Achieving the nitrite pathway using aeration phase length control and step-feed in an SBR removing nutrients from abattoir wastewater

Aeration phase length control and step-feed of wastewater are used to achieve nitrogen removal from wastewater via nitrite in sequencing batch reactors (SBR). Aeration is switched off as soon as ammonia oxidation is completed, which is followed by the addition of a fraction of the wastewater that the SBR receives over a cycle to facilitate denitrification. The end-point of ammonia oxidation is detected from the on-line measured pH and oxygen uptake rate (OUR). The method was implemented in an SBR achieving biological nitrogen and phosphorus removal from anaerobically pre-treated abattoir wastewater. The degree of nitrite accumulation during the aeration period was monitored along with the variation in the nitrite oxidizing bacteria (NOB) population using fluorescence in situ hybridization (FISH) techniques. It is demonstrated that the nitrite pathway could be repeatedly and reliably achieved, which significantly reduced the carbon requirement for nutrient removal. Model-based studies show that the establishment of the nitrite pathway was primarily the result of a gradual reduction of the amount of nitrite that is available to provide energy for the growth of NOB, eventually leading to the elimination of NOB from the system.     

Comparison of biological removal via nitrite with real-time control using aerobic granular sludge and flocculent activated sludge

The process of nitrification–denitrification via nitrite for nitrogen removal under real-time control mode was tested in two laboratory-scale sequencing batch reactors (SBRs) with flocculent activated sludge (R1) and aerobic granular sludge (R2) to compare operational performance and real-time control strategies. The results showed that the average ammonia nitrogen, total inorganic nitrogen (TIN), and chemical oxygen demand (COD) removal during aeration phase were 97.6%, 57.0%, and 90.1% in R2 compared with 98.6%, 48.7%, and 88.1% in R1. The TIN removed in both SBRs was partially due to the presence of simultaneous nitrification–denitrification via nitrite, especially in R2. The specific nitrification and denitrification rates in R2 were 0.0416 mgNH₄⁺–N/gSS-min and 0.1889 mgNO_X⁻–N/gSS-min, which were 1.48 times and 1.35 times that of R1. The higher rates for COD removal, nitrification, and denitrification were achieved in R2 than R1 with similar influent quality. Dissolved oxygen (DO), pH, and oxidization reduction potential, corresponding to nutrient variations, were used as diagnostic parameters to control the organic carbon degradation and nitrification–denitrification via nitrite processes in both SBRs. The online control strategy of granular SBR was similar to that of the SBR with flocculent activated sludge. However, a unique U-type pattern on the DO curve in granular SBR was different from SBR with flocculent activated sludge in aerobic phase.     

Nitrification and nitrate dissimilation in soil

Summary 1. Studies were made on the decomposition of a substrate containing glucose, ammonia, and nitrate in soil held under differing aeration conditions.2. When water slurries were incubated with substrate, the loss of total-N equalled the loss of nitrate plus nitrite nitrogen.3. Under percolation conditions, with small amounts of substrate and an oxygen partial pressure of 15.2 cm of mercury, there was little change in nitrate or nitrite concentrations. Loss of nitrate only occurred under conditions of reduced aeration but, when it did occur, the sum of nitrate plus atmospheric oxygen utilized by the soil was approximately the same, irrespective of the loss of nitrate. Under an atmosphere of oxygen-free nitrogen, gas output was proportional to loss of nitrate plus nitrite nitrogen. In all cases immobilisation of ammonia was similar.4. Soils which had been percolated under anaerobic conditions with substrate, when put under aerobic conditions and with fresh substrate added, did not lose nitrate. Soils that had been percolated under aerobic conditions, when put under anaerobic conditions and with fresh substrate added, lost nitrate after a lag phase. The period of the phase was decreased by using small amounts of substrate for the aerobic percolation.5. It is concluded that analyses for nitrate and nitrite, or measurements of oxygen uptake, can be used to give approximate measures of nitrate dissimilation.     

Effect of carbon to nitrogen (C:N) ratio on nitrogen removal from shrimp production waste water using sequencing batch reactor

The United States Marine Shrimp Farming Program (USMSFP) introduced a new technology for shrimp farming called recirculating raceway system. This is a zero-water exchange system capable of producing high-density shrimp yields. However, this system produces wastewater characterized by high levels of ammonia, nitrite, and nitrate due to 40% protein diet for the shrimp at a high density of 1,000 shrimp per square meter. The high concentrations of nitrate and nitrite (greater than 25 ppm) are toxic to shrimp and cause high mortality. So treatment of this wastewater is imperative in order to make shrimp farming viable. One simple method of treating high-nitrogen wastewater is the use of a sequencing batch reactor (SBR). An SBR is a variation of the activated sludge process, which accomplishes many treatment events in a single reactor. Removal of ammonia and nitrate involved nitrification and denitrification reactions by operating the SBR aerobically and anaerobically in sequence. Initial SBR operation successfully removed ammonia, but nitrate concentrations were too high because of carbon limitation in the shrimp production wastewater. An optimization study revealed the optimum carbon to nitrogen (C:N) ratio of 10:1 for successful removal of all nitrogen species from the wastewater. The SBR operated with a C:N ratio of 10:1 with the addition of molasses as carbon source successfully removed 99% of ammonia, nitrate, and nitrite from the shrimp aquaculture wastewater within 9 days of operation.     

Integration of a Coagulation/Flocculation step in a biological sequencing batch reactor for COD and nitrogen removal of supernatant of anaerobically digested piggery wastewater

The supernatant from mesophilic anaerobic digestion of piggery wastewater is characterised by a high amount of COD (4.1 g COD L(-1)), ammonium (2.3g NH(4)(+)-NL(-1)) and suspended solids (2.5 g SS L(-1)). This effluent can be efficiently treated by means of a Sequencing Batch Reactor (SBR) strategy for biological COD, SS and nitrogen removal including a Coagulation/Flocculation step. Total COD and SS reduction yields higher than 66% and 74%, respectively, and a total nitrogen removal (via nitrite) of more than 98% were reached when working with HRT 2.7 days, SRT 12 days, temperature 32 degrees C, three aerobic/anoxic periods, without external control of pH and under limited aeration flow. The inhibition of nitrite oxidizing biomass was achieved by the working free ammonia concentration and the restricted air supply (dissolved oxygen concentration below 1 mg O(2)L(-1)). Since a part of the total COD was colloidal and/or refractory, a Coagulation/Flocculation step was implemented inside the SBR operating strategy to meet a suitable effluent quality to be discharged. Several Jar-Tests demonstrated that the optimal concentration of FeCl(3) was 800 mg L(-1). A respirometric assay showed that this coagulant dosage did not affect the biological activity of nitrifying/denitrifying biomass.     

Metabolic profiles and aprE expression in anaerobic cultures of Bacillus subtilis using nitrate as terminal electron acceptor

Cultures using nitrate as the terminal electron acceptor were conducted in Schaeffer's medium to evaluate the growth performance and metabolic profiles of Bacillus subtilis, and its potential to express the aprE (subtilisin) gene under anoxic conditions. Nitrate was converted to ammonia through nitrite reduction; and different product profiles were observed during the growth phase when nitrate was added at various concentrations (4-24 mM) to Schaeffer's medium containing glucose (4 g l(-1)). If nitrate was not limiting, then acetic acid and acetoin were accumulated, suggesting a limitation of reduced cofactors but, if nitrate became limiting, then lactic acid and butanediol were accumulated, suggesting an excess of reduced cofactors. Due to a strong lysis at the onset of the end of the growth phase, sporulation frequency and aprE expression were negligible in anaerobic batch cultures. Fed-batch fermentation allowed the development of a stationary phase through a continuous supply of glucose and nitrate. In this case, sporulation frequency was almost null, but interestingly aprE expression was similar to that found in aerobic cultures.     

Aerobic granulation and nitrogen removal with the effluent of internal circulation reactor in start-up of a pilot-scale sequencing batch reactor

Aerobic granular sludge was successfully cultivated with the effluent of internal circulation (IC) reactor in a pilot-scale sequencing batch reactor (SBR) using activated sludge as seeding sludge. N removal was investigated in the start-up of aerobic granulation process. Initially, the phenomenon of partial nitrification was observed and nitrite accumulation rates (NO₂ ^?-N/NO _x ^? -N) were between 84.6 and 99.1?%. It was potentially caused by ammonium oxidizing bacteria (AOB) in the seeding activated sludge, high external environmental temperature (~32?°C) and free ammonia (FA) concentration. After 50?days’ running, the aerobic granules-based bioreactor demonstrated perfect performance in simultaneous removal of organic matter and ammonia nitrogen, and average removal efficiencies were maintained above 93 and 96?%, respectively. The maximum nitrogen removal efficiency of 83.1?% was achieved after the formation of aerobic granules. The average diameter of mature aerobic granular sludge mostly ranged from 0.5 to 1.0?mm. Furthermore, one typical cyclic test indicated that pH and DO profiles could be used as effective parameters for biological reactions occurring in the aerobic/anoxic process. The obtained results could provide further information on the cultivation of aerobic granular sludge with practical wastewater, especially with regard to nitrogen-rich industrial wastewater.     

Mixed culture of nitrifying bacteria and denitrifying bacteria for simultaneous nitrification and denitrification

A mixed culture containing nitrifying bacteria and denitrifying bacteria was investigated for aerobic simultaneous nitrification and denitrification. A mixture of NaHCO₃ and CH₃COONa was selected as the appropriate carbon source for cell growth and nitrogen removal, the concentrations of carbon and nitrogen sources were also examined. Ammonia could be oxidized aerobically to nitrite by the mixed culture, and the intermediate nitrite was then reduced to dinitrogen gas. No nitrite was detected during the process. 0.212 g of ammonia/l could be removed in 30 h and nitrate could not be utilized aerobically by the mixed culture. Nitrite could be degraded aerobically as well as anaerobically. Very little ammonia was degraded anaerobically, but the ability to degrade ammonia could be recovered even after oxygen had been supplied for 42 h.     

Identifying an interfering factor on chemical oxygen demand (COD) determination in piggery wastewater and eliminating the factor by an indigenous Pseudomonas stutzeri strain

AIMS: This study attempted to demonstrate nitrite interference on chemical oxygen demand (COD) determination in piggery wastewater, and the capability of aerobic denitrification of the SU2 strain which is capable of promoting the efficiency of nitrogen and COD removal from piggery wastewater. METHODS AND RESULTS: This study was performed in a 17-litre reactor with a 30% packing ratio, with a ratio of immobilized SU2 cells to sludge of 100:1. The ratio of aeration to nonaeration was 4 : 1.5. Removal efficiency of COD was 86.8%. Removal efficiency of BOD and SS was higher than 90%, and removal efficiency of NH4+-N and TKN was almost 100%. CONCLUSIONS: NO2- -N interference is significant when its concentration in piggery wastewater exceeds 100 mg l-1. COD in piggery wastewater can be indirectly reduced following nitrite reduction by SU2 strain. SIGNIFICANCE AND IMPACT OF THE STUDY: Utilizing immobilized SU2 cells in coordination with an SBR system simultaneously reduces nitrite and COD concentrations.     

Effect of anoxic/aerobic phase fraction on N2O emission in a sequencing batch reactor under low temperature

Laboratory scale anoxic/aerobic sequencing batch reactor (A/O SBR) was operated around 15 °C to evaluate the effect of anoxic/aerobic phase fraction (PF) on N₂O emission. The ammonia removal exhibited a decrease trend with the increase of PF, while the highest total nitrogen removal was achieved at PF = 0.5. Almost all the N₂O was emitted during the aerobic phase, despite of the PF value. However, the net emission of N₂O was affected by PF. Under the premise of completely aerobic nitrification, the lowest N₂O emission was achieved at PF = 0.5, with a N₂O-N conversion rate of 9.8%. At lower PF (PF = 0.2), N₂O emission was stimulated by residual nitrite caused by uncompleted denitrification during the anoxic phase. On the other hand, the exhaustion of the easily degradable carbon was the major cause for the high N₂O emission at higher PF (PF = 0.5). The N₂O emission increased with the decreasing temperature. The time-weighted N₂O emission quantity at 15 °C was 2.9 times higher than that at 25 °C.     

Stoichiometric and kinetic characterisation of Nitrobacter in mixed culture by decoupling the growth and energy generation processes

The growth, maintenance and lysis processes of Nitrobacter were characterised. A Nitrobacter culture was enriched in a sequencing batch reactor (SBR). Fluorescent in situ hybridisation showed that Nitrobacter constituted 73% of the bacterial population. Batch tests were carried out to measure the oxygen uptake rate and/or nitrite consumption rate when both nitrite and CO2 were in excess, and in the absence of either of these two substrates. The results obtained, along with the SBR performance data, allowed the determination of the maintenance coefficient and in situ cell lysis rate of Nitrobacter. Nitrobacter spends a significant amount of energy for maintenance, which varies considerably with the specific growth rate. At maximum growth, Nitrobacter consume nitrite at a rate of 0.042 mgN/mgCOD(biomass) . h for maintenance purposes, which increases more than threefold to 0.143 mgN/mgCOD(biomass) . h in the absence of growth. In the SBR, where Nitrobacter grew at 40% of its maximum growth rate, a maintenance coefficient of 0.113 mgN/mgCOD . h was found, resulting in 42% of the total amount of nitrite being consumed for maintenance. The above three maintenance coefficient values obtained at different growth rates appear to support the maintenance model proposed in Pirt (1982). The in situ lysis rate of Nitrobacter was determined to be 0.07/day under aerobic conditions at 22 degrees C and pH 7.3. Further, the maximum specific growth rate of Nitrobacter was estimated to be 0.02/h (0.48/day). The affinity constant of Nitrobacter with respect to nitrite was determined to be 1.50 mgNO2(-)-N/L, independent of the presence or absence of CO2.     

Free nitrous acid selectively inhibits and eliminates nitrite oxidizers from nitrifying sequencing batch reactor

In a complete nitrification sequencing batch reactor (CNSBR), where ammonium containing wastewater (200–1,000 mg N/L) is completely oxidized to nitrate up to 2.4 kg NH₄ ⁺–N/m³ d, both ammonia oxidizers and nitrite oxidizers were enriched in the sludge granules. Quantitative fluorescence in situ hybridization analyses of the sludge granules of the CNSBR showed that ammonia oxidizers and nitrite oxidizers occupied 31 and 4.2% of total bacteria, respectively. Most of the nitrite oxidizers were Nitrobacter species (95% of the nitrite oxidizers) and the remainder was Nitrospira species. The population of nitrite oxidizers was significantly higher than that of partial nitrification SBR (PNSBR) where most of the ammonium was oxidized to nitrite. The PNSBR had 37% (ammonia oxidizers) and 0.4% (nitrite oxidizers) of total bacteria. Comparative study with CNSBR and PNSBR revealed that free nitrous acid, rather than free ammonia, played a critical inhibition role to wash out nitrite oxidizers from the reactor. The concentrations of free ammonia and nitrite as well as free nitrous acid in the CNSBR selected Nitrobacter as the dominant nitrite oxidizers rather than Nitrospira.     

Effects of acetate and nitrite addition on fraction of denitrifying phosphate-accumulating organisms and nutrient removal efficiency in anaerobic/aerobic/anoxic process

The effects of acetate and nitrite on the performance of sequencing batch reactors (SBRs) employing an anaerobic/aerobic/anoxic (AOA) process were investigated. Three types of SBR operations were used: sodium acetate addition at the start of anoxic condition for heterotrophic denitrification (Type 1); sodium acetate addition at the start of aerobic condition for anoxic phosphate removal by denitrifying phosphate-accumulating organisms (DNPAOs) (Type 2: conventional AOA process); and nitrite addition at the start of aerobic condition for inhibition of phosphate-accumulating organisms (PAOs) (Type 3). A track experiment shows that Type 2 led to the best performance of SBRs among the three types. An analysis by fluorescence in situ hybridization (FISH) revealed that nitrite addition decreased the ratio of PAOs with a decrease in phosphorus removal efficiency. The fraction of DNPAOs in Type 2 was the highest at 13%, indicating that Type 2 is suitable for the simultaneous nitrogen and phosphorus removal in the AOA process.     

Identification and comparison of aerobic and denitrifying polyphosphate-accumulating organisms

Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic-aerobic or alternating anaerobic-anoxic modes, respectively. Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic-aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic-aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, "Candidatus Accumulibacter phosphatis" was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic-anoxic SBR enriched for DPAOs was converted to anaerobic-aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic-aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic-aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic-aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic-anoxic conditions.     

Nitrite reductase system involved in the terminal oxidation of the Streptomyces griseus respiratory particle.

A nitrite reductase system which was associated with the electron transfer system of the respiratory particle in Streptomyces griseus was studied. The electron transfer pathway consisted of the cytochrome oxidase and the nitrite reductase systems under aerobic and anaerobic conditions respectively, and these systems showed the exact opposite response to 2-n-heptyl-4-hydroxyquinoline-N-oxide and azide. Azide inhibited specifically the nitrite reductase system. It seems that cytochrome d works as the nitrite reductase and the reduced cytochrome b works as an intermediate electron donor for cytochrome d respectively. The respiratory particle also had a hydroxylamine reductase activity and ammonia was identified as the product of hydroxylamine reduction by the respiratory particle. A terminal electron transfer pathway in Streptomyces griseus was proposed.     

Biological treatment of shrimp production wastewater

Over the last few decades, there has been an increase in consumer demand for shrimp, which has resulted in its worldwide aquaculture production. In the United States, the stringent enforcement of environmental regulations encourages shrimp farmers to develop new technologies, such as recirculating raceway systems. This is a zero-water exchange system capable of producing high-density shrimp yields. The system also produces wastewater characterized by high levels of ammonia, nitrate, nitrite, and organic carbon, which make waste management costs prohibitive. Shrimp farmers have a great need for a waste management method that is effective and economical. One such method is the sequencing batch reactor (SBR). A SBR is a variation of the activated sludge biological treatment process. This process uses multiple steps in the same reactor to take the place of multiple reactors in a conventional treatment system. The SBR accomplishes equalization, aeration, and clarification in a timed sequence in a single reactor system. This is achieved through reactor operation in sequences, which includes fill, react, settle, decant, and idle. A laboratory scale SBR was successfully operated using shrimp aquaculture wastewater. The wastewater contained high concentrations of carbon and nitrogen. By operating the reactors sequentially, namely, aerobic and anoxic modes, nitrification and denitrification were achieved as well as removal of carbon. Ammonia in the waste was nitrified within 4 days. The denitrification of nitrate was achieved by the anoxic process, and 100% removal of nitrate was observed within 15 days of reactor operation.     

Dynamics of denitrification activity of Paracoccus denitrificans in continuous culture during aerobic-anaerobic changes.

Induction and repression of denitrification activity were studied in a continuous culture of Paracoccus denitrificans during changes from aerobic to anaerobic growth conditions and vice versa. The denitrification activity of the cells was monitored by measuring the formation of denitrification products (nitrite, nitric oxide, nitrous oxide, and dinitrogen), individual mRNA levels for the nitrate, nitrite, and nitrous oxide reductases, and the concentration of the nitrite reductase enzyme with polyclonal antibodies against the cd1-type nitrite reductase. On a change from aerobic to anaerobic respiration, the culture entered an unstable transition phase during which the denitrification pathway became induced. The onset of this phase was formed by a 15- to 45-fold increase of the mRNA levels for the individual denitrification enzymes. All mRNAs accumulated during a short period, after which their overall concentration declined to reach a stable value slightly higher than that observed under aerobic steady-state conditions. Interestingly, the first mRNAs to be formed were those for nitrate and nitrous oxide reductase. The nitrite reductase mRNA appeared significantly later, suggesting different modes of regulation for the three genes. Unlike the mRNA levels, the level of the nitrite reductase protein increased slowly during the anaerobic period, reaching a stable value about 30 h after the switch. All denitrification intermediates could be observed transiently, but when the new anaerobic steady state was reached, dinitrogen was the main product. When the anaerobic cultures were switched back to aerobic respiration, denitrification of the cells stopped at once, although sufficient nitrite reductase was still present. We could observe that the mRNA levels for the individual denitrification enzymes decreased slightly to their aerobic, uninduced levels. The nitrite reductase protein was not actively degraded during the aerobic period.     

The occurrence of chemolitho-autotrophic nitrifiers in water-saturated grassland soils

Relatively high most probable number (MPN) counts of chemolithotrophic nitrite oxidizers were present in water-saturated soils compared with MPNs and activity of ammonia oxidizers. These high numbers of nitrite oxidizers were confirmed by fluorescent antibody counts and potential activity measurements. Application of different nitrite concentrations in the MPN procedure discriminated within the community of nitrite oxidizers and revealed a large number of nitrite-sensitive nitrite oxidizers and a subcommunity of nitrite-insensitive nitrite oxidizers. The size of this subcommunity was small but corresponded with the low numbers of ammonium oxidizers. Numbers of nitrite-sensitive nitrite oxidizers outnumbered the ammonia oxidizing bacteria by 2–4 orders of magnitude in these soils. The possibility is discussed that the fraction of the nitrite-insensitive cells was active as aerobic nitrite oxidizers, whereas the nitrite-sensitive cells represented an inactive group of nitrite oxidizers growing as heterotrophs or as anaerobes reducing nitrite. In this situation, both MPN enumerations at a low nitrite concentration and activity measurements could give false information about the size of the in situ nitrite-oxidizing community.     

Genetic regulation of nitrate assimilation in Klebsiella pneumoniae M5al.

We isolated Mu dI1734 insertion mutants of Klebsiella pneumoniae that were unable to assimilate nitrate or nitrite as the sole nitrogen source during aerobic growth (Nas- phenotype). The mutants were not altered in respiratory (anaerobic) nitrate and nitrite reduction or in general nitrogen control. The mutations were linked and thus defined a single locus (nas) containing genes required for nitrate assimilation. beta-Galactosidase synthesis in nas+/phi(nas-lacZ) merodiploid strains was induced by nitrate or nitrite and was inhibited by exogenous ammonia or by anaerobiosis. beta-Galactosidase synthesis in phi(nas-lacZ) haploid (Nas-) strains was nearly constitutive during nitrogen-limited aerobic growth and uninducible during anaerobic growth. A general nitrogen control regulatory mutation (ntrB4) allowed nitrate induction of phi(nas-lacZ) expression during anaerobic growth. This and other results suggest that the apparent anaerobic inhibition of phi(nas-lacZ) expression was due to general nitrogen control, exerted in response to ammonia generated by anaerobic (respiratory) nitrate reduction.     

Nitrogen removal by tubular gel containing Nitrosomonas europaea and Paracoccus denitrificans.

A new bioreactor for the removal of nitrogen from wastewater is described which consists of a tubular polymeric gel containing Nitrosomonas europaea and Paracoccus denitrificans. The outer surface of the tube is in aerobic contact with wastewater containing ammonia, while the inside of the tube is in anaerobic contact with ethanol flowing through the tube. N. europaea oxidizes ammonia to nitrite in the gel, and then P. denitrificans reduces the nitrite to nitrogen gas in the same gel. This concept would be effective for simplifying nitrogen removal systems requiring aerobic and anaerobic operations.