Nitrite inhibition and limitation - the effect of nitrite spiking on anammox biofilm, suspended and granular biomass.

Anaerobic ammonium oxidation (anammox) has been studied extensively while no widely accepted optimum values for nitrite (both a substance and inhibitor) has been determined. In the current paper, nitrite spiking (abruptly increasing nitrite concentration in reactor over 20 mg NO-2-NL-1) effect on anammox process was studied on three systems: a moving bed biofilm reactor (MBBR), a sequencing batch reactor (SBR) and an upflow anaerobic sludge blanket (UASB). The inhibition thresholds and concentrations causing 50% of biomass activity decrease (IC50) were determined in batch tests. The results showed spiked biomass to be less susceptible to nitrite inhibition. Although the values of inhibition threshold and IC50 concentrations were similar for non-spiked biomass (81 and 98 mg NO-2-NL-1, respectively, for SBR), nitrite spiking increased IC50 considerably (83 and 240 mg NO-2-NL-1, respectively, for UASB). As the highest total nitrogen removal rate was also measured at the aforementioned thresholds, there is basis to suggest stronger limiting effect of nitrite on anammox process than previously reported. The quantitative polymerase chain reaction analysis showed similar number of anammox 16S rRNA copies in all reactors, with the lowest quantity in SBR and the highest in MBBR (3.98 × 108 and 1.04 × 109 copies g-1 TSS, respectively).

Nitric oxide for anammox recovery in a nitrite-inhibited deammonification system.

The anaerobic ammonium oxidation (anammox) process is widely used for N-rich wastewater treatment. In the current research the deammonification reactor in a reverse order (first anammox, then the nitrifying biofilm cultivation) was started up with a high maximum N removal rate (1.4 g N m(-2) d(-1)) in a moving bed biofilm reactor. Cultivated biofilm total nitrogen removal rates were accelerated the most by anammox intermediate - nitric oxide (optimum 58 mg NO-N L(-1)) addition. Furthermore, NO was added in order to eliminate inhibition caused by nitrite concentrations (>50 mg [Formula: see text]) increasing [Formula: see text] (2/1, respectively) along with a higher ratio of [Formula: see text] (0.6/1, respectively) than stoichiometrical for this optimal NO amount added during batch tests. Planctomycetales clone P4 sequences, which was the closest (98% and 99% similarity, respectively) relative to Candidatus Brocadia fulgida sequences quantities increase to 1 × 10(6) anammox gene copies g(-1) total suspended solids to till day 650 were determined by quantitative polymerase chain reaction.

Start-up of low-temperature anammox in UASB from mesophilic yeast factory anaerobic tank inoculum.

Robust start-up of the anaerobic ammonium oxidation (anammox) process from non-anammox-specific seeding material was achieved by using an inoculation with sludge-treating industrial [Formula: see text]-, organics- and N-rich yeast factory wastewater. N-rich reject water was treated at 20°C, which is significantly lower than optimum treatment temperature. Increasing the frequency of biomass fluidization (from 1-2 times per day to 4-5 times per day) through feeding the reactor with higher flow rate resulted in an improved total nitrogen removal rate (from 100 to 500 g m(-3)d(-1)) and increased anammox bacteria activity. As a result of polymerase chain reaction (PCR) tests, uncultured planctomycetes clone 07260064(4)-2-M13-_A01 (GenBank: JX852965) was identified from the biomass taken from the reactor. The presence of anammox bacteria after cultivation in the reactor was confirmed by quantitative PCR (qPCR); an increase in quantity up to ∼2×10(6) copies g VSS(-1) during operation could be seen in qPCR. Statistical modelling of chemical parameters revealed the roles of several optimized parameters needed for a stable process.

Comparison of sulfate-reducing and conventional Anammox upflow anaerobic sludge blanket reactors.

Autotrophic NH4(+) removal has been extensively researched, but few studies have investigated alternative electron acceptors (for example, SO4(2-)) in NH4(+) oxidation. In this study, sulfate-reducing anaerobic ammonium oxidation (SRAO) and conventional Anammox were started up in upflow anaerobic sludge blanket reactors (UASBRs) at 36 (±0.5)°C and 20 (±0.5)°C respectively, using reject water as a source of NH4(+). SO4(2-) or NO2(-), respectively, were applied as electron acceptors. It was assumed that higher temperature could promote the SRAO, partly compensating its thermodynamic disadvantage comparing with the conventional Anammox to achieve comparable total nitrogen (TN) removal rate. Average volumetric NH4(+)-N removal rate in the sulfate-reducing UASBR1 was however 5-6 times less (0.03 kg-N/(m(3) day)) than in the UASBR2 performing conventional nitrite-dependent autotrophic nitrogen removal (0.17 kg-N/(m(3) day)). However, the stoichiometric ratio of NH4(+) removal in UASBR1 was significantly higher than could be expected from the extent of SO4(2-) reduction, possibly due to interactions between the N- and S-compounds and organic matter of the reject water. Injections of N2H4 and NH2OH accelerated the SRAO. Similar effect was observed in batch tests with anthraquinone-2,6-disulfonate (AQDS). For detection of key microorganisms PCR-DGGE was used. From both UASBRs, uncultured bacterium clone ATB-KS-1929 belonging to the order Verrucomicrobiales, Anammox bacteria (uncultured Planctomycete clone Pla_PO55-9) and aerobic ammonium-oxidizing bacteria (uncultured sludge bacterium clone ASB08 "Nitrosomonas") were detected. Nevertheless the SRAO process was shown to be less effective for the treatment of reject water, compared to the conventional Anammox.

Nitritating-anammox biomass tolerant to high dissolved oxygen concentration and C/N ratio in treatment of yeast factory wastewater.

Maintaining stability of low concentration (< 1 g L(-1)) floccular biomass in the nitritation-anaerobic ammonium oxidation (anammox) process in the sequencing batch reactor (SBR) system for the treatment of high COD (> 15,000 mg O2 L(-1)) to N (1680 mg N L(-1)) ratio real wastewater streams coming from the food industry is challenging. The anammox process was suitable for the treatment of yeast factory wastewater containing relatively high and abruptly increased organic C/N ratio and dissolved oxygen (DO) concentrations. Maximum specific total inorganic nitrogen (TIN) loading and removal rates applied were 600 and 280 mg N g(-1) VSS d(-1), respectively. Average TIN removal efficiency over the operation period of 270 days was 70%. Prior to simultaneous reduction of high organics (total organic carbon > 600mg L(-1)) and N concentrations > 400 mg L(-1), hydraulic retention time of 15 h and DO concentrations of 3.18 (+/- 1.73) mg O2 L(-1) were applied. Surprisingly, higher DO concentrations did not inhibit the anammox process efficiency demonstrating a wider application of cultivated anammox biomass. The SBR was fed rapidly over 5% of the cycle time at 50% volumetric exchange ratio. It maintained high free ammonia concentration, suppressing growth of nitrite-oxidizing bacteria. Partial least squares and response surface modelling revealed two periods of SBR operation and the SBR performances change at different periods with different total nitrogen (TN) loadings. Anammox activity tests showed yeast factory-specific organic N compound-betaine and inorganic N simultaneous biodegradation. Among other microorganisms determined by pyrosequencing, anammox microorganism (uncultured Planctomycetales bacterium clone P4) was determined by polymerase chain reaction also after applying high TN loading rates.

Deammonification process start-up after enrichment of anammox microorganisms from reject water in a moving-bed biofilm reactor.

Deammonification via intermittent aeration in biofilm process for the treatment of sewage sludge digester supernatant (reject water) was started up using two opposite strategies. Two moving-bed biofilm reactors were operated for 2.5 years at 26 (+/- 0.5 degree C with spiked influent(and hence free ammonia (FA)) addition. In the first start-up strategy, an enrichment of anammox biomass was first established, followed by the development of nitrifying biomass in the system (R1). In contrast, the second strategy aimed at the enrichment of anammox organisms into a nitrifying biofilm (R2). The first strategy was most successful, reaching higher maximum total nitrogen (TN) removal rates over a shorter start-up period. For both reactors, increasing FA spiking frequency and increasing effluent concentrations of the anammox intermediate hydrazine correlated to decreasing aerobic nitrate production (nitritation). The bacterial consortium of aerobic and anaerobic ammonium oxidizing bacteria in the bioreactor was determined via denaturing gel gradient electrophoresis, polymerase chain reaction and pyrosequencing. In addition to a shorter start-up with a better TN removal rate, nitrite oxidizing bacteria (Nitrospira) were outcompeted by spiked ammonium feeding from R1.

Accelerating effect of hydroxylamine and hydrazine on nitrogen removal rate in moving bed biofilm reactor.

In biological nitrogen removal, application of the autotrophic anammox process is gaining ground worldwide. Although this field has been widely researched in last years, some aspects as the accelerating effect of putative intermediates (mainly N2H4 and NH2OH) need more specific investigation. In the current study, experiments in a moving bed biofilm reactor (MBBR) and batch tests were performed to evaluate the optimum concentrations of anammox process intermediates that accelerate the autotrophic nitrogen removal and mitigate a decrease in the anammox bacteria activity using anammox (anaerobic ammonium oxidation) biomass enriched on ring-shaped biofilm carriers. Anammox biomass was previously grown on blank biofilm carriers for 450 days at moderate temperature 26.0 (±0.5) °C by using sludge reject water as seeding material. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. With addition of 1.27 and 1.31 mg N L⁻¹ of each NH2OH and N2H4, respectively, into the MBBR total nitrogen (TN) removal efficiency was rapidly restored after inhibitions by NO2⁻. Various combinations of N2H4, NH2OH, NH4⁺, and NO2⁻ were used as batch substrates. The highest total nitrogen (TN) removal rate with the optimum N2H4 concentration (4.38 mg N L⁻¹) present in these batches was 5.43 mg N g⁻¹ TSS h⁻¹, whereas equimolar concentrations of N2H4 and NH2OH added together showed lower TN removal rates. Intermediates could be applied in practice to contribute to the recovery of inhibition-damaged wastewater treatment facilities using anammox technology.

Anammox enrichment from reject water on blank biofilm carriers and carriers containing nitrifying biomass: operation of two moving bed biofilm reactors (MBBR).

The anammox bacteria were enriched from reject water of anaerobic digestion of municipal wastewater sludge onto moving bed biofilm reactor (MBBR) system carriers-the ones initially containing no biomass (MBBR1) as well as the ones containing nitrifying biomass (MBBR2). Duration of start-up periods of the both reactors was similar (about 100 days), but stable total nitrogen (TN) removal efficiency occurred earlier in the system containing nitrifying biomass. Anammox TN removal efficiency of 70% was achieved by 180 days in both 20 l volume reactors at moderate temperature of 26.0°C. During the steady state phase of operation of MBBRs the average TN removal efficiencies and maximum TN removal rates in MBBR1 were 80% (1,000 g-N/m(3)/day, achieved by 308 days) and in MBBR2 85% (1,100 g-N/m(3)/day, achieved by 266 days). In both reactors mixed bacterial cultures were detected. Uncultured Planctomycetales bacterium clone P4, Candidatus Nitrospira defluvii and uncultured Nitrospira sp. clone 53 were identified by PCR-DGGE from the system initially containing blank biofilm carriers as well as from the nitrifying biofilm system; from the latter in addition to these also uncultured ammonium oxidizing bacterium clone W1 and Nitrospira sp. clone S1-62 were detected. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. Using previously grown nitrifying biofilm matrix for anammox enrichment has some benefits over starting up the process from zero, such as less time for enrichment and protection against severe inhibitions in case of high substrate loading rates.

Effect of HCO3- concentration on anammox nitrogen removal rate in a moving bed biofilm reactor.

Anammox biomass enriched in a moving bed biofilm reactor (MBBR) fed by actual sewage sludge reject water and synthetically added NO2- was used to study the total nitrogen (TN) removal rate of the anammox process depending on bicarbonate (HCO3-) concentration. MBBR performance resulted in the maximum TN removal rate of 1100 g N m(-3) d(-1) when the optimum HCO3- concentration (910 mg L(-1)) was used. The average reaction ratio of NO2- removal, NO3- production and NH4+ removal were 1.18/0.20/1. When the HCO3- concentration was increased to 1760mg L(-1) the TN removal rate diminished to 270 g N m(-3) d(-1). The process recovered from bicarbonate inhibition within 1 week. The batch tests performed with biomass taken from the MBBR showed that for the HCO3- concentration of 615 mg L(-1) the TN removal rate was 3.3 mg N L(-1) h(-1), whereas for both lower (120 mg L(-1)) and higher (5750 mg L(-1)) HCO3- concentrations the TN removal rates were 2.3 (+/- 0.15) and 1.6 (+/- 0.12) mg N L(-1) d(-1), respectively. PCR and DGGE analyses resulted in the detection of uncultured Planctomycetales bacterium clone P4 and, surprisingly, low-oxygen-tolerant aerobic ammonia oxidizers. The ability of anammox bacteria for mixotrophy was established by diminished amounts of nitrate produced when comparing the experiments with an organic carbon source and an inorganic carbon source.