Enriched Nitrifying Research Articles

Cycling batch vs continuous enrichment of endogenous nitrifiers in membrane bioreactors treating petrochemical wastewater

Petrochemical wastewater treatment is a new sector for the possible widespread application of membrane bioreactor technology. In this field, hard to degrade compounds such as amines and nitrogen residuals were generally the major issues. In addition, over the last years the irregular operation of the manufacturing plants involved drastic variability of the wastewater’s loadings and characteristics, so as to call for more flexible schemes of the treatment plants. This paper compares two different schemes of a continuously fed membrane bioreactor to adequate the nitrification potential to the influent loadings. These are: a) controlled and continuous external ammonia dosage; b) sequencing batch nitrifier enrichment. On the basis of lab and pilot-scale treatment of real petrochemical wastewater, the cyclic batch enrichment process proved to be reliable, cost-effective, and easy to control, being more flexible with respect to changeable influent loadings.

Effect of Ammonium Concentration on the Emission of N2O Under Oxygen-Limited Autotrophic Wastewater Nitrification

A significant amount of nitrous oxide (N(2)O), which is one of the serious greenhouse gases, is emitted from nitrification and denitrification of wastewater. Batch wastewater nitrifications with enriched nitrifiers were carried out under oxygen-limited condition with synthetic (without organic carbon) and real wastewater (with organic carbon) in order to find out the effect of ammonium concentration on N(2)O emission. Cumulated N(2)O-N emission reached 3.0, 5.7, 6.2, and 13.5 mg from 0.4 l of the synthetic wastewater with 50, 100, 200, and 500 mg/l NH(4)(+)-N, respectively, and 1.0 mg from the real wastewater with 125 mg/l NH(4)(+)-N. The results indicate that N(2)O emission increased with ammonium concentration and the load. The ammonium removal rate and nitrite concentration also increased N(2)O emission. Comparative analysis of N(2)O emission from synthetic and real wastewaters revealed that wastewater nitrification under oxygen-limited condition emitted more N(2)O than that of heterotrophic denitrification. Summarizing the results, it can be concluded that denitrification by autotrophic nitrifiers contributes significantly to the N(2)O emission from wastewater nitrification.

Mobilization of Metals from Pristine Mineral Soil by Nitrifying and Sulfur-Oxidizing Bacteria – The Leaching Potential of Indigenous Culture Enrichments

Indigenous chemolithotrophic nitrifying and sulfur-oxidizing culture enrichments mobilized metals from pristine mineral soil under conditions of ammonium or thiosulfate supplementation in a laboratory experiment carried out over a period of 40 days. The average mineralogical composition of the mineral soil was quartz (62%), feldspar (20%), muscovite (6%), chlorite (2%), hornblende (2%), dolomite (4%), and calcium carbonate (4%). The leaching efficiency of the nitrifying enrichment was calcium (27%), magnesium (15%), zinc (5.4%), manganese (0.6%), and cobalt (1.4%) after 40 days of incubation. In case of sulfur-oxidizing enrichment, leaching efficiency was calcium (56%), magnesium (36%), iron (0.8%), zinc (12%), manganese (2.1%), and cobalt (12%). The impact of these organisms on pristine mineral soil could be important in understanding primary colonization and the early stages of soil formation.

Change in ammonia-oxidizing microorganisms in enriched nitrifying activated sludge

In this study, sludge was taken from a municipal wastewater treatment plant that contained a nearly equal number of archaeal amoA genes (5.70 × 10⁶ ± 3.30 × 10⁵ copies mg sludge⁻¹) to bacterial amoA genes (8.60 × 10⁶ ± 7.64 × 10⁵ copies mg sludge⁻¹) and enriched in three continuous-flow reactors receiving an inorganic medium containing different ammonium concentrations: 2, 10, and 30 mM NH (4) (+) -N (28, 140, and 420 mg N l⁻¹). The abundance and communities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in enriched nitrifying activated sludge (NAS) were monitored at days 60 and 360 of the operation. Early on, between day 0 and day 60 of reactor operation, comparative abundance of AOA amoA genes to AOB amoA genes varied among the reactors depending on the ammonium levels found in the reactors. As compared to the seed sludge, the number of AOA amoA genes was unchanged in the reactor with lower ammonium level (0.06 ± 0.04 mgN l⁻¹), while in the reactors with higher ammonium levels (0.51 ± 0.33 and 0.25 ± 0.10 mgN l⁻¹), the numbers of AOA amoA genes were deteriorated. By day 360, AOA disappeared from the ammonia-oxidizing consortiums in all reactors. The majority of the AOA sequences from all NASs at each sampling period fell into a single AOA cluster, however, suggesting that the ammonium did not affect the AOA communities under this operational condition. This result is contradictory to the case of AOB, where the communities varied significantly among the NASs. AOB with a high affinity for ammonia were present in the reactors with lower ammonium levels, whereas AOB with a low affinity to ammonia existed in the reactors with higher ammonium levels.

Relationship between respirometric activity and community of entrapped nitrifying bacteria: Implications for partial nitrification

Activated sludge obtained from two municipal wastewater treatment facilities (WWTF) was used as seed sludge for enriched nitrifiers, which were later entrapped in polyvinyl alcohol. Seed sludge from one WWTF was acclimated to high ammonia level (1813 mg NH 3-N l −1) through the return of sludge digester supernatant back to primary clarifier while seed sludge from the other WWTF was un-acclimated. To elucidate on how to control partial nitrification by entrapped cells, which could be different from suspended cells, kinetics of entrapped enriched nitrifiers were studied using a respirometric assay. The community of nitrifiers within the entrapment matrix, which was observed by fluorescence in situ hybridization (FISH) technique, was related to the nitritation and nitratation kinetics based on oxygen uptake rate. Maximum oxygen uptake rate, and substrate and oxygen affinities of both ammonia oxidizing bacteria (AOB) for nitritation and nitrite oxidizing bacteria (NOB) for nitratation in entrapped cells were lower than those of corresponding suspended cells. Under dissolved oxygen (DO) limiting conditions, nitratation was more suppressed than nitritation for suspended cells, while for the entrapped cells, the results were the contrary. A free ammonia (FA) inhibition affected only the un-acclimated sludge. Either FA inhibition or DO limitation might not be a sole effective control parameter to achieve partial nitrification by entrapped cells. FISH results revealed that Nitrosomonas europaea was the dominant AOB while Nitrobacter species was the dominant NOB in all cases. Heterotrophs were also present in the entrapment at 22.8 ± 18.6% and 41.5 ± 4.3% of total bacteria for acclimated and un-acclimated originated sludge. The availability of substrate and oxygen governed the distributions of AOB, NOB and heterotrophs within the entrapment and nitritation kinetics of entrapped nitrifiers.

Biological removal of 17α-ethinylestradiol by a nitrifier enrichment culture in a membrane bioreactor

Increasing concern about the fate of 17α-ethinylestradiol (EE2) in the environment stimulates the search for alternative methods for wastewater treatment plant (WWTP) effluent polishing. The aim of this study was to establish an innovative and effective biological removal technique for EE2 by means of a nitrifier enrichment culture (NEC) applied in a membrane bioreactor (MBR). In batch incubation tests, the microbial consortium was able to remove EE2 from both a synthetic minimal medium and WWTP effluent. A maximum EE2 removal rate of 9.0 μg EE2 g −1 biomass-VSS h −1 was achieved (>94% removal efficiency). Incubation of the heterotrophic bacteria isolated from the NEC did not result in a significant EE2 removal, indicating the importance of nitrification as driving force in the mechanism. Application of the NEC in a MBR to treat a synthetic influent with an EE2 concentration of 83 ng EE2 L −1 resulted in a removal efficiency of 99% (loading rates up to 208 ng EE2 L −1 d −1; membrane flux rate: 6.9 L m −2 h −1). Simultaneously, complete nitrification was achieved at an optimal ammonium influent concentration of 1.0 mg NH 4 +-N L −1. This minimal NH 4 +-N input is very advantageous for effluent polishing since the concomitant effluent nitrate concentrations will be low as well and it offers opportunities for the nitrifying MBR as a promising add-on technology for WWTP effluent polishing.

Nitrifying bacterial communities in an aquaculture wastewater treatment system using fluorescence in situ hybridization (FISH), 16S rRNA gene cloning, and phylogenetic analysis

Aquaculture, especially shrimp farming, has played a major role in the growth of Thailand's economy in recent years, as well as in many South East Asian countries. However, the nutrient discharges from these activities have caused adverse impacts on the quality of the receiving waterways. In particular nitrogenous compounds, which may accumulate in aquaculture ponds, can be toxic to aquatic animals and cause environmental problems such as eutrophication. The mineralization process is well known, but certain aspects of the microbial ecology of nitrifiers, the microorganisms that convert ammonia to nitrate, are poorly understood. A previously reported enrichment of nitrifying bacteria (ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) from a shrimp farm inoculated in a sequencing batch reactor (SBR) was studied by molecular methods. The initial identification and partial quantification of the nitrifying bacteria (AOB and NOB) were carried out by fluorescence in situ hybridization (FISH) using previously published 16S rRNA-targeting oligonucleotide probes. The two dominant bacterial groups detected by FISH were from the Cytophaga-Flavobacterium-Bacteroides and Proteobacteria (beta subdivision) phyla. Published FISH probes for Nitrobacter and Nitrospira did not hybridize to any of the bacterial cells. Therefore it is likely that new communities of NOBs, differing from previously reported ones, exist in the enrichments. Molecular genetic techniques (cloning, sequencing, and phylogenetic analysis) targeting the 16S rRNA genes from the nitrifying enrichments were performed to identify putative AOBs and NOBs.

The Link between Nitrification and Biotransformation of 17α-Ethinylestradiol

Biological treatment processes are probably important for preventing the proliferation of steroidal compounds in the environment, and a growing number of reports suggest that nitrification may play a role in removing these chemicals from wastewater. The link between nitrification and biotransformation of 17alpha-ethinylestradiol (EE2) was investigated using enriched cultures of autotrophic ammonia-oxidizers. Batch experiments showed that ring A of EE2 is the site of electrophilic initiating reactions, including conjugation and hydroxylation. Ring A was also cleaved before any of the other rings were broken, which is likely because the frontier electron density of the ring A carbon units is higher than those of rings B, C, or D. EE2 and NH3 were degraded in the presence of an ammonium monooxygenase (AMO) containing protein extract, and the reaction stoichiometry was consistent with a conceptual model involving a binuclear copper site located at the AMO active site. Continuous tests showed a linear relationship between nitrification and EE2 removal in enriched nitrifying cultures. Taken together, these results support the notion that EE2 biotransformation can be cometabolically mediated under operating conditions that allow for enrichment of nitrifiers.

Enhanced nitrification in industrial wastewater after augmentation by soil nitrifying enrichments

Nitrification (the oxidation of ammonia to nitrate) is a key process in the removal of nitrogen from wastewater, a crucial step in wastewater purification before reuse or discharge to the environment. In this research, we examined a new strategy for ensuring significant nitrification in toxic industrial wastewater by using stress-tolerant nitrifying enrichment cultures harvested from desert soils. Plastic beads covered with biofilm of high salt-tolerant nitrifiers were used in bioaugmentation experiments of wastewater from different sources. The results showed that bead-attached nitrifiers were more active than suspended ones, and significant full nitrification was achieved in industrial wastewater that has no nitrifying activity at all. Our results indicate that using sludge from treatment plants or isolated strains is not always the right practice to enhance nitrification in wastewater. Specific environments can be the source of nitrifying consortia suitable for the treatment of wastewater with distinct properties.

Cometabolic degradation of TCE in enriched nitrifying batch systems

The aim of this study was to evaluate the effect of the trace pollutant trichloroethylene (TCE) on the nitrification process and to assess its cometabolic degradation. Nitrification was accomplished in batch suspended growth systems containing an enriched nitrifier culture. The presence of TCE resulted in both the inhibition of specific oxygen uptake rate (SOUR) and specific ammonium utilization rate (qNH 4–N). In both SOUR and qNH 4–N a 50% decrease was observed in a TCE concentration range of 1000–2000 ppb. TCE was cometabolically degraded by this enriched nitrifier culture. The cometabolic degradation of TCE was found to be dependent on initial TCE concentration. The results may be applicable in the treatment of TCE containing industrial wastewaters and contaminated groundwaters and soils.

Nitrification and occurrence of salt-tolerant nitrifying bacteria in the Negev desert soils

Ammonia oxidation potential, major ammonia oxidizers and occurrence of salt-tolerant nitrifying bacteria were studied in soil samples collected from diverse ecosystems along the northern Negev desert. Great diversity in ammonia oxidation potential was observed among the soil samples, and ammonia oxidizers were the rate-limiting step of nitrification. Denaturing gradient gel electrophoresis and partial 16S rRNA gene sequences indicate that members of the genus Nitrosospira are the major ammonia oxidizers in the natural desert soil samples. Upon enrichment with different salt concentrations, salt-tolerant nitrifying enrichments were established from several soil samples. In two enrichments, nitrification was not inhibited by 400 mM NaCl. Electrophoretic analysis and partial 16S rRNA gene sequences indicate that Nitrosomonas species were dominant in the 400 mM salt enrichment. The results point towards the potential of the desert ecosystem as a source of stress-tolerant nitrifying bacteria or other microorganisms with important properties.

순환여과시스템에서 온도가 질산화 반응에 미치는 영향

The effects of temperature on nitrification of enriched nitrifiers were investigated by using kinetics and thermodynamics method through the batch test. Aquaculture recirculating water, which was sampled at Chung Cheong Buk-Do Inland Fisheries Research Institute, was analized to observe the characteristics of nitrification. Temporal variation of ammonium, nitrite and nitrate concentration was measured at batch experiments. Activation energy was calculated using Arrhenius equation with the oxidation rates of specific ammonium or nitrite ion. These oxidation rates were measured at temperature range of <TEX>$6-35^{\circ}C$</TEX> and ammonium concentration range of 0.2-1.8 mg/L. Two distinct activation energy of Nitrosomonas sp. at temperature <TEX>$6-15^{\circ}C\;and\;15-35^{\circ}C$</TEX> was 93.1 and 25.0 KJ/mol, respectively. Nitrate accumulation was observed at temperature over <TEX>$15^{\circ}C.$</TEX>

Microbial community analysis by FISH for mathematical modelling of selective enrichment of gel-entrapped nitrifiers obtained from domestic wastewater

Nitrifying activated sludge from natural domestic sewage was entrapped in hydrogel beads, which were subsequently enriched for nitrifiers in a continuous stirred tank reactor (CSTR). Fluorescently labelled, 16S rRNA-targeted oligonucleotide probes specific for ammonia and nitrite oxidisers were used in combination with DAPI staining to monitor the selectivity of the enrichment process. The growth of both nitrifying and heterotrophic bacteria was more pronounced in the periphery of the beads, leading to a biofilm-like stratification of the biomass during the enrichment. Quantitatively, the relative number of nitrifiers increased from 20% immediately after immobilisation up to 64% after 30 days, but decreased again due to extensive heterotrophic growth. These changes were accompanied by an increase in nitrifying activity for about 30 days, whereupon it reached a stable level. This selective enrichment was mathematically modelled by applying finite difference techniques to the diffusion-reaction mass balances of all soluble substrates relevant in the nitrification process. To model biomass growth and spreading, balanced by both decay and detachment at the surface of the beads, the differential methods were combined with a descrete cellular automaton approach. The spatially two-dimensional model was used to calculate radial concentration profiles within a gel bead, as well as to estimate the corresponding total activity of the reactor. Qualitatively, this model could simulate all essential aspects observed experimentally. However, more and better population data as well as independent estimates of decay and hydrolysis rates are needed to refine and verify the quantitative model. In conclusion, even in the absence of an external carbon source and with excess ammonium, it was only possible to obtain a moderate enrichment of nitrifying cells compared to heterotrophs. Under long-term cultivation, the biofilm-like structure developed in the outer gel layers led to a vigorous competition between auto- and heterotrophs for space, and thereby, access to oxygen. FISH analysis in combination with mathematical modelling constitute a suitable toolbox for analysing the population dynamics and biocatalytic performance of such an ecosystem based on lithoautotrophic primary production.

Biodegradation of monohalogenated alkanes by soil NH(3)-oxidizing bacteria.

Although cooxidative biodegradation of monohalogenated hydrocarbons has been well studied in the model NH(3)-oxidizing bacterium, Nitrosomonas europaea, virtually no information exists about cooxidation of these compounds by native populations of NH(3)-oxidizing bacteria. To address this subject, nitrifying activity was stimulated to 125-400 nmol NO(3)(-) produced g(-1) soil h(-1) by first incubating a Ca(OH)(2)-amended, silt loam soil (pH 7.0+/-0.2) at field capacity (270 g H(2)O kg(-1) soil) with 10 micro mol NH(4)(+) g(-1) soil for 14 days, followed by another 10 days of incubation in a shaken slurry (2:1 water:soil, v/w) with periodic pH adjustment and maintenance of 10 mM NH(4)(+). These slurries actively degraded both methyl bromide (MeBr) and ethyl chloride (EtCl) at maximum rates of 20-30 nmol ml(-1) h(-1) that could be sustained for approximately 12 h. Although the MeBr degradation rates were linear for the first 10-12 h of incubation, they could not be sustained regardless of NH(4)(+) level and declined to zero over 20 h of incubation. The transformation capacity of the slurry enrichments (~1 micro mol MeBr ml(-1) soil slurry) was similar to the value measured previously in cell suspensions of N. europaea with similar NH(3)-oxidizing activity. Several MeBr-degrading characteristics of the nitrifying enrichments were found to be similar to those documented in the literature for MeBr-degrading methanotrophs and facultatively methylotrophic bacteria.

Biological Removal of Gaseous Ammonia in Biofilters: Space Travel and Earth-Based Applications

ABSTRACT Gaseous NH3 removal was studied in laboratory-scale biofilters (14-L reactor volume) containing perlite inoculated with a nitrifying enrichment culture. These biofilters received 6 L/min of airflow with inlet NH3 concentrations of 20 or 50 ppm, and removed more than 99.99% of the NH3 for the period of operation (101, 102 days). Comparison between an active reactor and an autoclaved control indicated that NH3 removal resulted from nitrification directly, as well as from enhanced absorption resulting from acidity produced by nitrification. Spatial distribution studies (20 ppm only) after 8 days of operation showed that nearly 95% of the NH3 could be accounted for in the lower 25% of the biofilter matrix, proximate to the port of entry. Periodic analysis of the biofilter material (20 and 50 ppm) showed accumulation of the nitrification product NO3 - early in the operation, but later both NO2 - and NO3 - accumulated. Additionally, the N-mass balance accountability dropped from near 100% early in the experiments to ~95 and 75% for the 20- and 50-ppm biofilters, respectively. A partial contributing factor to this drop in mass balance accountability was the production of NO and N2O, which were detected in the biofilter exhaust.

Inhibition of nitrifiers and methanotrophs from an agricultural humisol by allylsulfide and its implications for environmental studies.

Allylsulfide, an inhibitor of ammonia monooxygenase, was tested to determine its ability to inhibit nitrification and methane oxidation in pure cultures, in agricultural humisol enrichment cultures, and in humisol slurries. We confirmed that allylsulfide is a differential inhibitor of cultures of nitrifiers and methanotrophs at concentrations of 1 and 200 microM, respectively, which result in 50% inhibition. However, although a nitrifying enrichment culture added to sterilized humisol was inhibited 50% by 4 microM allylsulfide, 500 microM allylsulfide was necessary for 50% inhibition of the endogenous nitrifying activity in nonsterile humisol. We concluded that native nitrifiers were protected, possibly by being in colonial aggregates or sheltered microenvironments.

Estimation of nitrifying bacterial activities by measuring oxygen uptake in the presence of the metabolic inhibitors allylthiourea and azide

The effects of two metabolic inhibitors on an enriched nitrifying biomass during incubation for short periods of time were investigated by determining respirometric measurements. Allylthiourea (86 &mgr;M) and azide (24 &mgr;M) were shown to be strong, selective inhibitors of ammonia and nitrite oxidation, respectively. Consequently, a differential respirometry method for estimating nitrifying and heterotrophic bacterial activities within a mixed biomass is proposed.

Nitrogen transformations near urea in soil: Effects of nitrification inhibition, nitrifier activity and liming

A laboratory incubation experiment was conducted to gain a better understanding of N transformations which occur near large urea granules in soil and the effects of dicyandiamide (DCD), nitrifier activity and liming. Soil cores containing a layer of urea were used to provide a one-dimensional approach and to facilitate sampling. A uniform layer of 2 g urea or urea + DCD was placed in the centre of a 20 cm-long soil core within PVC tubing. DCD was mixed with urea powder at 50 mg kg−1 urea and enrichment of soil with nitrifiers was accomplished by preincubating Conestogo silt loam with 50 mg NH 4 + -N kg−1 soil. Brookston clay (pH 5.7) was limited with CaCO3 to increase the pH to 7.3. The cores were incubated at 15°C and, after periods of 10, 20, 35 and 45 days, were separated into 1-cm sections. The distribution of N species was similar on each side of the urea layer at each sampling. The pH and NH 4 + (NH3) concentration were very high near the urea layer but decreased sharply with distance from it. DCD did not influence urea hydrolysis significantly. Liming of Brookston clay increased urea hydrolysis. The rate of urea hydrolysis was greater in Conestogo silt loam than limed Brookston clay. Nitrite accumulate was relatively small with all the treatments and occurred near the urea layer (0–4 cm) where pH and NH 4 + (NH3) concentration were high. The nitrification occurred in the zone where NH 4 + (NH3) concentration was below 1000µgN g−1 and soil pH was below 8.0 and 8.7 in Brookston and Conestogo soils, respectively. DCD reduced the nitrifier activity (NA) in soil thereby markedly inhibiting nitrification of NH 4 + . Nitrification was increased significantly with liming of the Brookston soil or nitrifier enrichment of the Conestogo soil. There was a significant increase in NA during the nitrification of urea-N. The (NO 2 − + NO 3 − )-N concentration peaks coincided with the NA peaks in the soil cores. A practical implication of this work is that large urea granules will not necessarily result in NO 2 − phytotoxicity when applied near plants. A placement depth of about 5 cm below the soil surface may preclude NH3 loss from large urea granules. DCD is a potential nitrification inhibitor for use with large urea granules or small urea granules placed in nests.

Steady State Studies of Nitrification in Soil: Theoretical Considerations

AbstractEquations are set up for consecutive reactions in an idealized soil column and solved for concentrations of metabolites as functions of time and depth within the column under steady state conditions. The reactions NH+4 → NO‐2 → NO‐3 were so expressed for columns with maximum numbers of nitrifying organisms.These equations for an open system can be modified to accommodate rates of metabolism for reperfusion experiments for the condition of maximum enrichment of nitrifiers.