Changes in ammonia oxidiser population during transition to low pH in a biofilm reactor starting with Nitrosomonas europaea.

Recent experiments in our laboratory using both biofilm and suspended biomass reactors have demonstrated high rate nitrification at low pH with known autotrophic nitrifying bacteria originating from wastewater treatment plants refuting previous assumptions that nitrification is significantly inhibited at low pH. Since much of the earlier microbiological work regarding ammonia oxidising bacteria (AOB) physiology was carried out using Nitrosomonas europaea, this model bacterium's capability for high rate nitrification at low pH in a continuous biofilm reactor was tested. A biofilm reactor filled with sintered glass particles was inoculated with a pure culture of N. europaea. The reactor was first operated to high nitrification rates under conditions favourable to N. europaea (pH > 7; high ammonium concentrations). To eliminate inhibitory concentrations of nitrite at low pH, an enriched culture of Nitrospira (a nitrite oxidising bacterium) was then added. The transition from neutral to acidic conditions was attempted by sharply lowering the nitrification rate and by using a feeding solution containing insufficient buffer for complete nitrification. As opposed to other successful transitions, the pH in the N. europaea/Nitrospira reactor initially dropped only slightly and maintained pH > 6 for over two weeks. The reactor reached pH 4.5 only after four weeks. FISH results showed that while the percent of AOB and Nitrospira to eubacteria remained relatively constant at 51.1 +/- 8.2% and 40.8 +/- 6.4%, respectively, the AOB community changed completely in 60 days from 100% N. europaea to 100% Nitrosomonas oligotropha. Even though N. oligotropha was not intentionally introduced into the reactor, it is apparently much better adapted to conditions of low pH.     

High nitrification rate at low pH in a fluidized bed reactor with chalk as the biofilm carrier.

A typical steady state bulk pH of about 5 was established in a nitrifying fluidized bed with chalk as the only buffer agent. In spite of the low pH, high rate nitrification was observed with the nitrification kinetic parameters in the chalk reactor similar to those of biological reactors operating at pH>7. Various methods were used to determine the reasons for high rate nitrification at such low pH including (i) determination of bacterial species, (ii) microsensor measurements in the biofilm, and (iii) comparison of nitrification performance at low pH with a non-chalk fluidized bed reactor. Fluorescence in situ hybridization (FISH) using existing 16S rRNA-targeted oligonucleotide probes showed common nitrifying bacteria in the low pH chalk reactor. The prevalent nitrifying bacteria were identified in the Nitrosomonas oligotropha, Nitrosomonas europeae/eutropha, Nitrosospira and Nitrospira related groups, all well known nitrifiers. Microelectrode measurements showed that the pH in the biofilm was low and similar to that of the bulk pH. Finally, reactor performance using a non-chalk biofilm carrier (sintered glass) with the same bacterial inoculum also showed high rate nitrification below pH 5. The results suggest that inhibition of nitrification at low pH is highly overestimated.     

Influence of dissolved oxygen on nitrification kinetics in a circulating bed reactor

The influence of dissolved oxygen concentration in nitrification kinetics was studied in a new biofilm reactor, the circulating bed reactor (CBR). The study was carried out partly at laboratory scale with synthetic water containing inorganic carbon and nitrogen compounds, and partly at pilot scale for secondary and tertiary nitrification of municipal wastewater.The experimental results showed that either the ammonia or the oxygen concentration could be limiting for the nitrification rate. The transition from ammonia to oxygen limiting conditions occurred for an oxygen to ammonia concentration ratio of about 1.5 - 2 gO₂/gN-NH₄⁺ for both laboratory- and pilot-scale reactors. The nitrification kinetics of the laboratory-scale reactor was close to a half order function of the oxygen concentration, when oxygen was the rate limiting substrate.     

Nitrogen loss in a nitrifying rotating contactor treating ammonium rich leachate without organic carbon

Extended loss (up to 70%) of nitrogen is observed in a nitrifying rotating biological contactor (RBC) treating ammonium - rich leachate of a hazardous waste landfill. Due to pretreatment (flocculation, BOD-removal, activated carbon) DOC was less than 20 mg/l so heterotrophic denitrification can be excluded. The nitrification rate reaches 3-4 g NH₄-N m⁻² d⁻¹ at a pH of 7-7.3 in the first two of three RBC compartments. An increasing partial pressure of oxygen and ammonium concentration favor nitrogen removal over ammonium oxidation. The reduction of nitrite produced in the aerobic biofilm layer close to the surface might therefore be coupled with ammonium oxidation and takes place in the deeper or temporarily anoxic layer of the biofilm.     

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.     

Impact of an urban centre on the nitrogen cycle processes of epilithic biofilms during a summer low‐water period *

Nitrogen transformations in epilithic biofilms of a large gravel bed river, the Garonne, France, has been studied upstream (one site) and downstream (four sites) of a large urban centre (Toulouse, 740 000 inhabitants). High biomass, up to 49 g AFDM m^?2 (ashes free dry matter) and 300 mg chlorophyll a m^?2 (Chl. a), were recorded at 6 and 12 km downstream from the main wastewater treatment plant outlet. The lowest records upstream and larger downstream (less than 16 g AFDM m^?2 or 120 mg Chl. a m^?2) could be explained by recent water fall (early summer low‐water period). Measurements of nitrogen exchange at the biofilm–overlying water interface were performed in incubation chambers under light and dark conditions. The addition of acetylene at the mid‐incubation time allowed evaluation of both nitrification (variation in NH₄⁺ flux after the ammonium monooxygenase inhibition) and denitrification (N₂O accumulation related to the inhibition of N₂O reduction). Denitrification (D_w) and nitrification rates were maximum at sites close to the city discharges in dark conditions (up to 9.1 and 5.6 mg N m^?2 h^?1, respectively). Unexpected denitrification activities in light conditions (up to 1.4 mg N m^?2 h^?1) at these sites provided evidence for enhanced nitrogen self‐purification downstream. As confirmed by most probable number (MPN) counts, high nitrification rates in biofilm close downstream were related to enhanced (more than almost 3 log) nitrifying bacteria densities (up to 7.6×10⁹ MPN m^?2). Downstream of an urban centre, nitrogen transformations in the biofilm appeared to be influenced by the occurrence of an adapted microflora which is inoculated or stimulated by anthropic pollution. Copyright © 2002 John Wiley & Sons, Ltd.     

Performance and microbial ecology of the hybrid membrane biofilm process for concurrent nitrification and denitrification of wastewater.

We report on a novel process for total nitrogen (TN) removal, the hybrid membrane biofilm process (HMBP). The HMBP uses air-supplying hollow-fibre membranes inside an activated sludge tank, with suppressed aeration, to allow concurrent nitrification and denitrification. We hypothesised that a nitrifying biofilm would form on the membranes, and that the low bulk-liquid BOD concentrations would encourage heterotrophic denitrifying bacteria to grow in suspension. A nitrifying biofilm was initially established by supplying an influent ammonia concentration of 20 mgN/L. Subsequently, 120 mg/L acetate was added to the influent as BOD. With a bulk-liquid SRT of only 5 days, nitrification rates were 0.85 gN/m(2) per day and the TN removal reached 75%. The biofilm thickness was approximately 500 lim. We used DGGE to obtain a microbial community fingerprint of suspended and attached growth, and prepared a clone library. The DGGE results, along with the clone library and operating data, suggest that nitrifying bacteria were primarily attached to the membranes, while heterotrophic bacteria were predominant in the bulk liquid. Our results demonstrate that the HMBP is effective for TN removal, achieving high levels of nitrification with a low bulk-liquid SRT and concurrently denitrifying with BOD as the sole electron donor.     

Enhancement of biofilm formation onto surface-modified hollow-fiber membranes and its application to a membrane-aerated biofilm reactor.

Surface-modified hollow-fiber membranes were prepared by radiation-induced grafting of an epoxy-group-containing monomer, glycidylmethacrylate (GMA), onto a polyethylene-based fiber (PE-fiber). The epoxy ring of GMA was opened by introduction of diethylamine (DEA). The bacterial adhesivity to this material (DEA-fiber) was tested by immersion into a nitrifying bacterial suspension. The initial adhesion rates and the amount of attached bacteria of the DEA-fiber were 6-10-fold and 3-fold greater than those of the PE fiber, respectively. A membrane-aerated biofilm reactor (MABR) composed of DEA fibers was developed for partial nitrification with nitrite accumulation. Prior to the nitrification test, it was confirmed that the oxygen supply rate (OSR) was proportional to air pressure up to 100 kPa, allowing easy control of oxygen supply. Stable nitrite accumulation was observed in the partial nitrification test at a fixed oxygen supply throughout the operation period, indicating that oxygen was consumed only by ammonia oxidizers. Furthermore, it was demonstrated that oxygen utilization efficiency (OUE) in the ammonia oxidation process was nearly 100% after 300 h incubation.     

Formation and characteristics of nitritation granules cultivated in sequencing batch reactor by stepwise increase of N/C ratio

The cultivation of nitritation granules in sequencing batch reactor (SBR) by seeding conventional floccular activated sludge was investigated using ethanol-based synthetic wastewater. Reducing settling time offers selection pressure for aerobic granulation, and stepwise increase of influent N/C ratio can help to selectively enrich ammonia oxidizing bacteria (AOB) in aerobic granules. The spherical shaped granules were observed with the mean diameter of 1.25 mm, average settling velocity of 1.9 cm s(-1) and the sludge volume index (SVI) of 18.5-31.4 ml g(-1). After 25 days of operation, the nitrogen loading rate reached 0.0455 kg NH(4)(+)-N (kg MLSS·d)(-1), which was 4.55 times higher than that of the start-up period. The mature granules showed high nitrification ability. Ammonia removal efficiency was above 95% and nitrite accumulation ratio was in the range of 80-95%. The nitrifying bacteria were quantified by fluorescence in situ hybridization analysis, which indicated that AOB was 14.9 ± 0.5% of the total bacteria and nitrite oxidizing bacteria (NOB) was 0.89 ± 0.1% of the total bacteria. Therefore, AOB was the dominant nitrifying bacteria. It was concluded that the associated inhibition of free ammonia at the start of each cycle and free nitrous acid during the later phase of aeration may be the key factors to start up and maintain the stable nitritation.     

Application of biofilm reactors to improve ammonia oxidation in low nitrogen loaded wastewater

An airlift reactor using zeolite particles as carrier material was used for the nitrification of effluents from the aquaculture industry. During the start-up the nitrogen concentration was kept around 100 mg NH4(+)-N/L to develop the nitrifying population. Later it was decreased down to around 3 mg NH4(+)-N/L and the dilution rate was increased up to 4.8 d(-1) in order to simulate the conditions in a an aquaculture waster treatment system. A nitrogen loading rate (NLR) of 535 mg NH(+)-N/m2 d was fully oxidized to nitrate. Higher values of NLRs caused nitrite accumulation. A second biofilm reactor was fed with a synthetic medium containing 50 mg NH4(+)-N/L which simulated the effluents from anaerobic units treating domestic wastewater. A nitrogen loading rate of 400 mg NH4(+)-N/L d was oxidized into nitrate with an efficiency of 60% at a dilution rate of 8 d(-1). Both biofilm systems allowed the development of a nitrifying population to treat the studied types of wastewaters.     

High-rate nitrification using aerobic granular sludge.

The performance of nitrifying granules, which had been produced in an aerobic upflow fluidised bed (AUFB) reactor, was investigated in various types of ammonia-containing wastewaters. When pure oxygen was supplied to the AUFB reactor with a synthetic wastewater containing a high concentration of ammonia (500 g-N/m3), the ammonia removal rate reached 16.7 kg-N/m3/day with a sustained ammonia removal efficiency of more than 80%. The nitrifying granules possessing a high settling ability could be retained with a high density (approximately 10,000 g-MLSS/m3) in a continuous stirring tank reactor (CSTR) even under a short hydraulic retention time (44 min), which enabled a high-rate and stable nitrification for an inorganic wastewater containing low concentrations of ammonia (50 g-N/m3). Moreover, the nitrifying granules exhibited sufficient performance in the nitrification of real industrial wastewater containing high concentrations of ammonia (1000-1400 g-N/m3) and salinity (1.2-2.2%), which was discharged from metal-refinery processes. When the nitrifying granules were used in cooperation with activated sludge to treat domestic wastewater containing organic pollutants as well as ammonia, they fully contributed to nitrification even though a part of activated sludge adhered onto the granule surfaces to form biofilms. These results show the wide applicability of nitrifying granules to various cases in the nitrification step of wastewater treatment plants.     

The expanded bed biofilter: combined nitrification, solids destruction, and removal of bacteria.

Developed for tertiary nitrification, this biofilter also removed carbonaceous BOD (cBOD) and (SS). Because the biofilter is expanded, it cannot clog, and therefore does not require backflushing; yet, it removed a significant proportion of the influent SS. This unanticipated capability was due to the activities of heterotrophic bacteria, protozoa, and metazoa (nematode and oligochaete worms). The expanded bed is an intensified process, which is based on natural immobilization of microbes to small support particles. Using glassy coke as the support material, an attached layer of microbes develops, forming particulate biofilms having a superficial surface area of 1 800 m2 m(-3)(expandedbed). Autotrophic nitritifiers (Nitrosomonas spp.) were detected in the biofilm using rRNA-based molecular methods and were likely responsible, at least in part, for reducing the ammonia concentration by up to 99% (to 0.1 mg L(-1)), while the other organisms reduced cBOD and SS by up to 56% and 62%, respectively. Furthermore, the influent concentrations of Escherichia coli, coliform and heterotrophic bacteria were reduced by over 80%. It thereby provides a single process solution for combined tertiary nitrification and solids removal. Operating the process to consistently achieve < 0.5mg NH3N L(-1) and at the same time removing a significant fraction of cBOD and SS, it can replace processes such as SAFs or NTFs followed by a sandfilter.     

污染河道水质强化脱氮生化工艺研究

应用生化工艺对河道污染水体进行修复是目前最经济的一条途径,但其面临的一个突出问题是在生物脱氮过程中可利用碳源不足,从而影响其处理效果。本研究采用分段进水生物接触氧化工艺来强化受污染水脱氮性能,与传统单点进水方式相比,两段进水对有机物和总氮去除率有显著提升,CODMn平均去除率从50.6%提升到66.3%;总氮平均去除率从31.4%提升到60.9%。沿程统计硝化细菌和反硝化细菌数量,硝化细菌主要集中在曝气区,数量为5.58×106,反硝化细菌主要集中在非曝气区的中后段,数量为6.49×105。同时检测沿程溶解氧和各氮素浓度,溶解氧浓度沿程降低,最后出水仅为0.2 mg/L;氨氮在曝气区转化为硝态氮,在非曝气区硝态氮还原成氮气,其结果进一步证实了硝化细菌和反硝化细菌的分布特征。     

Inhibitory effect of the xenobiotic 1,2-dichloroethane in a nitrifying biofilm reactor.

The aim was to investigate the inhibitory effect of the xenobiotic 1,2-DCA on nitrification during the cometabolic degradation in a packed bed nitrifying biofilm reactor. This xenobiotic inhibited primarily the conversion of NH4-N to hydroxylamine by binding to the AMO enzyme. It had no inhibitory effect on the conversion of nitrite to nitrate. At high NH4-N loadings, the presence of 1,2-DCA inhibited NH4-N utilisation more severely than at low loadings. The suppressing effect of 1,2-DCA on NH4-N utilisation was found to be reversible due to the ability of cells to recover from inhibition. These results could fill a gap in the literature about the potential use of nitrifying biofilm systems for cometabolic treatment of 1,2-DCA and could be useful in the design of engineered 1,2-DCA remediation/treatment in biofilm reactors.     

Evaluation of hybrid processes for nitrification by comparing MBBR/AS and IFAS configurations.

An integrated fixed-film activated sludge (IFAS) pilot plant and a moving bed biofilm reactor coupled with an activated sludge process (MBBR/AS) were operated under different temperatures, carbon loadings and solids retention times (SRTs). These two types of hybrid systems were compared, focusing on the nitrification capacity and the nitrifiers population of the media and suspended biomass alongside other process performances such as carbonaceous and total nitrogen (TN) removal rates. At high temperatures and loadings rates, both processes were fully nitrifying and achieved similarly high carbonaceous removal rates. However, under these conditions, the IFAS configuration performed better in terms of TN removal. Lower temperatures and carbon loadings led to lower carbonaceous removal rates for the MBBR/AS configuration, whereas the IFAS configuration was not affected. However, the nitrification capacity of the IFAS process decreased significantly under these conditions and the MBBR/AS process was more robust in terms of nitrification. Ammonia oxidising bacteria (AOB) and nitrite oxidising bacteria (NOB) population counts accurately reflected the changes in nitrification capacity. However, significantly less NOBs than AOBs were observed, without noticeable nitrite accumulation, suggesting that the characterisation method used was not as sensitive for NOBs and/or that the NOBs had a higher activity than the AOBs.     

Analysis of nitrifying bacterial communities in aerobic biofilm reactors with different DO conditions using molecular techniques

In order to assess the relationship between the dissolved oxygen (DO) concentration and the characteristics of nitrifying bacterial communities in an aerobic biofilm reactor, molecular techniques including denaturing gradient gel electrophoresis (DGGE)/cloning based on PCR targeting 16S rRNA and the amoA gene and fluorescence in situ hybridisation (FISH) were conducted. The D-1, D-2, D-3 and D-4 reactors with different DO concentrations (1, 3, 5 and 7 mg/L, respectively) were set up in the thermostat and acclimated. The optimal DO concentration with stable nitrification efficiency was above 5.0 mg/L. As was shown by the results of DGGE and cloning, the community of ammonia-oxidising bacteria (AOB) and the ratio of Nitrosomonas sp. changed only slightly despite their differing nitrification efficiencies. The results of FISH indicated that higher DO concentrations resulted in an increase in AOB and nitrite-oxidising bacteria (NOB), and a reduction in heterotrophic microorganisms. The INT-dehydrogenase activity (DHA) test demonstrated that the activity of AOB decreased with reductions in the DO concentration. This means that the DO concentration does not influence the community of AOB, but rather the activity of AOB. In the relationship between the attached biomass and the nitrification efficiency, only the active biomass affected the nitrification efficiencies.     

Development and optimisation of VFA driven DEAMOX process for treatment of strong nitrogenous anaerobic effluents.

The recently proposed DEAMOX (DEnitrifying AMmonium OXidation) process combines the anammox reaction with autotrophic denitrifying conditions using sulphide as an electron donor for the production of nitrite from nitrate within an anaerobic biofilm. This paper firstly presents a feasibility study of the DEAMOX process using synthetic (ammonia + nitrate) wastewater where sulphide is replaced by volatile fatty acids (VFA) as a more widespread electron donor for partial denitrification. Under the influent N-NH+4/N-NO3(-) and COD/N-NO3(-) ratios of 1 and 2.3, respectively, the typical efficiencies of ammonia removal were around 40% (no matter whether a VFA mixture or only acetate were used) for nitrogen loading rates (NLR) up to 1236 mg N/l/d. This parameter increased to 80% by increasing the influent COD/N-NO3(-) ratio to 3.48 and decreasing the influent N-NH4 +/N-NO3(-) ratio to 0.29. As a result, the total nitrogen removal increased to 95%. The proposed process was further tested with typical strong nitrogenous effluent such as reject water (total N, 530-566 mg N/l; total COD, 1530-1780 mg/l) after thermophilic sludge anaerobic digestion. For this, the raw wastewater was split and partially ( approximately 50%) fed to a nitrifying reactor (to generate nitrate) and the remaining part ( approximately 50%) was directed to the DEAMOX reactor where this stream was mixed with the nitrified effluent. Stable process performance up to NLR of 1,243 mg N/l/d in the DEAMOX reactor was achieved resulting in 40, 100, and 66% removal of ammonia, NOx(-), and total nitrogen, respectively.     

Rapid start-up of a nitrifying reactor using aerobic granular sludge as seed sludge

In this study, the effectiveness of aerobic granular sludge as seed sludge for rapid start-up of nitrifying processes was investigated using a laboratory-scale continuous stirred-tank reactor (CSTR) fed with completely inorganic wastewater which contained a high concentration of ammonia. Even when a large amount of granular biomass was inoculated in the reactor, and the characteristics of influent wastewater were abruptly changed, excess biomass washout was not observed, and biomass concentration was kept high at the start-up period due to high settling ability of the aerobic granular sludge. As a result, an ammonia removal rate immediately increased and reached more than 1.0 kg N/m(3)/d within 20 days and up to 1.8 kg N/m(3)/d on day 39. Subsequently, high rate nitritation was stably attained during 100 days. However, nitrite accumulation had been observed for 140 days before attaining complete nitrification to nitrate. Fluorescence in situ hybridization analysis revealed the increase in amount of ammonia-oxidizing bacteria which existed in the outer edge of the granular sludge during the start-up period. This microbial ecological change would make it possible to attain high rate ammonia removal.     

In situ structure/function studies in wastewater treatment systems

As an example of the importance and the potential of in situ structure/function studies, nitrifying aggregates from different zones of a lab-scale fluidised bed reactor were analysed by microelectrode measurements of O₂ NH₄⁺, NO₂⁻, and NO₃⁻ and in situ hybridisation targeting the 16S rRNA of the nitrifying bacteria. A shift trom an ammonia oxidising to a nitrite oxidising community is present along the reactor. In the single aggregates an active nitrifying shell of about 100 gm could be related to the maximum abundance of nitrifiers in this zone. Interestingly, the main actors in this system are not representatives of the well-described genera Nitrosomonas and Nitrobacter but some other ammonia oxidisers from the beta subclass of Protcobacteria and a thus far unknown nitrite oxidising population.