Kinetics, diffusional limitation and microscale distribution of chemistry and organisms in a CANON reactor

In the Completely Autotrophic Nitrogen removal Over Nitrite (CANON) process, aerobic and anaerobic ammonia oxidizing bacteria cooperate to remove ammonia in one oxygen-limited reactor. Kinetic studies, microsensor analysis, and fluorescence in situ hybridization on CANON biomass showed a partial differentiation of processes and organisms within and among aggregates. Under normal oxygen-limited conditions ( approximately 5 microM O2), aerobic ammonia oxidation (nitrification) was restricted to an outer shell (<100 microm) while anaerobic ammonia oxidation (anammox) was found in the central anoxic parts. Larger type aggregates (>500 microm) accounted for 68% of the anammox potential whereas 65% of the nitrification potential was found in the smaller aggregates (<500 microm). Analysis with O2 and NO2- microsensors showed that the thickness of the activity zones varied as a function of bulk O2 and NO2- concentrations and flow rate.     

厌氧氨氧化菌特性及其在生物脱氮中的应用

在无分子氧环境中,同时存在NH4^＋和NO2^-时,NH4^＋作为反硝化的无机电子供体,NO2^-作为电子受体,生成氮气,这一过程称为厌氧氨氧化。目前已经发现了3种厌氧氨氧化菌（Brocadia anammoxidans,Kuenenia stuttgartiensis,Scalindua sorokinii）;对厌氧氨氧化菌的细胞色素、营养物质、抑制物、结构特征和生化反应机理的研究表明,厌氧氨氧化菌具有多种代谢能力。基于部分硝化至亚硝酸盐,然后与氨一起厌氧氨氧化,以及厌氧氨氧化菌与好氧氨氧化菌或甲烷菌的协同耦合作用,提出了几种生物脱氮的新工艺（ANAMMOX、SHARON—ANAMMOX、CANON和甲烷化与厌氧氨氧化耦合工艺）。     

Applications of Anammox based processes to treat anaerobic digester supernatant at room temperature

The supernatant of an anaerobic digester was treated at 20 °C in two systems. The first one is a two units configuration, conformed by two sequencing batch reactors (SBR), carrying out partial nitrification and Anammox processes, respectively. Partial nitrification was achieved by granular biomass with a mean diameter of 3 mm, operating at a dissolved oxygen concentration of 2.7 mg/L. The combined system allowed the removal of nitrogen loading rates around 0.08 g N/(L d).     

Reactivation of aerobic and anaerobic ammonium oxidizers in OLAND biomass after long-term storage

The biomass of an oxygen-limited autotrophic nitrification/denitrification (OLAND) biofilm reactor was preserved in various ways to find a storage method for both aerobic and anaerobic ammonium-oxidizing bacteria (AerAOB and AnAOB). Storage occurred at −20°C with and without glycerol as cryoprotectant and at 4 and 20°C with and without nitrate as redox buffer. After 2 and 5 months, reactivation of AerAOB and AnAOB was achieved with the biomass stored at 4°C with and without nitrate and at 20°C with nitrate. Moreover, the presence of the AerAOB and AnAOB was confirmed with fluorescent in situ hybridization (FISH). Preservation in a nitrate environment resulted in a lag phase for the AnAOB reactivation. The supplied nitrate was denitrified during storage, and a real-time polymerase chain reaction with nitrifying and denitrifying genes allowed to estimate that at least 1.0 to 6.0% of the OLAND biofilm consisted of denitrifiers. It was concluded that reactivation after long-term storage is possible and that preservation at 4°C without nitrate addition is the recommended storage technique. The possibility to store OLAND biomass will facilitate research on AnAOB and can overcome larger-scale start-up and inhibition problems of novel nitrogen processes involving AnAOB.     

Assessment of the positive effect of salinity on the nitrogen removal performance and microbial composition during the start-up of CANON process

In this study, a non-woven rotating biological contactor reactor was operated for the start-up of completely autotrophic nitrogen removal over nitrite (CANON) process. In this perfectly attached growth system, nitrite oxidizing was identified, which interfered with the nitrogen removal performance. Batch tests indicated that 10 g NaCl per liter salinity was a preferable definite level to stand out ammonium-oxidizing activity and anammox activity, and selectively suppress nitrite-oxidizing activity under oxygen-limited conditions. Reactor operation showed that the maximum TN removal rate was increased from 425 mg N l(-1) day(-1) to 637 mg N l(-1) day(-1) after the addition of 10 g NaCl per liter salinity on analogous technological parameters. Microbiological community analysis revealed that bacteria strains similar to the genus Nitrospira sp. were specialized nitrite oxidizers existing in CANON reactor, which were then eliminated under salinity exposure for their no salinity-tolerant relative. However, anammox bacteria belonging to Planctomycetes and some aerobic ammonium oxidizers belonging to Nitrosomonas could be highly enriched under this oxygen-limited salinity conditions. Salinity-contained high ammonium wastewater will be so considered as suitable influent for CANON process in further industrial application.     

Nitrogen removal performance of a hybrid anammox reactor

The anaerobic ammonium oxidation (anammox) process has attracted considerable attention in recent years as an alternative to conventional nitrogen removal technologies. In this study, an innovative hybrid reactor combining fluidized and fixed beds for anammox treatment was developed. The fluidized bed was mechanically stirred and the gaseous product could be rapidly released from the anammox sludge to prevent washout of the sludge caused by floatation. The fixed bed comprising a non-woven biomass carrier could efficiently catch sludge to reduce washout. During the operation, nitrogen loading rates to the reactor were increased to 27.3 kg N/m³/d, with total nitrogen removal efficiencies of 75%. The biomass concentration in the fluidized bed reached 26-g VSS/L. Anammox granules were observed in the reactors, with settling velocities and sludge volumetric index of 27.3 ± 6.5 m/h and 23 mL/g, respectively. Quantification of extracellular polymeric substances revealed the anammox granules contained a significant amount of extracellular proteins.     

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.     

Stability of the ANAMMOX process in a gas-lift reactor and a SBR

In the last years, the ANAerobic AMMonium OXidation (ANAMMOX) process has been put forward as a promising alternative to treat ammonium rich wastewaters. An ANAMMOX gas-lift reactor and a sequential batch reactor (SBR) were operated during around 200 days in this study, reaching nitrogen loading rates (NLRs) of 2.0 and 0.75 g l(-1) per day, respectively. The efficiency in the nitrite (limiting substrate) removal was 99%. The ammonium and nitrite influent concentrations were increased stepwise until biomass in the reactors started to float. These flotation events coincided with periods when the NLR exceeded the maximum specific ANAMMOX activity (MSAA) of the sludge. The MSAA, determined in batch experiments, was 0.9 and 0.44 g g(-1) per day for biomasses from the gas-lift reactor and the SBR, respectively. Flotation of the biomass occurred most likely due to a granule density decrease caused by dinitrogen gas accumulation inside the granules and an apparent breakage of the granules. Further research is needed to understand this phenomenon and to optimise the corresponding strategies to counteract the flotation.     

土壤氮素转化的关键微生物过程及机制

微生物是驱动土壤元素生物地球化学循环的引擎.氮循环是土壤生态系统元素循环的核心之一,其四个主要过程,即生物固氮作用、氨化作用、硝化作用、反硝化作用,均由微生物所驱动.近10年来,随着免培养的分子生态学技术和高通量测序技术等的发展,在硝化微生物多样性及其作用机理、厌氧氨氧化过程和机理等研究方面取得了突破性进展.本文重点阐述了我国有关土壤硝化微生物方面的研究进展,在此基础上,简要介绍了反硝化微生物和厌氧氨氧化及硝酸盐异化还原成铵作用的研究进展,并对今后的研究工作提出了展望.今后土壤氮素转化微生物生态学的研究,应瞄准国际微生生态学发展的前沿,加强新技术新方法的应用,结合我国农业可持续发展、资源环境保护和全球变化研究的重大需求,重点开展以下几方面的工作:(1)开展大尺度上土壤硝化作用及氨氧化微生物分布的时空演变特征及驱动因子的研究;(2)加强氮素转化关键微生物过程与机理的研究,并与相关过程的通量(如氨挥发、N2O释放)和反应速率(如矿化速率、硝化速率)关联起来;(3)在特定生态系统中系统研究各个氮转化过程的耦合关系,构建相关氮素转化和氮素平衡模型,为定向调控土壤氮素转化过程,提高氮素利用效率并减少其负面效应提供科学依据.     

Enrichment of Anammox from Activated Sludge and Its Application in the CANON Process

A microbial culture capable of actively oxidizing ammonium to dinitrogen gas in the absence of oxygen, using nitrite as the electron acceptor, was enriched from local activated sludge (Western Australia) in <14 weeks. The maximum anaerobic ammonium oxidation (i.e., anammox) activity achieved by the anaerobic culture was 0.26 mmol NH ₄ ⁺ (g biomass)⁻¹ h⁻¹ (0.58 kg total-N m⁻³ day⁻¹). Qualitative FISH analysis (fluorescence in situ hybridization) confirmed the phylogenetic position of the enriched microorganism as belonging to the order Planctomycetales, in which all currently identified anammox strains fall. Preliminary FISH analysis suggests the anammox strain belongs to the same phylogenetic group as the Candidatus ‘Brocadia anammoxidans’ strain discovered in the Netherlands. However, there are quite a few differences in the target sites for the more specific probes of these organisms and it is therefore likely to represent a new species of anammox bacteria. A small amount of aerobic ammonium-oxidizing biomass was inoculated into the anammox reactor (10% v/v) to initiate completely autotrophic nitrogen removal over nitrite (the CANON process) in chemostat culture. The culture was always under oxygen limitation and no organic carbon was added. The CANON reactor was operated as an intermittently aerated system with 20 min aerobiosis and 30 min anaerobiosis, during which aerobic and anaerobic ammonium oxidation were performed in sequential fashion, respectively. Anammox was not inhibited by repeated intermittent exposure to oxygen, allowing sustained, completely autotrophic ammonium removal (0.08 kg N m⁻³ day⁻¹) for an extended period of time.     

氮形态转化途径研究的新进展—厌气铵氧化及其应用前景

20世纪90年代初在污泥处理系统中发现了氮素形态转化的新途径－厌气铵氧化过程,厌气铵氧化过程是铵以亚硝酸根为电子受体在自养细菌参数下氧化成氮气的过程,但目前尚无土壤,河,湖,海底泥等自然系统中是否存在厌氧铵氧化过程的报道,由于该过程无需外加有机碳,耗氧和处理产生污泥少,用于污泥脱氮成本较低,具有很大潜力。     

Vertical migration of aggregated aerobic and anaerobic ammonium oxidizers enhances oxygen uptake in a stagnant water layer

Ammonium can be removed as dinitrogen gas by cooperating aerobic and anaerobic ammonium-oxidizing bacteria (AerAOB and AnAOB). The goal of this study was to verify putative mutual benefits for aggregated AerAOB and AnAOB in a stagnant freshwater environment. In an ammonium fed water column, the biological oxygen consumption rate was, on average, 76 kg O₂ ha⁻¹ day⁻¹. As the oxygen transfer rate of an abiotic control column was only 17 kg O₂ ha⁻¹ day⁻¹, biomass activity enhanced the oxygen transfer. Increasing the AnAOB gas production increased the oxygen consumption rate with more than 50% as a result of enhanced vertical movement of the biomass. The coupled decrease in dissolved oxygen concentration increased the diffusional oxygen transfer from the atmosphere in the water. Physically preventing the biomass from rising to the upper water layer instantaneously decreased oxygen and ammonium consumption and even led to the occurrence of some sulfate reduction. Floating of the biomass was further confirmed to be beneficial, as this allowed for the development of a higher AerAOB and AnAOB activity, compared to settled biomass. Overall, the results support mutual benefits for aggregated AerAOB and AnAOB, derived from the biomass uplifting effect of AnAOB gas production.     

Effect of ferrous sulphate on nitrification during simultaneous phosphorus removal from domestic wastewater using a laboratory scale anoxic/oxic reactor

In the present study a laboratory scale anoxic/oxic reactor was used to remove the important eutrophication nutrients such as phosphorus and nitrogen from synthetic domestic wastewater. Phosphorus was removed through simultaneous precipitation and was carried out using the coagulant ferrous sulphate FeSO₄ · 7H₂O. Total phosphorus in the effluent was controlled to below 1 mg/l using a ferrous to phosphorus molar ratio of 2.1. pH after the addition of coagulant plays a major role in determining the molar ratio of the precipitant. Nitrogen was removed biologically in the anoxic/oxic system and the effect of simultaneous precipitation on nitrification and denitrification was investigated. The nitrification rate of the system remained unaffected during simultaneous precipitation and varied from 0.046 to 0.059 g N–NH₄ ⁺/g VSS/day. Denitrification was complete and was not affected by the coagulation process. The nitrogen removal efficiency varied from 78% to 85%. COD removal efficiency was not affected during simultaneous precipitation and was varied from 94% to 98%. The highly efficient nitrogen removal in the presence of simultaneous precipitant ferrous sulphate makes the process an ideal option for nutrient removal.     

Removal of carbonaceous and nitrogenous pollutants from a synthetic wastewater using a membrane-coupled bioreactor

Two modified Ludzack-Ettinger (MLE)-type membrane-coupled bioreactors (MBRs) were investigated in this study for the purpose of removing both nitrogenous and carbonaceous pollutants from a synthetic wastewater. During the first MBR experiment, removal efficiencies were high (>90%) for chemical oxygen demand (COD) and ammonia, but total nitrogenous pollutant removal efficiency was poor (~25%). Bacterial community analysis of ammonia oxidizing bacteria (AOB) by a nested PCR-DGGE approach detected two Nitrosomonas-like populations and one Nitrosospira-like population. During the initial portion of the second MBR experiment, COD and ammonia removal efficiencies were similar to the first MBR experiment until the COD of the influent wastewater was increased to provide additional electron donors to support denitrification. Total nitrogen removal efficiencies eventually exceeded 90%, with a hydraulic residence time (HRT) of 24 h and a recirculation ratio of 8. When the HRT of the MBR experiment was decreased to 12 h, however, ammonia removal efficiency was adversely affected. A subsequent increase in the HRT to 18 h helped improve removal efficiencies for both ammonia (>85%) and total nitrogenous compounds (~70%). Our research demonstrates that MBRs can be effectively designed to remove both carbonaceous and nitrogenous pollutants. The ability of the microbial community to switch between anoxic (denitrifying) and oxic (nitrifying) conditions, however, represents a critical process constraint for the application of MLE-type MBR systems, such that little benefit is gained compared to conventional designs.     

Nitrification and ammonia volatilisation losses from urea and dicyandiamide-treated urea in a sandy loam soil

Summary Nitrification and ammonia volatilisation losses from urea and dicyandiamide (DCD)-treated urea were studied in a sandy loam soil. Laboratory experiments indicated that 20 ppm (of soil) DCD effectively inhibited nitrification of urea over sixty days. If the urea was treated with DCD (20 ppm), ammonia emission from the soil was extended over 105 days; with urea alone, it was negligible after 15 days. A field study indicated that DCD treatment increased volatilisation losses of ammonia tremondously if urea was applied to the soil surface; these losses were minimised if the urea was placed at 5 cm depth. It would seem that nitrification inhibitors must be combined with a placement technique.     

Development of a simultaneous partial nitrification and anaerobic ammonia oxidation process in a single reactor

Up-flow oxygen-controlled biofilm reactors equipped with a non-woven fabric support were used as a single reactor system for autotrophic nitrogen removal based on a combined partial nitrification and anaerobic ammonium oxidation (anammox) reaction. The up-flow biofilm reactors were initiated as either a partial nitrifying reactor or an anammox reactor, respectively, and simultaneous partial nitrification and anammox was established by careful control of the aeration rate. The combined partial nitrification and anammox reaction was successfully developed in both biofilm reactors without additional biomass inoculation. The reactor initiated as the anammox reactor gave a slightly higher and more stable mean nitrogen removal rate of 0.35 (± 0.19) kg-N m⁻³ d⁻¹ than the reactor initiated as the partial nitrifying reactor (0.23 (± 0.16) kg-N m⁻³ d⁻¹). FISH analysis revealed that the biofilm in the reactor started as the anammox reactor were composed of anammox bacteria located in inner anoxic layers that were surrounded by surface aerobic AOB layers, whereas AOB and anammox bacteria were mixed without a distinguishable niche in the biofilm in the reactor started as the partial nitrifying reactor. However, it was difficult to efficiently maintain the stable partial nitrification owing to inefficient aeration in the reactor, which is a key to development of the combined partial nitrification and anammox reaction in a single biofilm reactor.     

Start-up and inhibition analysis of the Anammox process seeded with anaerobic granular sludge

The longer start-up period of the Anammox process is due to the very low cellular yield and growth rates of Anammox bacteria. Nitrite inhibition is considered to be the key factor in the instability of the Anammox process during the operation. However, little attention was paid to the inhibitory effect of pH and free ammonia. This paper presents start-up and inhibition analysis of an Anammox biofilm reactor seeded with anaerobic granular sludge. Results showed that the start-up period could be divided into the sludge lysis phase, lag phase, propagation phase, stationary phase and inhibition phase. Optimization control could be implemented correspondingly to accelerate the start-up of Anammox bioreactors. Effluent pH increased to 8.7–9.1 when the nitrogen removal rate was higher than 1,200 mg l⁻¹ day⁻¹. The free ammonia concentration was accompanied with a higher level of 64–73 mg l⁻¹. Inhibitory effects of high pH and free ammonia on Anammox bacteria contributed to the destabilization of the Anammox bioreactor during the first 125 days with influent KHCO₃ of 0.5 g l⁻¹. Increasing the suffering capacity in the inlet by dosing 1.25 g KHCO₃ l⁻¹ effectively reduced the pH variation, and the nitrogen removal performance of the reactor was further developed.     

Construction of a highly bioluminescent Nitrosomonas as a probe for nitrification conditions

Cloned luciferase-encoding operons were transferred by conjugation to a natural isolate of the ammonia-oxidizing bacterial strain Nitrosomonas sp. RST41–3, thereby establishing conjugation as a tool for gene transfer into Nitrosomonas strains. Luminescence was dependent on the pH of the medium and the concentration of the substrate ammonium chloride. Moreover, the luminescence of the transconjugants was reduced immediately by micromolar concentrations of nitrapyrin and allylthiourea, which are specific inhibitors of nitrification. Our results indicate that luminescent Nitrosomonas strains may be useful as a probe to detect nitrification conditions in the natural environment as well as in sewage plants.