relatively constant at a level of 1.5-3.5 mgN mstreambed(-2) h(-1). Streambed nitrification could establish only during periods with average an daily concentration of at least 0.3 g(NH4-N) m(-3) in river water for several weeks. The maximum nitrification rate was 35 mgN mstreambed(-2) h(-1) for 3 g(NH4-N) m(-3). The effects of reduced nitrification in the WWTP and of river banks shading on a sudden ammonium peak were simulated. A river reach endangered by ammonium spills should be kept open to sun to favour ammonium uptake by algae. In-stream nitrification reduces ammonium peaks efficiently but leads to toxic nitrite concentrations.

Bacteria, Aerobic↗

A new process for enriching nitrifiers in activated sludge through separate heterotrophic wasting from biofilm carriers.

A new process, the biofilm-activated sludge innovative nitrification (BASIN) process, consisting of a moving-bed biofilm reactor (MBBR) with separate heterotrophic wasting, followed by an activated-sludge process, has been proposed to reduce the volumetric requirements of the activated-sludge process for nitrification. The basic principle is to remove chemical oxygen demand on the biofilm carriers by heterotrophic organisms and then to waste a portion of the heterotrophic biomass before it can be released into the activated-sludge reactor. By this means, the amount of heterotrophic organisms grown in the activated-sludge reactor is reduced, thereby reducing the volume of that tank needed for nitrification. For nitrification applications, the simplest method for stripping biomass was to use an in-tank technique using high shearing rates with aeration. Bench-scale testing showed sludge yields in the BASIN process were one-half of that in a control activated-sludge process and twice that of a process line with intermediate settling between the MBBR and activated-sludge stage. Critical washout solids retention times for nitrifiers were the same for all three lines, so activated-sludge volumes for the BASIN process could be reduced by 50% compared with the control. Originally conceived process concepts for the BASIN process were confirmed by the experimental work.

Algorithms↗

Nitrifying biofilm development with time: activity versus phylogenetic composition.

The development of nitrifying biofilms collected from a full-scale nitrifying trickling filter was evaluated through the application of fluorescent in situ hybridization (FISH) and by quantification of nitrification rates in bench-scale reactors. Two sampling campaigns were conducted to evaluate the structure and function of biofilms between 14 and 70 days old. The structure, or number of ammonia-oxidizing bacteria, was quantified with Nso190. The function was quantified with bench-scale nitrification rates. The two assays were compared by calculating correlation coefficients by simple linear regression of the two data sets. The number of ammonia-oxidizing bacteria closely tracked activity data (linear correlation, r2 > 0.500). Changes in ammonia-oxidation capacity with time (7-day intervals) were mirrored by shifts in the percent of ammonia-oxidizing bacteria present. Nitrification rates did not correlate to the EUB338-probe stained area (r2 < 0.500), suggesting that nonnitrifying bacteria play a larger role in nitrifying biofilm ecology than previously thought. Dry-weight biomass accumulations did not correlate to either the EUB338-probe stained area or the bench-scale nitrification rates. This suggests that inert materials accumulate in the biofilms over time.

Ammonia↗

Acetate injection into anaerobic settled sludge for biological P-removal in an intermittently aerated reactor.

Injecting acetate into the sludge layer during the settling and decanting periods was adopted to enhance phosphorus release inside the sludge layer during those periods and phosphorus uptake during the subsequent aeration period in a KIST Intermittently Decanted Extended Aeration (KIDEA) process. The relationship among nitrification, denitrification and phosphorus removal was investigated in detail and analyzed with a qualitative floc model. Dependencies of nitrification on the maximum DO level during the aerobic phase and phosphorus release on residual nitrate concentration during the settling phase were significant. High degree of nitrification resulted that phosphorus release inside the sludge layer was significantly interfered with nitrate due to the limitation of available acetate and the carbon sources from influent. Such limitation was related to the primary utilization of organic substance for denitrification in the outer layer of the floc and the retarded mass transfer into the inner layer of the floc. Nevertheless, effects of acetate injection on both denitrification and phosphorus release during the settling phase were significant. Denitrification rate after acetate injection was two times as high as that before acetate injection, and phosphorus release reached about 14 mg PO4(3-)-P/g MLVSS/hr during the decanting phase after the termination of denitrification inside the sludge layer. Extremely low level of maximum DO (around 0.5 mg/L) during the aerobic phase may inhibited nitrification, considerably, and thus nearly no nitrate was present. However, the absence of nitrate increased when the phosphorus release rate was reached up to 33 mg PO4(3-)-P/g MLVSS/hr during the settling and decanting phase, and nearly all phosphorus was taken up during subsequent aerobic phase. Since the sludge layer could function as a blocking layer, phosphorus concentrations in the supernatant was not influenced by the released phosphorus inside the sludge layer during the settling and decanting period. Phosphorus removal was directly (for uptake) and indirectly (for release) dependent on the median and maximum DO concentration during the aerobic phase, and those optimal values may exist within the range from 0.2 to 0.6 mg/L and 0.4 to 1.2 mg/L, respectively.

Acetates↗

Molecular analysis of ammonia-oxidizing bacterial populations in aerated-anoxic orbal processes.

Aerated-anoxic processes operate under the principle that small additions of oxygen to an anoxic reactor induce simultaneous nitrification and denitrification. In these systems, ammonia oxidation in the anoxic zone can easily account for 30-50% of the total nitrification in the reactor, even though the dissolve oxygen concentration is usually below detection limit. To investigate whether the nitrification efficiency in aerated-anoxic processes was due to the presence of specialized ammonia-oxidizing bacteria (AOB), an analysis of the AOB population in an aerated-anoxic Orbal process and a conventional nitrogen removal process was carried out using phylogenetic analyses based on the ammonia monooxygenase A (amoA) gene. Terminal restriction fragment length polymorphism (TRFLP) analyses revealed that Nitrosospira-like organisms were one of the major contributors to ammonia oxidation in a full-scale aerated-anoxic Orbal reactor. However, the relative populations of Nitrosospira-like and Nitrosomonas-like AOB were not constant and appeared to have seasonal variability. Cloning and sequence comparison of amoA gene fragments demonstrated that most of the AOB in the aerated-anoxic Orbal process belonged to the Nitrosospira sp. and Nitrosomonas oligotropha lineages. The abundance of Nitrosospira-like organisms in aerated-anoxic reactors is significant, since this group of AOB has not been usually associated with nitrification in wastewater treatment plants.

Ammonia↗

Simultaneous removal of nitrogen and phosphorus with A2/O process using immobilized media.

It is necessary to introduce nitrogen and phosphorus removal processes in the existing wastewater treatment plants of Fukuoka City to improve the water quality of Hakata Bay. An A2/O (anaerobic-anoxic-oxic) process using immobilized media is proposed here, as a high-end technology to meet stricter discharge standards when faced with treatment space limitations. In order to investigate the applicability of the process, a pilot-scale study using municipal wastewater was conducted. Fluidized immobilization media were added to the aerobic reactor to maintain a high concentration of nitrifying bacteria and nitrification rate. Relationships between nutrient removal performance and varied operating conditions were examined. The results of the pilot plant test indicated that the process performs well for removal of nitrogen and phosphorus. Under the condition of HRT = 8 hr, it was possible to obtain an effluent water quality as follows: T-N<8.0 mg/L; T-P<1.0 mg/L. It was observed that reaction time needed for a complete nitrification was shortened by the addition of immobilized media to the aerobic reactor, and longer reaction time would be needed for a completed phosphorus uptake process other than complete nitrification in an aerobic tank. Therefore, the phosphorus uptake rate, as well as nitrification process, should be considered as one of the important factors affecting the design parameters of an aerobic reactor.

Bacteria, Aerobic↗

Oxygen flux implications of observed nitrogen removal rates in subsurface-flow treatment wetlands.

Nitrification, an oxygen-requiring microbial process, is generally considered the rate-limiting step for N removal in subsurface-flow constructed wetlands treating organic wastewaters. We used a simplified model of sequential N transformations and sinks to infer required rates of oxygen supply at 5 stages along experimental wetland mesocosms supplied with four different organic wastewaters with contrasting ratios of COD: N and forms of N. Mass balances of water-borne organic, ammoniacal and nitrate N, and plant and sediment N uptake showed average net rates of N mineralisation ranging from 0.22-0.53 g m(-2) d(-1), nitrification 0.56-2.15 g m(-2) d(-1), denitrification 0.47-1.99 g m(-2) d(-1) (60-84% of measured N removal) and plant assimilation 0.28-0.47 g m(-2) d(-1). The nitrogenous oxygen demand (NOD) required to support the observed nitrification rates alone was high compared to expected fluxes from surficial and plant-mediated oxygen transfer. In the presence of high levels of degradable organic matter (COD removal rates up to 66 g m(-2) d(-1)), heterotrophs with significantly higher oxygen affinities and energy yields are expected to outcompete nitrifiers for available oxygen. Problems with commonly held assumptions on the nature of coupled nitrification-denitrification in treatment wetlands are discussed.

Biodegradation, Environmental↗

Advanced nitrogen elimination by encapsulated nitrifiers.

By introducing a mixed population of nitrifiers encapsulated in gel lens beads a more selective nitrification process was found in treatment of settled sewage in lab scale at a hydraulic retention time (HRT) of about 30 to 60 minutes. The reaction rates for oxidation of soluble chemical oxygen demand (SCOD) were found to vary between 25 to 150 mg/L x h while nitrification takes place around 50 mg nitrogen per hour and litre reaction volume. However, based on this SCOD removal in the nitrification step, a consequent post-denitrification process without nitrate recycle and dosage of external carbon sources has been proven to reach substantial nitrate elimination of up to 20 mg nitrogen per litre at COD/N-ratios of approx. 6 in settled sewage. At such COD/N-ratios, suitable nitrogen elimination seems to be possible, because the bioflocs of settled sewage, produced so far by SCOD oxidation and entrapment of particulate COD, are passing through the nitrification process having a substantial contribution to the denitrification rate additionally to the remaining SCOD.

Bioreactors↗

Nitrite accumulation followed by denitrification using sequencing batch reactor.

Biological ammonia-nitrogen removal utilizes two distinct processes, nitrification and denitrification. In nitrification, ammonia oxidizes to nitrite then to nitrate. In this study, elimination of nitrite oxidation to nitrate step was attempted in order to directly remove nitrite to nitrogen gas by denitrification. For this study the supernatant from an anaerobic digester was used as an ammonia source and a sequencing batch reactor (SBR) was employed. Emphasis was given to the evaluation of the operational factors affecting nitrite accumulation and the elucidation of kinetics for biological nitrification and denitrification. Accumulation of nitrite in the nitrification process was achieved by suppressing the growth of Nitrobacter, a nitrite oxidizer, by loading high concentration ammonia supernatant immediately after all ammonia in the previous loading was oxidized to nitrite. Nitrite oxidation was taking place as the solid retention time (SRT) was increased from 2.5 days to 3.0 days in a continuously aerated SBR mode with daily feeding. However, nitrite accumulation was achieved even at longer SRT of 5 days when the aeration and non-aeration periods were appropriately combined and the non-aeration period can be used for denitrification of the accumulated nitrite with a carbon source supplied.

Ammonia↗

Fluorescence in situ hybridization analysis of nitrifiers in piggery wastewater treatment reactors.

Fluorescence in situ hybridization (FISH) was performed to analyze the nitrifying microbial communities in an activated sludge reactor (ASR) and a fixed biofilm reactor (FBR) for piggery wastewater treatment. Heterotrophic oxidation and nitrification were occurring simultaneously in the ASR and the COD and nitrification efficiencies depend on the loads. In the FBR nitrification efficiency also depends on ammonium load to the reactor and nitrite was accumulated when free ammonia concentration was higher than 0.2 mg NH3-N/L. FISH analysis showed that ammonia-oxidizing bacteria (NSO1225) and denitrifying bacteria (RRP1088) were less abundant than other bacteria (EUB338) in ASR. Further analysis on nitrifying bacteria in the FBR showed that Nitrosomonas species (NSM156) and Nitrospira species (NSR1156) were the dominant ammonia-oxidizing and nitrite-oxidizing bacteria, respectively, in the piggery wastewater nitrification system.

Animals↗

[Treatment performance of submerged membrane bioreactor treating brewery wastewater].

Under the condition of keeping the influent COD: TN: TP = 100:5:1, submerged MBR has an excellent treatment performance for COD and NH4+ -N, and the removals for COD and NH4+ -N are both beyond 90% under steady state, in addition, MBR has a strong adaptation ability for shock organics loading rate. When the organic loading rate was increased from 0.27g/(g x d) to 0.54g/(g x d) suddenly, there was no big fluctuation for COD in effluent. According to the results of GC/MS, the remaining organics in effluent was mainly alkyl hydrocarbon, and the membrane modules played a main role in stabling permeation quality. When the sludge in MBR was at the multiplication stage, the system has a removal of about 40% for TN because of biosynthesis and simultaneous nitrification and de-nitrification, in addition, a certain removal for TP was also observed. When the sludge was at the steady stage, the removal for TN decreased to about 30% due to simultaneous nitrification and de-nitrification, whereas, the removal efficiency for TP was very little, and sometimes even below zero.

Beer↗

Enhancement of oxygen transfer and nitrogen removal in a membrane separation bioreactor for domestic wastewater treatment.

Biological nitrogen removal in a membrane separation bioreactor developed for on-site domestic wastewater treatment was investigated. The bioreactor employed hollow fiber membrane modules for solid-liquid separation so that the biomass could be completely retained within the system. Intermittent aeration was supplied with 90 minutes on and off cycle to achieve nitrification and denitrification reaction for nitrogen removal. High COD and nitrogen removal of more than 90% were achieved under a moderate temperature of 25 degrees C. As the temperature was stepwise decreased from 25 to 5 degrees C, COD removal in the system could be constantly maintained while nitrogen removal was deteriorated. Nevertheless, increasing aeration supply could enhance nitrification at low temperature with benefit from complete retention of nitrifying bacteria within the system by membrane separation. At low operating temperature range of 5 degrees C, nitrogen removal could be recovered to more than 85%. A mathematical model considering diffusion resistance of limiting substrate into the bio-particle is applied to describe nitrogen removal in a membrane separation bioreactor. The simulation suggested that limitation of the oxygen supply was the major cause of inhibition of nitrification during temperature decrease. Nevertheless, increasing aeration could promote oxygen diffusion into the bio-particle. Sufficient oxygen was supplied to the nitrifying bacteria and the nitrification could proceed. In the membrane separation bioreactor, biomass concentration under low temperature operation was allowed to increase by 2-3 times of that of moderate temperature to compensate for the loss of bacterial activities so that the temperature effect was masked.

Biomass↗

[Ability of obligate methylotrophs to perform nitrogen fixation].

The ability for nitrification was studied among mesophilic and thermophilic cultures of obligate methylotrophs Methylobacter ucrainicus, Methylomonas methanica, and Methylococcus thermophilus. The strains were almost incapable of nitrification under autotrophic conditions. In the presence of methane, however, they oxidized NH+4 TO NO-2: over 150 mg/litre NO-2 nitrogen was found in the cultural broth. Therefore, obligate methylotrophs are capable of heterotrophic nitrification. The level of nitrification suggests that it is not a side reaction but a necessary stage of the metabolism of obligate methylotrophs. The phenotype relation between obligate methylotrophs and nitrifying organisms is discussed.

Bacteria↗

Contrasting rhizosphere nitrogen dynamics in Andropogoneae grasses.

Nitrogen (N) fertilization in crop production significantly impacts ecosystems, often disrupting natural plant-microbe-soil interactions and causing environmental pollution. This study tested the hypothesis that diverse species adapting independently to various environments might exhibit a wide range of rhizosphere nutrient management strategies, and some of them may be conducive to an efficient N economy for crops. We analyzed the N cycle in the rhizospheres of 36 Andropogoneae grass species related to maize and sorghum and observed significant phylogenetic variation among their impacts on N availability and losses. All three annual species examined, including sorghum and maize, function as N 'Conservationists', reducing soil nitrification potential and conserving NH4 +. In contrast, seven of the assayed perennial species enhance nitrification and leaching ('Leachers'). Four other species exhibit similar nitrification stimulation effects but limited NO3 - losses ('Nitrate Keepers'). We complemented the controlled phenotypic evaluation with an evolutionary-ecological analysis of the same species. We identified several soil characteristics associated with the phylogenetic variation in rhizosphere N dynamics across grasses and highlighted the crucial roles of a few transporter genes in soil N management and utilization. In addition to the ecological and genetic insights, these findings offer valuable guidelines for future maize breeding efforts to enhance agricultural N efficiency and sustainability.

Rhizosphere↗