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.     

Population dynamics and biofilm composition in a new three-phase circulating bed reactor

The biofilm characteristics of a novel three-phase reactor, the circulating bed reactor (CBR), were studied using industrial prototype fed with primary and secondary settled effluent in conditions of tertiary N and secondary C+N nitrification. The results showed a high nitrification rate close to the intrinsic values for N and C+N conditions: up to 2 and 0.6 kgN-NH₄ m^-3 d^-1, or 1.88±0.26 and 0.22±0.07 gN g^-1 PR d^-1, respectively. The application of an integrated approach for biofilm analysis enabled the better understanding of biofihn dynamics. The biofilm remained relatively thin, below 100 μm, indicating an effective control of the biofilm development. Protein, measured by the conventional colometric method and pyrolysis-GCMS, was the major fraction accounting for up to 35% of the biomass dry weight and 58% of the biopolymer content. The polysaccharide's fraction remained very low (<3%). The ribosomal RNA probes analysis confirmed the predominance of bacterial cells in the CBR biofilm (80–86% of bacteria versus the universal probe) showing a high proportion of nitrifying bacteria accounting for up to 50% and 27% in the N and C+N removal respectively. Nitrosomonas predominated in tertiary nitrification whereas carbon input led to the appearance of other ammonia oxidizers. This particular composition was characterized by a high state of oxidation of the biomass, expressed by the low COD/DW ratio of about 0.85. In conclusion, it can be stated that this new three-phase bioreactor ensures a high nitrification rate through an effective biofilm control promoting the development of bacterial cells, especially nitrifying bacteria, and minimizing exopolysaccharides production.     

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.     

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.     

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.     

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.     

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.     

水生经济植物根际氮循环细菌及其作用的研究

利用生态浮床系统,在室内研究了浮床植物单作和混作对氮的去除效果及其根际周围硝化与反硝化菌种群数量以及硝化与反硝化作用速率的变化规律.结果表明,浮床植物混作系统对氮的去除效果优于植物单作和无植物系统,而植物系统又优于无植物系统;植物单作与混作根际硝化细菌种群数量和硝化作用速率、反硝化细菌种群数量和反硝化作用速率均具有明显的差异;处理15 d后,硝化细菌种群数量在水芹系统中的减少较比豆瓣菜和混作系统多,硝化作用速率在水芹系统中的降低较豆瓣菜和混作系统明显(P <0.05);反硝化细菌种群数量由多到少的顺序分别为水芹系统,豆瓣菜系统,混作系统,无植物系统;且反硝化作用速率由高到低的顺序分别为水芹系统,豆瓣菜系统,混作系统,无植物系统.     

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.     

Formation mechanism of nitrifying granules observed in an aerobic upflow fluidized bed (AUFB) reactor.

The influences of trace metals in the wastewater and shear stress by aeration were particularly examined to clarify the formation mechanism of nitrifying granules in an aerobic upflow fluidized bed (AUFB) reactor. It was found that Fe added as a trace element to the inorganic wastewater accumulated at the central part of the nitrifying granules. Another result obtained was that suitable shear stress by moderate aeration (0.07-0.20 L/min/L-bed) promoted granulation. Furthermore, it was successfully demonstrated that pre-aggregation of seed sludge using hematite promoted core formation, leading to rapid production of nitrifying granules. From these results, a nitrifying granulation mechanism is proposed: 1) as a first step, nitrifying bacteria aggregate along with Fe precipitation, and then the cores of granules are formed; 2) as a second step, the aggregates grow to be spherical or elliptical in form due to multiplication of the nitrifying bacteria and moderate shear stress in the reactor, and then mature nitrifying granules are produced. Fluorescence in situ hybridization (FISH) analysis successfully visualized the change in the spatial distribution of nitrifying bacteria in the granules, which supports the proposed granulation mechanism.     

Multistage wastewater treatment using separated storage driven denitrification and nitrification biofilms.

The feasibility of combining a previously reported storage driven denitrification biofilm, where 80% of influent acetate can be converted to poly-beta-hydroxybutyrate (PHB), with a suitable nitrification reactor, either submerged or trickling filter design, to achieve complete biological nitrogen removal was tested. The reactor system showed the potential of complete biological nitrogen removal of waste streams with a C/N ratio as low as 3.93 kg COD/kg N-NH3 at an overall nitrogen removal rate of 1.1 mmole NH3/L/h. While the efficiency and the rates of nitrogen removal were higher than what is observed in traditional or simultaneous nitrification and denitrification (SND) systems, there were two problems that require further development: (a) the incomplete draining of the reactor caused ammonia retention and release in the effluent, limiting the overall N-removal and (b) pH drifts in the nitrification step slowed down the rate of nitrification if not corrected by appropriate pH adjustment or buffering.     

Biological granulated activated carbon fluidized bed reactor for atrazine remediation.

To show that an adsorbing biofilm carrier (GAC) can be advantageous for atrazine bioremediation over a non-adsorbing carrier, fluidized bed (FB) reactors were operated under atrazine limiting concentrations using Pseudomonas sp. strain ADP as the atrazine degrading bacteria. The following interrelated subjects were investigated: 1) atrazine adsorption to GAC under conditions of atrazine partial penetration in the biofilm, 2) differences in atrazine degradation rates and 3) stability of atrazine biodegradation under non-sterile anoxic conditions in the GAC reactor versus a reactor with a non-adsorbing biofilm carrier. Results from batch adsorption tests together with modeling best described the biofilm as patchy in nature with covered and non-biofilm covered areas. Under conditions of atrazine partial penetration in the biofilm, atrazine adsorption occurs in the non-covered areas and is consequently desorbed at the base of the biofilm substantially increasing the active biofilm surface area. The double flux of atrazine to the biofilm in the GAC reactor results in lower effluent atrazine concentrations as compared to a FB reactor with a non-adsorbing carrier. Moreover, under non-sterile denitrification conditions, atrazine degradation stability was found to be much higher (several months) using GAC as a biofilm carrier while non-adsorbing carrier reactors showed sharp deterioration within 30 days due to contamination of non-atrazine degrading bacteria.     

Study of a three-stage fluidized bed process treating acrylic synthetic-fiber manufacturing wastewater containing high-strength nitrogenous compounds.

Polyacrylonitrile (PAN) is one of the major synthetic fibers commonly used in the mass production of clothing. The chemical synthesis of PAN is carried out by polymerization of the acrylonitrile (AN) monomers with co-monomers such as vinyl acetate, methyl acrylate and cyclohexyl acrylate. Using water quality analysis of the PAN wastewater, high concentration of organic nitrogen was found and the TKN/COD ratios achieved were 0.15-0.26, indicating the complicated biodegradation characteristics for the PAN wastewater. In order to enhance biodegradation of nitrogenous compounds in PAN wastewater, a combined three-stage process of thermophilic anaerobic/anoxic denitrification/aerobic nitrification fluidized bed reactors was employed. The results indicated that the concentration of effluent in the three-stage process of OD and organic nitrogen was 175 mg/L and 13 mg/L, respectively. Furthermore, molecular biotechnology was applied to study the microbial population in the thermophilic anaerobic fluidized bed reactor. From the results of denaturing gradient gel electrophoresis, the diversity of PAN-degrading bacteria would change in different volumetric loading. Furthermore, the bacteria communities in the thermophilic anaerobic fluidized bed reactor were also studied by fluorescence in situ hybridization and confocal laser scanning microscopy. Alpha and delta-Proteobacteria were dominant in the bacteria population, and some high G+C content bacteria and Clostridium could be characterized in this system.     

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.     

Use of immobilised bacteria for the wastewater treatment--examples from the sugar industry.

This work focuses on the implementation of high performance systems to the wastewater treatment of sugar factories. For this purpose, systems with immobilised bacteria were studied. For the hydrolysis of organic matter and denitrification, fluidized bed reactors were used. The nitrification was studied with an airlift reactor system. Both hydrolysis and nitrogen elimination were investigated on laboratory and pilot scales in sugar factories. Although with porous materials higher biomass concentrations are attainable for the hydrolysis (up to 55 kg/m3), for economical reasons sand was used (22.5 kg/m3) for the pilot scale-study. With a pilot-scale reactor (volume 1 m3) the maximum sucrose conversion rate achieved with sand in the first campaign was 52 kg/(m3 d). For the nitrogen elimination on the pilot scale, a system with denitrification and nitrification was combined. The highest performance for the nitrification (reactor volume: 0.68 m3) with pumice as support material was 1.2 kg NH4-N/(m3 d), limiting the whole system. The denitrification rate (reactor volume: 0.12 m3) was four times higher (3.5-5 kg NO3-N/(m3 d). Rules of the modelling of the system are discussed.     

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.     

Modeling response of nitrifying biofilm to inhibitory shock loads.

To study the response of nitrifying biofilm to inhibitory shock loads, a lab-scale nitrifying biofilm reactor was operated in ambient conditions. Shock loads of various concentrations of inhibitory compound were applied to the biofilm. Aniline was used as an inhibitory compound. The experimental results were utilized to develop a model for predicting the variation of effluent nitrate concentration from the biofilm reactor for given shock loads of aniline concentration and exposure time both in exposure as well as in recovery phase. Close agreement between model and experimental observation of bulk aniline concentration and effluent nitrate concentration was obtained which indicates the usefulness of the model to estimate bulk aniline concentration and to predict the response of inhibitory shock loads on nitrifying biofilm.     

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.     

Full scale fluidized bed anaerobic reactor for domestic wastewater treatment: performance, sludge production and biofilm.

This paper describes the performance, sludge production and biofilm characteristics of a full scale fluidized bed anaerobic reactor (32 m3) for domestic wastewater treatment. The reactor was operated with 10.5 m x h(-1) upflow velocity, 3.2 h hydraulic retention time, and recirculation ratio of 0.85 and it presented removal efficiencies of 71+/-8% of COD and 77+/-14% of TSS. During the apparent steady-state period, specific sludge production and sludge age in the reactor were (0.116+/-0.033) kgVSS. kgCOD(-1) and (12+/-5)d, respectively. Biofilm formed in the reactor presented two different patterns: one of them at the beginning of the colonization and the other of mature biofilm. These different colonization patterns are due to bed stratification in the reactor, caused by the difference in local-energy dissipation rates along the reactor's height, and density, shape, etc. of the bioparticles. The biofilm population is formed mainly of syntrophic consortia among sulfate reducing bacteria, methanogenic archaea such as Methanobacterium and Methanosaeta-like cells.     

Treatment of nitrogen and phosphorus in highly concentrated effluent in SBR and SBBR processes.

Various sludge treatment processes produced supernatant with high ammonia concentration from 500 to 2,000 mgN/L and generally high phosphate concentration. Conversion of ammonia into nitrite via partial nitrification has proven to be an economic way, reducing oxygen and external COD requirements during the nitrification/denitrification process. Two processes with biomass retention are studied simultaneously: the sequencing batch reactor (SBR) and the sequencing batch biofilm reactor (SBBR). At a temperature of 30 degrees C, the inhibition of nitrite-oxidizing bacteria due to high ammonia concentration has been studied in order to obtain a stable nitrite accumulation. This work has confirmed the effect of pH and dissolved oxygen on nitrite accumulation performance. During a two month starting period, both processes led to nitrite accumulation without nitrate production when pH was maintained above 7.5. From a 500 mgN/L effluent, the performance of the SBR, and the SBBR, reached respectively about 0.95gN-NO2-/gN-NH4+, and 0.4gN-NO2-/gN-NH4+. The SBBR appears to be more stable facing disturbances in dissolved oxygen conditions. Finally, the maximal phosphate removal rates obtained in the SBR reached 90%, and 70% in the SBBR, depending on ammonium accumulation in the reactor. Ammonium phosphate precipitation is likely to occur, as was suggested by crystals observation in the reactor.