Nitrogen removal with the anaerobic ammonium oxidation process

Anaerobic ammonium-oxidizing (anammox) bacteria convert ammonium to N₂ with nitrite as the terminal electron acceptor in the absence of O₂. Nitritation–anammox bioreactors provide a cost-effective and environment-friendly alternative to conventional nitrification/denitrification nitrogen removal systems. Currently, this process is only applied for ammonium removal from wastewater with high ammonium load and temperature. Nevertheless, recent results obtained with laboratory-scale bioreactors suggest new possible routes of application of the Nitritation–anammox technology including (1) municipal wastewater treatment, removal of (2) methane in combination with nitrite-reducing methane-oxidizing bacteria, (3) nitrate coupled to organic acid oxidation and (4) nitrogen oxides. The current review summarizes the state-of-the-art of the application of Nitritation–anammox systems and discusses the possibilities of utilizing these recent results for wastewater treatment.     

Nitrogen removal performance using anaerobic ammonium oxidation at low temperatures

An anaerobic ammonium oxidation (anammox) process for ammonia-rich wastewater treatment has not been reported at temperatures below 15 degrees C. This study used a gel carrier with entrapped anammox bacteria to obtain a stable nitrogen removal performance at low temperatures. In a continuous feeding test, a high nitrogen conversion rate (6.2 kg N m(-3) day(-1)) was confirmed at 32 degrees C. Nitrogen removal activity decreased gradually with decreasing operation temperature; however, it still occurred at 6 degrees C. Nitrogen conversion rates at 22 and 6.3 degrees C were 2.8 and 0.36 kg N m(-3) day(-1), respectively. Moreover, the stability of anammox activity below 20 degrees C was confirmed for more than 130 days. In batch experiments, anammox gel carriers were characterized with respect to temperature. The optimum temperature for anammox bacteria was found to be 37 degrees C. Furthermore, it was clear that the temperature dependence changed at about 28 degrees C. The apparent activation energy in the temperature range from 22 to 28 degrees C was calculated as 93 kJ mol(-1), and that in the range from 28 to 37 degrees C was 33 kJ mol(-1). This value agrees with the result of a continuous feeding test (94 kJ mol(-1), between 6 and 22 degrees C). The nitrogen removal performance demonstrated at the low temperatures used in this study will open the door for the application of anammox processes to many types of industrial wastewater treatment.     

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.

     

厌氧氨氧化菌群体感应系统研究

厌氧氨氧化(Anammox)是以铵为电子供体将亚硝酸盐转化为氮气的生物过程。厌氧氨氧化菌(AAOB)生理代谢和细胞结构均十分特殊,且在氮素循环中起着十分重要的作用。厌氧氨氧化已成为环境学、微生物学、海洋学等领域的研究热点。但是,至今人们未能对厌氧氨氧化菌进行纯培养,这严重限制了对厌氧氨氧化菌的深入研究。群体感应是一种普遍存在于微生物细胞之间的通讯机制,它具有根据菌群密度和周围环境变化调节基因表达,以控制细菌群体行为的功能。厌氧氨氧化菌活性的细胞密度效应和生物团聚行为与细菌中普遍存在的群体感应现象相符。探讨了厌氧氨氧化菌群体感应系统存在的可能性、工作机制及其生态学意义,以期为厌氧氨氧化菌的分离培养、团聚体培育等提供理论指导。     

Spatial separation of anaerobic ammonium oxidation and nitrite-dependent anaerobic methane oxidation in permeable riverbeds

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) play important roles in nitrogen and carbon cycling in fresh waters but we do not know how these two processes compete for their common electron acceptor, nitrite. Here, we investigated the spatial distribution of anammox and n-damo across a range of permeable riverbed sediments. Anammox activity and gene abundance were detected in both gravel and sandy riverbeds and showed a simple, common vertical distribution pattern, while the patterns in n-damo were more complex and n-damo activity was confined to the more reduced, sandy riverbeds. Anammox was most active in surficial sediment (0–2 cm), coincident with a peak in hzsA gene abundance and nitrite. In contrast, n-damo activity peaked deeper down (4–8 cm) in the sandy riverbeds, coincident with a peak in n-damo 16S rRNA gene abundance and higher methane concentration. Pore water nitrite, methane and oxygen were key factors influencing the distribution of these two processes in permeable riverbeds. Furthermore, both anammox- and n-damo- activity were positively correlated with denitrification activity, suggesting a role for denitrification in supplying both processes with nitrite. Our data reveal spatial separation between anammox and n-damo in permeable riverbed sediments that potentially avoids them competing for nitrite.     

Key physiology of anaerobic ammonium oxidation.

The physiology of anaerobic ammonium oxidizing (anammox) aggregates grown in a sequencing batch reactor was investigated quantitatively. The physiological pH and temperature ranges were 6.7 to 8.3 and 20 to 43 degrees C, respectively. The affinity constants for the substrates ammonium and nitrite were each less than 0.1 mg of nitrogen per liter. The anammox process was completely inhibited by nitrite concentrations higher than 0.1 g of nitrogen per liter. Addition of trace amounts of either of the anammox intermediates (1. 4 mg of nitrogen per liter of hydrazine or 0.7 mg of nitrogen per liter of hydroxylamine) restored activity completely.     

利用EGSB反应器富集高纯度厌氧氨氧化菌

以储存60 d的ANAMMOX污泥为种泥,采用EGSB反应器富集高活性、高纯度的ANAMMOX菌富集培养物,考察反应器的脱氮能力,同时采用分子生物学方法鉴定培养前后ANAMMOX富集培养物中的优势菌种及ANAMMOX菌的相对丰度。在反应器运行的185 d中,最高氮负荷达3.0 kg N/(m3·d),氨氮与亚硝酸盐氮的去除效率均大于85%。培养后ANAMMOX富集培养物中的优势菌种由待定荧光布罗卡地菌Candidatus Brocadia fulgid和待定巴西布罗卡地菌Candidatus Brocadia brasiliensis变为待定亚洲杰特氏菌Candidatus Jettenia asiatica。FISH结果表明ANAMMOX菌所占总细菌的相对丰度明显增加,由(57.69±4.79)%提高到(83.32±4.40)%。定量PCR结果证实了培养后的富集培养物内ANAMMOX菌的拷贝数由1.14×1011拷贝/g湿重提高到3.69×1011拷贝/g湿重。     

Effects of nitrite inhibition on anaerobic ammonium oxidation

In order to assess the stability of nitrogen removal systems utilizing anaerobic ammonium oxidation (anammox), it is necessary to study the toxic effects of nitrite on these biochemical reactions. In this study, the effects of nitrite on anammox bacteria entrapped in gel carriers were investigated using batch and continuous feeding tests. The results showed that the nitrite concentration in a reactor must be less than 274-mg N/L in order to prevent a decrease in the anammox activity, which occurred when the gel carriers were soaked in nitrite solutions with concentrations greater than 274-mg N/L in a batch test. In a continuous feeding test, nitrite inhibition was not observed at low concentrations of nitrite. However, the anammox activity decreased to 10% when the nitrite concentration increased to 750-mg N/L over a 7-day period in the reactor. In addition, it was shown that the effects of nitrogen on the anammox reaction were reversible because the anammox activity completely recovered within 3 days when the influent nitrite concentration was decreased to less than 274-mg N/L.     

Trending topics and open questions in anaerobic ammonium oxidation

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厌氧氨氧化细菌的研究进展

厌氧氨氧化是指微生物在无氧条件下,以NO2-为电子受体,将NH4+氧化成N2的过程,该过程主要由浮霉菌门下的厌氧氨氧化细菌参与.厌氧氨氧化细菌广泛存在于海洋生态系统、淡水生态系统、陆地生态系统及其他一些特殊生境中,其在废水生物脱氮和地球氮循环中扮演着重要角色.本文从厌氧氨氧化细菌的发现历程、种类、特性、代谢途径、分布、...     

Simultaneous nitrite-dependent anaerobic methane and ammonium oxidation processes

Nitrite-dependent anaerobic oxidation of methane (n-damo) and ammonium (anammox) are two recently discovered processes in the nitrogen cycle that are catalyzed by n-damo bacteria, including "Candidatus Methylomirabilis oxyfera," and anammox bacteria, respectively. The feasibility of coculturing anammox and n-damo bacteria is important for implementation in wastewater treatment systems that contain substantial amounts of both methane and ammonium. Here we tested this possible coexistence experimentally. To obtain such a coculture, ammonium was fed to a stable enrichment culture of n-damo bacteria that still contained some residual anammox bacteria. The ammonium supplied to the reactor was consumed rapidly and could be gradually increased from 1 to 20 mM/day. The enriched coculture was monitored by fluorescence in situ hybridization and 16S rRNA and pmoA gene clone libraries and activity measurements. After 161 days, a coculture with about equal amounts of n-damo and anammox bacteria was established that converted nitrite at a rate of 0.1 kg-N/m(3)/day (17.2 mmol day(-1)). This indicated that the application of such a coculture for nitrogen removal may be feasible in the near future.     

Involvement of a novel hydroxylamine oxidoreductase in anaerobic ammonium oxidation

In this study a novel hydroxylamine oxidoreductase (HAO) was purified and characterized from an anaerobic ammonium-oxidizing (Anammox) enrichment culture. The enzyme, which constituted about 9% of the protein mass in the soluble fraction of the cell extract, was able to oxidize hydroxylamine and hydrazine. When phenazine methosulfate and methylthiazolyltetrazolium bromide were used as electron acceptors, a V(max) [21 and 1.1 micromol min(-)(1) (mg of protein)(-)(1)] and K(m) (26 and 18 microM) for hydroxylamine and hydrazine were determined, respectively. The hydroxylamine oxidoreductase is a trimer and contains about 26 hemes per 183 kDa. As deduced from UV/vis spectra, hydroxylamine reduced more and different cytochromes than hydrazine. The dithionite-reduced spectrum showed an unusual 468 nm peak. Inhibition experiments with H(2)O(2) showed that hydroxylamine bound to this P-468 cytochrome, which is assumed to be the putative substrate binding site. Cyanide and hydrazine inhibited the oxidation of hydroxylamine. The amino acid sequences of several peptide fragments of HAO from Anammox showed a clear difference with the deduced amino acid sequence of HAO from the aerobic ammonia-oxidizing bacterium Nitrosomonas europaea. In EPR spectra of the Anammox HAO, two g-values (g(z)() = 2.37 and 2.42) were observed, which were not present in HAO of N. europaea.     

Engineering application of anaerobic ammonium oxidation process in wastewater treatment

Anaerobic ammonium oxidation (Anammox), a promising biological nitrogen removal process, has been verified as an efficient, sustainable and cost-effective alternative to conventional nitrification and denitrification processes. To date, more than 110 full-scale anammox plants have been installed and are in operation, treating industrial NH₄ ⁺-rich wastewater worldwide, and anammox-based technologies are flourishing. This review the current state of the art for engineering applications of the anammox process, including various anammox-based technologies, reactor selection and attempts to apply it at different wastewater plants. Process control and implementation for stable performance are discussed as well as some remaining issues concerning engineering application are exposed, including the start-up period, process disturbances, greenhouse gas emissions and especially mainstream anammox applications. Finally, further development of the anammox engineering application is proposed in this review.     

Factors controlling anaerobic ammonium oxidation with nitrite in marine sediments

Factors controlling the anaerobic oxidation of ammonium with nitrate and nitrite were explored in a marine sediment from the Skagerrak in the Baltic-North Sea transition. In anoxic incubations with the addition of nitrite, approximately 65% of the nitrogen gas formation was due to anaerobic ammonium oxidation with nitrite, with the remainder being produced by denitrification. Anaerobic ammonium oxidation with nitrite exhibited a biological temperature response, with a rate optimum at 15 degrees C and a maximum temperature of 37 degrees C. The biological nature of the process and a 1:1 stoichiometry for the reaction between nitrite and ammonium indicated that the transformations might be attributed to the anammox process. Attempts to find other anaerobic ammonium-oxidizing processes in this sediment failed. The apparent K(m) of nitrite consumption was less than 3 microM, and the relative importance of ammonium oxidation with nitrite and denitrification for the production of nitrogen gas was independent of nitrite concentration. Thus, the quantitative importance of ammonium oxidation with nitrite in the jar incubations at elevated nitrite concentrations probably represents the in situ situation. With the addition of nitrate, the production of nitrite from nitrate was four times faster than its consumption and therefore did not limit the rate of ammonium oxidation. Accordingly, the rate of this process was the same whether nitrate or nitrite was added as electron acceptor. The addition of organic matter did not stimulate denitrification, possibly because it was outcompeted by manganese reduction or because transport limitation was removed due to homogenization of the sediment.     

厌氧氨氧化生物脱氮技术的研究进展

厌氧氨氧化是指在厌氧条件下,厌氧氨氧化混合菌直接以NH4 为电子供体,以NO3-或NO2-为电子受体,将NH4^ 、NO3-或NO2-转变成N2的过程。厌氧氨氧化作为一种新型的污水处理工艺具有较高的理论意义和良好的应用前景。本文主要阐述了厌氧氨氧化生物脱氮技术原理、厌氧氨氧化的可能途径、方法及其应用现状,并且讨论了厌氧氨氧化反应的微生物学机理和厌氧氨氧化工艺的开发,提出了今后研究的主要方向。     

Spatial distribution and co-occurrence of aerobic ammonia oxidation and anaerobic ammonium oxidation activities in the water-soil interface,bulk, and rhizosphere regions of paddy soil

Plant and Soil - Aerobic ammonia oxidation often couples with anaerobic ammonium oxidation (anammox) in natural ecosystems; however, their in situ activities and relative contributions are still...