Enrichment and granulation of Anammox biomass started up with methanogenic granular sludge

A sequencing batch reactor (SBR) seeded with methanogenic granular sludge was started up to enrich Anammox (Anaerobic Ammonium Oxidation) bacteria and to investigate the feasibility of granulation of Anammox biomass. Research results showed that hydraulic retention time (HRT) was an important factor to enrich Anammox bacteria. When the HRT was controlled at 30 days during the initial cultivation, the SBR reactor presented Anammox activity at t = 58 days. Simultaneously, the methanogenic granular sludge changed gradually from dust black to brown colour and its diameter became smaller. At t = 90 days, the Anammox activity was further improved. NH₄⁺-N and NO₂⁻N were removed simultaneously with higher speed and the maximum removal rates reached 14.6 g NH₄⁺-N /(m³ _reactor·day) and 6.67 g NO₂⁻-N /(m³ _reactor·day), respectively. Between t = 110 days and t = 161 days, the nitrogen load was increased to a HRT of 5 days (70 mg/l NH₄⁺ and 70 mg/l NO₂), the removal rates of ammonium and nitrite were 60.6% and 62.5% respectively. The sludge changed to red and formed Anammox granulation with high nitrogen removal activity.     

Two ways to achieve an anammox influent from real reject water treatment at lab-scale: Partial SBR nitrification and SHARON process

A comparative study to produce the correct influent for Anammox process from anaerobic sludge reject water (700–800 mg NH₄⁺-N L⁻¹) was considered here. The influent for the Anammox process must be composed of NH₄⁺-N and NO₂⁻-N in a ratio 1:1 and therefore only a partial nitrification of ammonium to nitrite is required. The modifications of parameters (temperature, ammonium concentration, pH and solid retention time) allows to achieve this partial nitrification with a final effluent only composed by NH₄⁺-N and NO₂⁻-N at the right stoichiometric ratio. The equal ratio of HCO₃⁻/NH₄⁺ in reject water results in a natural pH decrease when approximately 50% of NH₄⁺ is oxidised. A Sequencing batch reactor (SBR) and a chemostat type of reactor (single-reactor high activity ammonia removal over nitrite (SHARON) process) were studied to obtain the required Anammox influent. At steady state conditions, both systems had a specific conversion rate around 40 mg NH₄⁺-N g⁻¹ volatile suspended solids (VSS) h⁻¹, but in terms of absolute nitrogen removal the SBR conversion was 1.1 kg N day⁻¹ m⁻³, whereas in the SHARON chemostat was 0.35 kg N day⁻¹ m⁻³ due to the different hydraulic retention time (HRT) used. Both systems are compared from operational (including starvation experiments) and kinetic point of view and their advantages/disadvantages are discussed.     

Effect of influent substrate ratio on anammox granular sludge: performance and kinetics

Effect of influent substrate ratio on anammox process was studied in sequencing batch reactor. Operating temperature was fixed at 35 ± 1 °C. Influent pH and hydraulic retention time were 7.5 and 6 h, respectively. When influent NO₂ ^?-N/NH₄ ⁺-N was no more than 2.0, total nitrogen removal rate (TNRR) increased whereas NH₄ ⁺-N removal rate stabilized at 0.32 kg/(m³ d). ΔNO₂ ^?-N/ΔNH₄ ⁺-N increased with enhancing NO₂ ^?-N/NH₄ ⁺-N. When NO₂ ^?-N/NH₄ ⁺-N was 4.5, ΔNO₂ ^?-N/ΔNH₄ ⁺-N was 1.98, which was much higher than theoretical value (1.32). The IC₅₀ of NO₂ ^?-N was 289 mg/L and anammox activity was inhibited at high NO₂ ^?-N/NH₄ ⁺-N ratio. With regard to influent NH₄ ⁺-N/NO₂ ^?-N, the maximum NH₄ ⁺-N removal rate was 0.36 kg/(m³ d), which occurred at the ratio of 4.0. Anammox activity was inhibited when influent NH₄ ⁺-N/NO₂ ^?-N was higher than 5.0. With influent NO₃ ^?-N/NH₄ ⁺-N of 2.5–6.5, NH₄ ⁺-N removal rate and NRR were stabilized at 0.33 and 0.40 kg/(m³ d), respectively. When the ratio was higher than 6.5, nitrogen removal would be worsened. The inhibitory threshold concentration of NO₂ ^?-N was lower than NH₄ ⁺-N and NO₃ ^?-N. Anammox bacteria were more sensitive to NO₂ ^?-N than NH₄ ⁺-N and NO₃ ^?-N. TNRR would be enhanced with increasing nitrogen loading rate, but sludge floatation occurred at high nitrogen loading shock. The Han-Levenspiel could be applied to simulate nitrogen removal resulting from NO₂ ^?-N inhibition.     

High-rate nitrogen removal by the Anammox process at ambient temperature

The purpose of this study is to investigate the nitrogen removal performance of the anaerobic ammonium oxidation (Anammox) process and the microbial community that enables the Anammox system to function well at ambient temperatures. A reactor with a novel spiral structure was used as the gas-solid separator. The reactor was fed with synthetic inorganic wastewater composed mainly of NH₄⁺-N and NO₂⁻-N, and operated for 92 days. Stable nitrogen removal rates (NRR) of 16.3 and 17.5 kg-N m⁻³ d⁻¹ were obtained at operating temperatures of 33 ± 1 and 23 ± 2 °C, respectively. To our knowledge, such a high NRR at ambient temperatures has not been reported previously. In addition, the experiments presented herein confirm that high influent NO₂⁻-N concentration of 460 mg L⁻¹ did not noticeably inhibit the Anammox activity. Furthermore, the freshwater Anammox bacterium KU2, which was identified as the dominant bacterial species in the consortium by 16S rRNA gene analysis, is considered to be responsible for the stable nitrogen removal performance at ambient temperatures.     

Nitrogen source effects on the denitrifying anaerobic methane oxidation culture and anaerobic ammonium oxidation bacteria enrichment process

The co-culture system of denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (Anammox) has a potential application in wastewater treatment plant. This study explored the effects of permutation and combination of nitrate, nitrite, and ammonium on the culture enrichment from freshwater sediments. The co-existence of NO₃ ⁻, NO₂ ⁻, and NH₄ ⁺ shortened the enrichment time from 75 to 30 days and achieved a total nitrogen removal rate of 106.5 mg/L/day on day 132. Even though ammonium addition led to Anammox bacteria increase and a higher nitrogen removal rate, DAMO bacteria still dominated in different reactors with the highest proportion of 64.7% and the maximum abundance was 3.07 ± 0.25 × 10⁸ copies/L (increased by five orders of magnitude) in the nitrite reactor. DAMO bacteria showed greater diversity in the nitrate reactor, and one was similar to M. oxyfera; DAMO bacteria in the nitrite reactor were relatively unified and similar to M. sinica. Interestingly, no DAMO archaea were found in the nitrate reactor. This study will improve the understanding of the impact of nitrogen source on DAMO and Anammox co-culture enrichment.

     

A control strategy of partial nitritation in a fixed bed biofilm reactor

To achieve an appropriate mixture of ammonium and nitrite for anaerobic ammonium oxidation (ANAMMOX), 50% partial nitritation was optimized in a fixed bed biofilm reactor treating synthetic wastewater. Results suggested that 50% partial nitritation could be achieved by stepwise increases of influent NH₄⁺-N at pH of 7.8 ± 0.2, temperature of 30 ± 1 °C and dissolved oxygen (DO) of 0.5-0.8 mg l⁻¹. Hydraulic retention time (HRT) and influent alkalinity did significantly affect partial nitritation. At HRT 12 h, 50% partial nitritation could be kept stable, regardless of influent NH₄⁺-N variation, by controlling the influent HCO₃⁻/NH₄⁺ molar ratio at 1:1. The fluorescent in situ hybridization (FISH) results indicated the abundance of evolution of ammonia-oxidizing bacteria (AOB) and the nitrite-oxidizing bacteria (NOB) coincided well with the performance of partial nitritation. Furthermore, the AOB were highly affiliated with Nitrosomonas spp. and Nitrosospira spp. dominated (64.1%) in the biofilm with a compact structure during the stable 50% partial nitritation period.     

Effects of inorganic carbon limitation on anaerobic ammonium oxidation (anammox) activity

Anammox bacteria are chemoautotrophic bacteria that oxidize ammonium with nitrite as the electron acceptor and with CO₂ as the main carbon source. The effects of inorganic carbon (IC) limitation on anammox bacteria were investigated using continuous feeding tests. In this study, a gel carrier with entrapped anammox sludge was used. It was clearly shown that the anammox activity deteriorated with a decrease in the influent IC concentration. The relationship between the influent IC concentration and the anammox activity was analyzed using Michaelis-Menten kinetics, and the apparent K_m was determined to be 1.2 mg-C/L. The activity could be recovered by adding IC to the influent. The consumption ratio of IC to ammonium was not constant and mainly depended on the influent ratio of the IC to ammonium concentrations (inf.IC/inf.NH₄-N). The results indicated that an inf.IC/inf.NH₄-N ratio of 0.2 in the anammox reactor was ideal for the anammox process using gel cubes.     

Long-term stability of partial nitritation of swine wastewater digester liquor and its subsequent treatment by Anammox

Partial nitritation using inhibition of free ammonia and free nitric acid is an effective technique for the treatment of high concentrations of ammonium in wastewaters. This technique was applied to the digester liquor of swine wastewater and the stability of its long-term operation was investigated. Partial nitritation was successfully maintained at a nitrogen loading rate (NLR) of 1.0 kg N m(-3)d(-1) for 120 days without acclimatization of nitrite oxidizing bacteria (NOB) to the inhibitory compounds (free ammonia and free nitric acid). The conversion efficiencies of NH(4)-N to NO(2)-N and to NO(3)-N were determined to be around 58% and <5%, respectively. After the establishment of partial nitritation, the influence of swine wastewater on the Anammox reaction was examined using continuous flow treatment experiments. Consistent nitrogen removal was achieved for 70 days at a nitrogen removal rate (NRR) of 0.22 kg N m(-3)d(-1) and the color of Anammox bacteria changed from red to greyish black. The NO(2)-N consumption and the NO(3)-N production increased concurrently and the Anammox reaction ratio was estimated to be 1:1.67:0.53, which is different from that reported previously (1:1.32:0.26).     

Start-up and stabilization of an Anammox process from a non-acclimatized sludge in CSTR

Development of an Anammox (anaerobic ammonium oxidation) process using non-acclimatized sludge requires a long start-up period owing to the very slow growth rate of Anammox bacteria. This article addresses the issue of achieving a shorter start-up period for Anammox activity in a well-mixed continuously stirred tank reactor (CSTR) using non-acclimatized anaerobic sludge. Proper selection of enrichment conditions and low stirring speed of 30 ± 5 rpm resulted in a shorter start-up period (82 days). Activity tests revealed the microbial community structure of Anammox micro-granules. Ammonia-oxidizing bacteria (AOB) were found on the surface and on the outer most layers of granules while nitrite-oxidizing bacteria (NOB) and Anammox bacteria were present inside. Fine-tuning of influent NO₂ ⁻/NH₄ ⁺ ratio allowed Anammox activity to be maintained when mixed microbial populations were present. The maximum nitrogen removal rate achieved in the system was 0.216 kg N/(m³ day) with a maximum specific nitrogen removal rate of 0.434 g N/(g VSS day). During the study period, Anammox activity was not inhibited by pH changes and free ammonia toxicity.     

Studies in nitrification of municipal sewage in an upflow biofilter

The upflow aerated biofilter with polyurethane foam cubes as the supporting medium was used for the investigation of nitrification studies on municipal sewage (secondary treated as well as untreated domestic sewage). In case of secondary treated sewage effluent, a synthetic composition of NH₄ ⁺-N and COD of each 50?mg/l was studied for a HRT variation of 24, 12, 8 and 6 hours. The ammonium removal efficiencies were found to be in the range of 98 to 100% with the steady-state effluent concentrations of NH₄ ⁺-N and NO₂ ^?-N in the range of 1–4 mg/l and 0.1–0.2?mg/l respectively. In case of domestic sewage system, nitrification studies along with suspended solids removal study was carried-out on untreated sewage for a HRT variation of 24, 12 and 6 hours. The ammonium removal efficiencies of 100% were observed for all the three HRT values at very high COD/NH₄ ⁺-N ratio of 15. The suspended solids removal efficiencies of 95 to 98% were observed with the average effluent suspended solids concentration of 5.9 to 15.9?mg/l. The experiments were conducted in non-backwash conditions of the biofilter. The study has revealed the best use of the upflow biofilter system for nitrification applications and suspended solids removal.     

Screening and characterization of an aerobic nitrifying-denitrifying bacterium from activated sludge

Taking advantage of the good biocompatibility and high efficiency of nitrogen removal with microbes, nitrifying and denitrifying bacteria, are becoming increasingly more widely used for wastewater treatment and prevention of eutrophication. In this research, an aerobic nitrifying-denitrifying bacterium was successfully screened from activated sludge and identified as Pseudomonas sp. (CCTCC No M2010209) by the 16S rDNA sequence. The activity verification confirmed its nitrifying-denitrifying capability of removing ammonium, nitrate and nitrite nitrogen. The types of carbon sources and carbon-nitrogen ratio greatly influenced the removal efficiency of NH₄ ⁺-N and NO₃ ⁻-N. When the initial concentrations of NH₄ ⁺-N and NO₃ ⁻-N in synthetic wastewater were less than 70 and 50 mg/L, the nitrogen removal rates reached 94 and 90% in 9 h, respectively. Preliminary comparisons of nitrogen removal capacity between this isolate and other commercial preparations in the treatment of synthetic wastewater revealed its promising potential to be used in the actual wastewater treatment.     

Enhanced nitrite build-up in proportion to increasing alklinity/NH4+ ratio of influent in biofilm reactor

When the alkalinity/NH₄ ⁺ratio increased from 4.1 to 9.4, the ammonium removal rate increased from 45 to 90 mg NO_x-N l^–1 h^–1. An increase in alkalinity/NH₄ ⁺ratio was a major reason for higher pH and free ammonia (FA) concentration in the reactor. The high concentration of FA showed a selective inhibition for Nitrobacter, which caused enhanced nitrite build-up in a biofilm reactor.     

Anaerobic ammonium oxidation process in an upflow anaerobic sludge blanket reactor with granular sludge selected from an anaerobic digestor

The purpose of this work was to evaluate the development of the anammox process by the use of granular sludge selected from a digestion reactor as a potential seed source in a lab-scale UASB (upflow anaerobic sludge blanket) reactor system. The reactor was operated for approximately 11 months and was fed by synthetic wastewater. After 200 days of feeding with NH₄ ⁺ and NO₂ ⁻ as the main substrates, the biomass showed steady signs of ammonium consumption, resulting in over 60% of ammonium nitrogen removal. This report aims to present the results and to more closely examine what occurs after the onset of anammox activity, while the previous work described the start-up experiment and the presence of anammox bacteria in the enriched community using the fluorescencein situ hybridization (FISH) technique. By the last month of operation, the consumed NO₂ ⁻N/NH₄ ⁺-N ratio in the UASB reactor was close to 1.32, the stoichiometric ratio of the anammox reaction. The obtained results from the influentshutdown test suggested that nitrite concentration would be one key parameter that promotes the anammox reaction during the start-up enrichment of anammox bacteria from granular sludge. During the study period, the sludge color gradually changed from black to red-brownish.     

Innovative treatment system for digester liquor using anammox process

This study demonstrated that partial nitritation using nitrifying activated sludge entrapped in a polyethylene glycol (PEG) gel carrier, as a pretreatment to anammox process, could be successfully applied to digester liquor of biogas plant at a nitrogen loading rate of 3.0 kg-N/m³/d. The nitritation process produced an effluent with a NO₂–N/NH₄–N ratio between 1.0 and 1.4, which was found to be suitable for the subsequent anammox process. A high SS concentration (2000–3000 mg/l) in the digester liquor did not affect partial nitritation treatment performances. Effluent from this partial nitritation reactor was successfully treated in the anammox reactor using anammox sludge entrapped in the PEG gel carrier with T-N removal rates of greater than 4.0 kg-N/m³/d. Influent BOD and SS contents did not inhibit anammox activity of the anammox gel carrier. The combination of partial nitritation and anammox reactors using PEG entrapped nitrifying and anammox bacteria was shown to be effective for the removal of high concentration ammonium in the digester liquor of a biogas plant.     

Partial nitritation at elevated loading rates: design curves and biofilm characteristics

There is a need to develop low operational intensity, cost-effective, and small-footprint systems to treat wastewater. Partial nitritation has been studied using a variety of control strategies, however, a gap in passive operation is evident. This research investigates the use of elevated loading rates as a strategy for achieving low operational intensity partial nitritation in a moving bed biofilm reactor (MBBR) system. The effects of loading rates on nitrification kinetics and biofilm characteristics were determined at elevated, steady dissolved oxygen concentrations between 5.5 and 7.0 mg O₂/L and ambient temperatures between 19 and 21 °C. Four elevated loading rates (3, 4, 5 and 6.5 g NH₄⁺-N/m² days) were tested with a distinct shift in kinetics being observed towards nitritation at elevated loadings. Complete partial nitritation (100% nitrite production) was achieved at 6.5 g NH₄⁺-N/m² days, likely due to thick biofilm (572 µm) and elevated NH₄⁺-N load, which resulted in suppression of nitrite oxidation.

     

Removal of nitrogen by heterotrophic nitrification–aerobic denitrification of a novel halotolerant bacterium Pseudomonas mendocina TJPU04

Excess inorganic nitrogen in water poses a severe threat to enviroment. Removal of inorganic nitrogen by heterotrophic nitrifying–aerobic denitrifying microorganism is supposed to be a promising and applicable technology only if the removal rate can be maintained sufficiently high in real wastewater under various conditions, such as high concentration of salt and wide range of different nitrogen concentrations. Here, a new heterotrophic nitrifying–aerobic denitrifying bacterium was isolated and named as Pseudomonas mendocina TJPU04, which removes NH₄⁺-N, NO₃⁻-N and NO₂⁻-N with average rate of 4.69, 5.60, 4.99 mg/L/h, respectively. It also maintains high nitrogen removal efficiency over a wide range of nitrogen concentrations. When concentration of NH₄⁺-N, NO₃⁻-N and NO₂⁻-N was up to 150, 150 and 50 mg/L, 98%, 93%, and 100% removal efficiency could be obtained, respectively, after 30-h incubation under sterile condition. When it was applied under non-sterile condition, the ammonia removal efficiency was slightly lower than that under sterile condition. However, the nitrate and nitrite removal efficiencies under non-sterile condition were significantly higher than those under sterile condition. Strain TJPU04 also showed efficient nitrogen removal performance in the presence of high concentration of salt and nitrogen. In addition, the removal efficiencies of NH₄⁺-N, NO₃⁻-N and TN in real wastewater were 91%, 52%, and 75%, respectively. These results suggest that strain TJPU04 is a promising candidate for efficient removal of inorganic nitrogen in wastewater treatment.

     

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD¹ and nitrogen species^2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH₄⁺ and NO₃^- concentrations in the feed did not have an effect on NH₄⁺ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH₄⁺ feed concentration was found to be negatively related with NO₃^- removal (n=44, p=0.000) and NO₃^- feed concentration was found to be positively correlated with NO₃^- removal (n=44, p=0.000). Consistent removal of NH₄⁺, combined with the accumulation of oxidized nitrogen species at high NH₄⁺ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.     

Partial nitrification in an upflow biological aerated filter by O2 limitation

A laboratory scale upflow biological aerated filter (BAF) packed with a porous expanded polyurethane medium was developed to nitrify ammonium to nitrite selectively. Greater than 95% removal of ammonium was achieved up to 2 kg NH₄ ⁺-N m^–3 d. NO₂ ^–-N was accumulated up to 60% of the total (NO₂ ^– + NO₃ ^–)-N when oxygen was limited.     

Ammonium Removal by the Oxygen-Limited Autotrophic Nitrification-Denitrification System

The present lab-scale research reveals the potential of implementation of an oxygen-limited autotrophic nitrification-denitrification (OLAND) system with normal nitrifying sludge as the biocatalyst for the removal of nitrogen from nitrogen-rich wastewater in one step. In a sequential batch reactor, synthetic wastewater containing 1 g of NH₄⁺-N liter⁻¹ and minerals was treated. Oxygen supply to the reactor was double-controlled with a pH controller and a timer. At a volumetric loading rate (B_v) of 0.13 g of NH₄⁺-N liter⁻¹ day⁻¹, about 22% of the fed NH₄⁺-N was converted to NO₂⁻-N or NO₃⁻-N, 38% remained as NH₄⁺-N, and the other 40% was removed mainly as N₂. The specific removal rate of nitrogen was on the order of 50 mg of N liter⁻¹ day⁻¹, corresponding to 16 mg of N g of volatile suspended solids⁻¹ day⁻¹. The microorganisms which catalyzed the OLAND process are assumed to be normal nitrifiers dominated by ammonium oxidizers. The loss of nitrogen in the OLAND system is presumed to occur via the oxidation of NH₄⁺ to N₂ with NO₂⁻ as the electron acceptor. Hydroxylamine stimulated the removal of NH₄⁺ and NO₂⁻. Hydroxylamine oxidoreductase (HAO) or an HAO-related enzyme might be responsible for the loss of nitrogen.     

Nitrogen removal from secondary effluent by using integrated constructed wetland system

The treatment capacity of an integrated constructed wetland system (CWS) that was designed to reduce nitrogen (N) from secondary effluent was explored. The integrated CWS consisted of vertical-flow constructed wetland, floating bed and sand filter. The vertical-flow wetland was filled with gravel, steel slag and peat from the bottom to the top. Vetiver zizanioides was selected to grow in the vertical-flow constructed wetland and Coix lacrymajobi L. was grown in the floating bed. The results showed that the integrated CWS displayed superior removal efficiency for nitrate nitrogen (NO₃⁻-N), ammonia nitrogen (NH₄⁺-N), nitrite nitrogen (NO₂⁻-N), and total nitrogen (TN). The average NO₃⁻-N, NO₂⁻-N, NH₄⁺-N and TN removal efficiencies of the integrated CWS were 98.83%, 95.60%, 98.05% and 92.41%, respectively, during the whole experimental operation. The integrated CWS may have a good potential for removing N from secondary effluent.