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Effect of volumetric organic loading on the nitrogen removal rate by immobilised activated sludge.

Activated sludge was immobilised in a porous ceramic carrier to create a stationary core of a bio-reactor. Municipal wastewater was treated in this reactor under varied conditions of volumetric organic loading rate (expressed by chemical oxygen demand (COD)) that were the following: 6.5, 8.0, 20.8, 48.8 g COD l(-1) d(-1). The rate constants of ammonification, nitrification and denitrification under aerobic conditions were determined. All rate constants increased with a growth in volumetric loading rate, but the highest loading value of 48.8 g COD l(-1) d(-1) limited the ammonification and nitrification rates.

Carbon↗

A novel design for anaerobic chemical oxygen demand and nitrogen removal from leachate in a semiaerobic landfill.

The removal capacity of carbon and nitrogen from an artificial leachate was evaluated by using laboratory-scale columns, and a design was proposed to remove nitrogen more efficiently from a semiaerobic landfill. Five columns (i.e., two artificial municipal waste columns under anaerobic and semiaerobic conditions, an artificial construction waste column under semiaerobic conditions, and two crushed stone columns under anaerobic and semiaerobic conditions) were used. The influent load rates of organics [g chemical oxygen demand (COD)/m3 x day], NH4+, NO3- and aeration conditions for the columns were varied, and the removal capacities of the columns for COD, NH4+-N, and NO3--N were measured. Among the packed column materials, crushed stone was shown to be most effective in removing COD, NH4+ N, and NO3--N from artificial leachate. Average removal rates of crushed column under the semiaerobic condition (column D) for COD and NH4+-N were estimated at about 150 g COD/m3 x day and 20 g COD/m3 x day, while those of crushed column under anaerobic condition (column E) for COD and NO3--N at about 400 and 150 g COD/m3 x day, respectively. It also was found that denitrification and nitrification reactions in column D occurred at the same time, and the ratio of denitrification to nitrification was estimated to be about 80%. Therefore, an anaerobic structure, which could be attached to the bottom of a main pipe in a semiaerobic landfill, is suggested to remove nitrogen and organic substances more effectively.

Bacteria, Anaerobic↗

Upgrading of a small overloaded activated sludge plant using a MBBR system.

The aim of this research was the application of a biofilm system for the upgrading of a full-scale overloaded activated sludge MWWTP using the MBBR (Moving Bed Biofilm Reactor) technology. The choice of this fixed biomass system appeared appropriate because it offers several advantages including good potential in nitrification process, easiness of management and above all, the possibility to use the existing tank with very few modifications. MBBR system counts only few full-scale plants in Italy at the moment, thus a pilot-scale experimentation was preliminarily carried out. The acquired parameters were used for the fullscale MWWTP upgrading. The upgrading of the activated sludge reactor in the MBBR system has given (1) a relevant increase in the flowrate treated up to 60%; (2) a good efficiency in organic carbon removal and nitrification, equal to 88% and 90% respectively, with HRTs of 5.5-7 h; (3) the overcoming of the hydraulic overload of the secondary settler, applying a lamellar settler. It was observed a good correlation between the results obtained at pilot-scale and those observed in the full-scale plant.

Biofilms↗

Nitrogen removal via nitrite from seawater contained sewage.

Under the control of both pH and the concentration of free ammonia (FA), the nitrification-denitrification via nitrite pathway was accomplished in SBR to achieve enhanced biological nitrogen removal from seawater contained wastewater, which is used to flush toilet, under relatively high salinity. Several parameters including salinity, temperature, pH, and NH4+-N loading rate were studied to evaluate their effects. The results indicate that at different salinity the nitrogen removal efficiency is relative to ammonia-nitrogen loading rate. The nitrogen removal efficiency reaches above 90% when the NH4+-N loading does not exceed 0.15 kg NH4+-N/kg MLSS d. With the salinity increasing, the ammonia-nitrogen loading rate should be lowered to obtain high removal efficiency. The evaluation of temperature effect shows that nitrogen removal efficiency is promoted twice when reaction temperature is elevated from 20 to 30 degrees C. Moderately high pH in the range of 7.5-8.5 has advantage to achieve effective nitrification-denitrification via nitrite, the process of which is caused by the selective inhibition of free ammonia (FA).

Bioreactors↗

Response of the bacteria and fungi of two soils to the sulfonylurea herbicide cinosulfuron.

Changes in aerobic bacteria and autotrophic nitrifier numbers, and in respiration and nitrification in two soils treated with cinosulfuron at 42 (field rate) and 4200 microg/kg were studied after 1 and 4 weeks of incubation under laboratory conditions. Only nitrification at 1 week was slightly inhibited by the cinosulfuron treatment, even at the field rate. In vitro toxicity tests carried out in agar media on representative aerobic bacteria, fungi and Azotobacter strains isolated from the two soils, as well as on nine collection soil bacteria, showed that only a very high cinosulfuron concentration (100 mg/l) can have negative effects on the growth of a limited number of soil heterotrophic microorganisms, under conditions similar to those of soil environment. The absence of three branched-chain amino acids increased bacterial sensitivity, thus showing the importance of the chemical conditions and suggesting acetolactate synthase enzyme blockage as the toxicity mechanism. It is concluded that cinosulfuron has a negative effect on only a few aspects of the microbial community in soil ecosystems, even at concentrations higher that those currently in use.

Acetolactate Synthase↗

Effects of butachlor on microbial populations and enzyme activities in paddy soil.

This paper reports the influences of the herbicide butachlor (n-butoxymethlchloro -2', 6'-diethylacetnilide) on microbial populations, respiration, nitrogen fixation and nitrification, and on the activities of dehydrogenase and hydrogen peroxidase in paddy soil. The results showed that the number of actinomycetes declined significantly after the application of butachlor at different concentrations ranging from 5.5 microg g(-1) to 22.0 microg g(-1) dried soil, while that of bacteria and fungi increased. Fungi were easily affected by butachlor compared to the bacteria. The growth of fungi was retarded by butachlor at higher concentrations. Butachlor however, stimulated the growth of anaerobic hydrolytic fermentative bacteria, sulfate-reducing bacteria (SRB) and denitrifying bacteria. The increased concentration of butachlor applied resulted in the higher number of SRB. Butachlor inhibited the growth of hydrogen-producing acetogenic bacteria. The effect of butachlor varied on methane-producing bacteria (MPB) at different concentrations. Butachlor at the concentration of 1.0 microg g(-1) dried soil or less than this concentration accelerated the growth of MPB, while at 22.0 microg g(-1) dried soil showed an inhibition. Butachlor enhanced the activity of dehydrogenase at increasing concentrations. The soil dehydrogenase showed the highest activity on the 16th day after application of 22.0 microg g(-1) dried soil of butachlor. The hydrogen peroxidase could be stimulated by butachlor. The soil respiration was depressed during the period from several days to more than 20 days, depending on concentrations of butachlor applied. Both the nitrogen fixation and nitrification were stimulated in the beginning but reduced greatly afterwards in paddy soil.

Acetanilides↗

In situ substrate conversion and assimilation by nitrifying bacteria in a model biofilm.

Local nitrification and carbon assimilation activities were studied in situ in a model biofilm to investigate carbon yields and contribution of distinct populations to these activities. Immobilized microcolonies (related to Nitrosomonas europaea/eutropha, Nitrosomonas oligotropha, Nitrospira sp., and to other Bacteria) were incubated with [14C]-bicarbonate under different experimental conditions. Nitrifying activity was measured concomitantly with microsensors (oxygen, ammonium, nitrite, nitrate). Biofilm thin sections were subjected to fluorescence in situ hybridization (FISH), microautoradiography (MAR), and local quantification of [14C]-bicarbonate uptake (beta microimaging). Nitrifying activity and tracer assimilation were restricted to a surface layer of different thickness in the various experiments (substrate or oxygen limitation). Excess oxygen uptake under all conditions revealed heterotrophic activity fuelled by decay or excretion products during active nitrification. Depth limits and intensity of tracer incorporation profiles were in agreement with ammonia-oxidation activity (measured with microsensors), and distribution of incorporated tracer (detected with MAR). Microautoradiography revealed a sharp individual response of distinct populations in terms of in-/activity depending on the (local) environmental conditions within the biofilm. Net in situ carbon yields on N, expressed as e- equivalent ratios, varied between 0.005 and 0.018, and, thus, were in the lower range of data reported for pure cultures of nitrifiers.

Ammonia↗

Long-term monitoring of the succession of a microbial community in activated sludge from a circulation flush toilet as a closed system.

The microbial diversity and community succession of a circulation flush toilet were investigated by terminal restriction fragment length polymorphism and cloning analyses. Clonal libraries of 16S rRNA gene on day 3 and day 127 were constructed. On day 3, 102 clones were sequenced; Proteobacteria and Bacteroidetes accounted for 27% and 45%, respectively. On day 127, Proteobacteria had increased to 43% and Bacteroidetes had decreased to 26% of a total of 100 clones. Terminal restriction fragment length polymorphism peaks were identified by in silico analysis of clone libraries. The relative abundances of Nitrosomonas increased from 1% to 6% with commencement of nitrification and denitrification. Similarly, the relative abundance of terminal restriction fragments generated from Xanthomonas increased from 3% to 10%. Therefore, these bacteria could play a prominent role in this process. To reveal the relationship between stability of the microbial community and performance of the system, microbial community succession was visualized by multidimensional scaling analysis. The microbial community structure changed markedly, particularly during the start-up period of the system. The plots then became stable after the start of nitrification and denitrification. This result suggests that the succession of microbial community structure had a correlation with the performance of the system.

Bacteroidetes↗

The anaerobic oxidation of ammonium.

From recent research it has become clear that at least two different possibilities for anaerobic ammonium oxidation exist in nature. 'Aerobic' ammonium oxidizers like Nitrosomonas eutropha were observed to reduce nitrite or nitrogen dioxide with hydroxylamine or ammonium as electron donor under anoxic conditions. The maximum rate for anaerobic ammonium oxidation was about 2 nmol NH4+ min-1 (mg protein)-1 using nitrogen dioxide as electron acceptor. This reaction, which may involve NO as an intermediate, is thought to generate energy sufficient for survival under anoxic conditions, but not for growth. A novel obligately anaerobic ammonium oxidation (Anammox) process was recently discovered in a denitrifying pilot plant reactor. From this system, a highly enriched microbial community with one dominating peculiar autotrophic organism was obtained. With nitrite as electron acceptor a maximum specific oxidation rate of 55 nmol NH4+ min-1 (mg protein)-1 was determined. Although this reaction is 25-fold faster than in Nitrosomonas, it allowed growth at a rate of only 0.003 h-1 (doubling time 11 days). 15N labeling studies showed that hydroxylamine and hydrazine were important intermediates in this new process. A novel type of hydroxylamine oxidoreductase containing an unusual P468 cytochrome has been purified from the Anammox culture. Microsensor studies have shown that at the oxic/anoxic interface of many ecosystems nitrite and ammonia occur in the absence of oxygen. In addition, the number of reports on unaccounted high nitrogen losses in wastewater treatment is gradually increasing, indicating that anaerobic ammonium oxidation may be more widespread than previously assumed. The recently developed nitrification systems in which oxidation of nitrite to nitrate is prevented form an ideal partner for the Anammox process. The combination of these partial nitrification and Anammox processes remains a challenge for future application in the removal of ammonium from wastewater with high ammonium concentrations.

Anaerobiosis↗

Diversity of ammonia-oxidizing archaea and bacteria in the sediments of a hypernutrified subtropical estuary: Bahía del Tóbari, Mexico.

Nitrification within estuarine sediments plays an important role in the nitrogen cycle, both at the global scale and in individual estuaries. Although bacteria were once thought to be solely responsible for catalyzing the first and rate-limiting step of this process, several recent studies have suggested that mesophilic Crenarchaeota are capable of performing ammonia oxidation. Here we examine the diversity (richness and community composition) of ammonia-oxidizing archaea (AOA) and bacteria (AOB) within sediments of Bahía del Tóbari, a hypernutrified estuary receiving substantial amounts of ammonium in agricultural runoff. Using PCR primers designed to specifically target the archaeal ammonia monooxygenase alpha-subunit (amoA) gene, we found AOA to be present at five sampling sites within this estuary and at two sampling time points (January and October 2004). In contrast, the bacterial amoA gene was PCR amplifiable from only 40% of samples. Bacterial amoA libraries were dominated by a few widely distributed Nitrosomonas-like sequence types, whereas AOA diversity showed significant variation in both richness and community composition. AOA communities nevertheless exhibited consistent spatial structuring, with two distinct end member assemblages recovered from the interior and the mouths of the estuary and a mixed assemblage from an intermediate site. These findings represent the first detailed examination of archaeal amoA diversity in estuarine sediments and demonstrate that diverse communities of Crenarchaeota capable of ammonia oxidation are present within estuaries, where they may be actively involved in nitrification.

Ammonia↗

Nitrate accumulation in aerobic hypolimnia: relative importance of benthic and planktonic nitrifiers in an oligotrophic lake.

Both nitrate and nitrous oxide accumulate in the hypolimnion of the oligotrophic Lake Taupo, New Zealand, throughout stratification. The two forms of oxidized nitrogen increase in concentration with increasing depth toward the sediments, where the dissolved concentrations of reduced nitrogen are two orders of magnitude higher than concentrations in the overlying water. Nitrification rates were measured by dark [C]CO(2) assays with and without the inhibitor nitrapyrin. The fastest rates were recorded for planktonic nitrifiers in the epilimnion and benthic species in the surficial 2.5 mm of the sediments. Nitrifying bacteria were least active in the deep hypolimnion. Deepwater accumulation of NO(3) in Lake Taupo must therefore be a product of benthic rather than planktonic nitrification.

Journal Article↗

N Kinetic Analysis of N(2)O Production by Nitrosomonas europaea: an Examination of Nitrifier Denitrification.

A series of N isotope tracer experiments showed that Nitrosomonas europaea produces nitrous oxide only under oxygen-limiting conditions and that the labeled N from nitrite, but not nitrate, is incorporated into nitrous oxide, indicating the presence of the "denitrifying enzyme" nitrite reductase. A kinetic analysis of the m/z 44, 45, and 46 nitrous oxide produced by washed cell suspensions of N. europaea when incubated with 4 mM ammonium (99% N) and 0.4 mM nitrite (99% N) was performed. No labeled nitrite was reduced to ammonium. All labeled material added was accounted for as either nitrite or nitrous oxide. The hypothesis that nitrous oxide is produced directly from nitrification was rejected since (i) it does not allow for the large amounts of double-labeled (m/z 46) nitrous oxide observed; (ii) the observed patterns of m/z 44, 45, and 46 nitrous oxide were completely consistent with a kinetic analysis based on denitrification as the sole mechanism of nitrous oxide production but not with a kinetic analysis based on both mechanisms; (iii) the asymptotic ratio of m/z 45 to m/z 46 nitrous oxide was consistent with denitrification kinetics but inconsistent with nitrification kinetics, which predicted no limit to m/z 45 production. It is concluded that N. europaea is a denitrifier which, under conditions of oxygen stress, uses nitrite as a terminal electron acceptor and produces nitrous oxide.

Journal Article↗

Contributions of Autotrophic and Heterotrophic Nitrifiers to Soil NO and N(2)O Emissions.

Soil emission of gaseous N oxides during nitrification of ammonium represents loss of an available plant nutrient and has an important impact on the chemistry of the atmosphere. We used selective inhibitors and a glucose amendment in a factorial design to determine the relative contributions of autotrophic ammonium oxidizers, autotrophic nitrite oxidizers, and heterotrophic nitrifiers to nitric oxide (NO) and nitrous oxide (N(2)O) emissions from aerobically incubated soil following the addition of 160 mg of N as ammonium sulfate kg. Without added C, peak NO emissions of 4 mug of N kg h were increased to 15 mug of N kg h by the addition of sodium chlorate, a nitrite oxidation inhibitor, but were reduced to 0.01 mug of N kg h in the presence of nitrapyrin [2-chloro-6-(trichloromethyl)-pyridine], an inhibitor of autotrophic ammonium oxidation. Carbon-amended soils had somewhat higher NO emission rates from these three treatments (6, 18, and 0.1 mug of N kg h after treatment with glucose, sodium chlorate, or nitrapyrin, respectively) until the glucose was exhausted but lower rates during the remainder of the incubation. Nitrous oxide emission levels exhibited trends similar to those observed for NO but were about 20 times lower. Periodic soil chemical analyses showed no increase in the nitrate concentration of soil treated with sodium chlorate until after the period of peak NO and N(2)O emissions; the nitrate concentration of soil treated with nitrapyrin remained unchanged throughout the incubation. These results suggest that chemoautotrophic ammonium-oxidizing bacteria are the predominant source of NO and N(2)O produced during nitrification in soil.

Journal Article↗

Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Chemostats.

The enhanced mineralization of organic nitrogen by bacteriophagous protozoa is thought to favor the nitrification process in soils, in which nitrifying bacteria have to compete with heterotrophic bacteria for the available ammonium. To obtain more insight into this process, the influence of grazing by the bacteriovorous flagellate Adriamonas peritocrescens on the competition for limiting amounts of ammonium between the ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis was studied in the presence of Nitrobacter winogradskyi in continuous cultures at dilution rates of 0.004 and 0.01 h. The ammonium concentration in the reservoir was maintained at 2 mM, whereas the glucose concentration was increased stepwise from 0 to 7 mM. A. globiformis won the competition for limiting amounts of ammonium when the glucose concentration in the reservoirs increased, in agreement with previously described experiments in which the flagellates were not included. The numbers of nitrifying bacteria decreased as the numbers of heterotrophic bacteria rose with increasing glucose concentrations. Critical C/N ratios, i.e., ratios between glucose and ammonium in the reservoirs at which no nitrate was found in the culture vessels, of 12.5 and 10.5 were determined at dilution rates of 0.004 and 0.01 h, respectively. Below these critical values, coexistence of the competing species was found. The numbers of nitrifying bacteria decreased more in the presence of flagellates than in their absence, presumably by selective predation on the nitrifying bacteria, either in the liquid culture or on the glass wall of the culture vessels. Despite this, the rate of nitrate production did not decrease more in the presence of flagellates than in their absence. This demonstrates that no correlation has to be expected between numbers of nitrifying bacteria and their activity and that a constant nitrification rate per cell cannot be assumed for nitrifying bacteria. Above the critical C/N ratios, low numbers of nitrifying bacteria were still found in the culture vessels, probably because of attachment of the nitrifying bacteria to the glass wall of the culture vessels. Like the numbers of heterotrophic bacteria, the numbers of flagellates increased when the glucose concentrations in the reservoirs increased. Numbers of 2 x 10 and 12 x 10 flagellates ml were found at 7 mM glucose at dilution rates of 0.004 and 0.01 h, respectively. It was concluded that the critical C/N ratios were practically unaffected by the presence of protozoa. Although nitrate production rates were equal in the presence and absence of flagellates, the numbers of nitrifying bacteria decreased more strongly in their presence. This indicates a higher activity per nitrifying cell in the presence of flagellates.

Journal Article↗

Evaluation of methyl fluoride and dimethyl ether as inhibitors of aerobic methane oxidation.

Methyl fluoride (MF) and dimethyl ether (DME) were effective inhibitors of aerobic methanotrophy in a variety of soils. MF and DME blocked consumption of CH(4) as well as the oxidation of CH(4) to CO(2), but neither MF nor DME affected the oxidation of [C]methanol or [C]formate to CO(2). Cooxidation of ethane and propane by methane-oxidizing soils was also inhibited by MF. Nitrification (ammonia oxidation) in soils was inhibited by both MF and DME. Production of N(2)O via nitrification was inhibited by MF; however, MF did not affect N(2)O production associated with denitrification. Methanogenesis was partially inhibited by MF but not by DME. Methane oxidation was approximately 100-fold more sensitive to MF than was methanogenesis, indicating that an optimum concentration could be employed to selectively block methanotrophy. MF inhibited methane oxidation by cell suspensions of Methylococcus capsulatus; however, DME was a much less effective inhibitor.

Journal Article↗

Mechanisms for soil moisture effects on activity of nitrifying bacteria.

Moisture may limit microbial activity in a wide range of environments including salt water, food, wood, biofilms, and soils. Low water availability can inhibit microbial activity by lowering intracellular water potential and thus reducing hydration and activity of enzymes. In solid matrices, low water content may also reduce microbial activity by restricting substrate supply. As pores within solid matrices drain and water films coating surfaces become thinner, diffusion path lengths become more tortuous, and the rate of substrate diffusion to microbial cells declines. We used two independent techniques to evaluate the relative importance of cytoplasmic dehydration versus diffusional limitations in controlling rates of nitrification in soil. Nitrification rates in shaken soil slurries, in which NH(inf4)(sup+) was maintained at high concentrations and osmotic potential was controlled by the addition of K(inf2)SO(inf4), were compared with rates in moist soil incubations, in which substrate supply was controlled by the addition of NH(inf3) gas. Comparison of results from these techniques demonstrated that diffusional limitation of substrate supply and adverse physiologic effects associated with cell dehydration can explain all of the decline in activity of nitrifying bacteria at low soil water content. However, the relative importance of substrate limitation and dehydration changes at different water potentials. For the soil-microbial system we worked with, substrate limitation was the major inhibiting factor when soil water potentials were greater than -0.6 MPa, whereas adverse physiological effects associated with cell dehydration were more inhibiting at water potentials of less than -0.6 MPa.

Journal Article↗

Nitrogen Transformations in Wetland Soil Cores Measured by (sup15)N Isotope Pairing and Dilution at Four Infiltration Rates.

The effect of water infiltration rate (IR) on nitrogen cycling in a saturated wetland soil was investigated by applying a (sup15)N isotope dilution and pairing method. Water containing [(sup15)N]nitrate was infiltrated through 10-cm-long cores of sieved and homogenized soil at rates of 72, 168, 267, and 638 mm day(sup-1). Then the frequencies of (sup30)N(inf2), (sup29)N(inf2), (sup15)NO(inf3)(sup-), and (sup15)NH(inf4)(sup+) in the outflow water were measured. This method allowed simultaneous determination of nitrification, coupled and uncoupled denitrification, and nitrate assimilation rates. From 3% (at the highest IR) to 95% (at the lowest IR) of nitrate was removed from the water, mainly by denitrification. The nitrate removal was compensated for by the net release of ammonium and dissolved organic nitrogen. Lower oxygen concentrations in the soil at lower IRs led to a sharper decrease in the nitrification rate than in the ammonification rate, and, consequently, more ammonium leaked from the soil. The decreasing organic-carbon-to-nitrogen ratio (from 12.8 to 5.1) and the increasing light A(inf250)/A(inf365) ratio (from 4.5 to 5.2) indicated an increasing bioavailability of the outflowing dissolved organic matter with increasing IR. The efflux of nitrous oxide was also very sensitive to IR and increased severalfold when a zone of low oxygen concentration was close to the outlet of the soil cores. N(inf2)O then constituted 8% of the total gaseous N lost from the soil.

Journal Article↗

Contribution of methanotrophic and nitrifying bacteria to CH4 and NH4+ oxidation in the rhizosphere of rice plants as determined by new methods of discrimination

Methanotrophic and nitrifying bacteria are both able to oxidize CH4 as well as NH4+. To date it is not possible to estimate the relative contribution of methanotrophs to nitrification and that of nitrifiers to CH4 oxidation and thus to assess their roles in N and C cycling in soils and sediments. This study presents new options for discrimination between the activities of methanotrophs and nitrifiers, based on the competitive inhibitor CH3F and on recovery after inhibition with C2H2. By using rice plant soil as a model system, it was possible to selectively inactivate methanotrophs in soil slurries at a CH4/CH3F/NH4+ molar ratio of 0.1:1:18. This ratio of CH3F to NH4+ did not affect ammonia oxidation, but methane oxidation was inhibited completely. By using the same model system, it could be shown that after 24 h of exposure to C2H2 (1,000 parts per million volume), methanotrophs recovered within 24 h while nitrifiers stayed inactive for at least 3 days. This gave an "assay window" of 48 h when only methanotrophs were active. Applying both assays to model microcosms planted with rice plants demonstrated a major contribution of methanotrophs to nitrification in the rhizosphere, while the contribution of nitrifiers to CH4 oxidation was insignificant.

Journal Article↗