厌氧氨氧化菌脱氮机理及其在污水处理中的应用

厌氧氨氧化细菌(anammox)可以将亚硝酸盐和氨氮转化为氮气从而缩短氨氮转化的过程,它已经成为新型生物污水脱氮技术研究的热点之一。当前,有关厌氧氨氧化菌特有的生理结构特点、种群分类及其功能酶等方面的研究取得了一定突破,为实现其工业应用奠定了良好的理论基础;同时分子生物学技术在厌氧氨氧化细菌种群分布、群落多样性及其共生关系等方面的应用也大大促进了污水生物脱氮技术的革新和进步。总结了厌氧氨氧化菌主要的生理生化特点、细胞结构特点、脱氮机理、污水处理体系中的应用以及分子生物学方法对污水处理体系中厌氧氨氧化菌种群分析的研究现状,并指出未来anammox细菌在生物特性及在污水脱氮处理实际应用的研究中的热点问题。生物特性方面的主要研究热点有：（1）anammox细菌除厌氧氨氧化作用外,其它新陈代谢途径有待探索;（2）anammox细菌在不同环境中分布的倾向性问题;（3）新型anammox细菌的确定。污水处理的实际应用方面的主要研究热点有：（1）anammox污泥的快速高效富集问题;（2）设计高特异性引物;（3）anammox细菌和其他微生物的共生关系。     

Adaptation of a freshwater anammox population to high salinity wastewater

For the successful application of anaerobic ammonium oxidation (anammox) in wastewater practice it is important to know how to seed new anammox reactors with biomass from existing reactors. In this study, a new high salinity anammox reactor was inoculated with biomass from a freshwater system. The changes in activity and population shifts were monitored. It was shown that freshwater anammox bacteria could adapt to salt concentrations as high as 30 gl(-1) provided the salt concentration was gradually increased. Higher salt concentrations reversibly inhibited anammox bacteria. The nitrogen removal efficiency and maximum anammox activity of the salt adapted sludge was very similar to the reference freshwater sludge. Fluorescence in situ hybridization analysis revealed that the freshwater anammox species Candidatus "Kuenenia stuttgartiensis" was the dominant in both salt adapted sludge and freshwater sludge. These results show that gradual adaptation may be the key to successful seeding of anammox bioreactors.     

Microbial community analysis of a full-scale DEMON bioreactor

Full-scale applications of autotrophic nitrogen removal technologies for the treatment of digested sludge liquor have proliferated during the last decade. Among these technologies, the aerobic/anoxic deammonification process (DEMON) is one of the major applied processes. This technology achieves nitrogen removal from wastewater through anammox metabolism inside a single bioreactor due to alternating cycles of aeration. To date, microbial community composition of full-scale DEMON bioreactors have never been reported. In this study, bacterial community structure of a full-scale DEMON bioreactor located at the Apeldoorn wastewater treatment plant was analyzed using pyrosequencing. This technique provided a higher-resolution study of the bacterial assemblage of the system compared to other techniques used in lab-scale DEMON bioreactors. Results showed that the DEMON bioreactor was a complex ecosystem where ammonium oxidizing bacteria, anammox bacteria and many other bacterial phylotypes coexist. The potential ecological role of all phylotypes found was discussed. Thus, metagenomic analysis through pyrosequencing offered new perspectives over the functioning of the DEMON bioreactor by exhaustive identification of microorganisms, which play a key role in the performance of bioreactors. In this way, pyrosequencing has been proven as a helpful tool for the in-depth investigation of the functioning of bioreactors at microbiological scale.     

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.     

厌氧氨氧化细菌及其群落内细菌互作关系研究进展

厌氧氨氧化细菌具有的厌氧氨氧化反应是在厌氧条件下将氨氮和亚硝氮或一氧化氮转化为硝氮、生成氮气的过程,因其能够高效低能地处理低碳氮比废水而广受关注。目前,厌氧氨氧化细菌仍未实现纯培养,借助宏组学手段研究厌氧氨氧化细菌及其群落内细菌之间的互作关系是近年来的研究趋势。本文介绍了厌氧氨氧化细菌的种类和特性,综述了厌氧氨氧化细菌的代谢多样性及其群落内细菌间的物质协同利用和信息交流方式,这对厌氧氨氧化群落的稳定具有重要影响,最后对值得进一步深入研究的问题做了总结和展望,以期为后续深入研究细菌间的互作关系提供参考,进而为构建稳定高效的厌氧氨氧化脱氮体系提供思路。     

Versatility and application of anaerobic ammonium-oxidizing bacteria

With the unique cell compartmentalization and the ability to simultaneously oxidize ammonium and reduce nitrite into nitrogen gas, anaerobic ammonium-oxidizing (anammox) bacteria have challenged our recognitions of microorganism. The research conducted on these bacteria has been extended from bench-scale tryouts to full-scale reactor systems. This review addresses the recently discovered versatile properties of anammox bacteria and the applications and obstacles of implementing the anammox process in ammonia-rich wastewater treatment. We also discuss the merits and drawbacks of traditional and anammox-based processes for nitrogen removal and suggest areas for improvement.     

Rapid startup and high rate nitrogen removal from anaerobic sludge digester liquor using a SNAP process

In this study, a single-stage autotrophic nitrogen removal reactor, packed with a novel acrylic fiber biomass carrier material (Biofix), was applied for nitrogen removal from sludge digester liquor. For rapid start-up, conventional activated sludge was added to the reactor soon after the attachment of anammox biomass on the Biofix carriers, which allowed conventional activated sludge to form a protective layer of biofilm around the anammox biomass. The Nitrogen removal efficiency reached 75% within 1 week at a nitrogen loading rate of 0.46 kg-N/m³/day for synthetic wastewater treatment. By the end of the synthetic wastewater treatment period, the maximum nitrogen removal rate had increased to 0.92 kg-N/m³/day at a nitrogen loading rate of 1.0 kg-N/m³/day. High nitrogen removal rate was also achieved during the actual raw digester liquor treatment with the highest nitrogen removal rate being 0.83 kg-N/m³/day at a nitrogen loading rate of 0.93 kg-N/m³/day. The thick biofilm on Biofix carriers allowed anammox bacteria to survive under high DO concentration of 5–6 mg/l resulting in stable and high nitrogen removal performance. FISH and CLSM analysis demonstrated that anammox bacteria coexisted and surrounded by ammonium oxidizing bacteria.     

Mainstream partial nitritation–anammox in municipal wastewater treatment: status,bottlenecks, and further studies

Driven by energy neutral/positive of wastewater treatment plants, significant efforts have been made on the research and development of mainstream partial nitritation and anaerobic ammonium oxidation (anammox) (PN/A) (deammonification) process since the early 2010s. To date, feasibility of mainstream PN/A process has been demonstrated and proven by experimental results at various scales although with the low loading rates and elevated nitrogen concentration in the effluent at low temperatures (15–10 °C). This review paper provides an overview of the current state of research and development of mainstream PN/A process and critically analyzes the bottlenecks for its full-scale application. The paper discusses the following: (i) the current status of research and development of mainstream PN/A process; (ii) the interactions among aerobic ammonium-oxidizing bacteria, aerobic nitrite-oxidizing bacteria, anammox bacteria, and heterotrophic bacteria; (iii) the suppression of aerobic nitrite-oxidizing bacteria; (iv) process and bioreactors; and (v) suggested further studies including efficient and robust carbon concentrating pretreatment, deepening of understanding competition between autotrophic nitrogen-converting organisms, intensification of biofilm anammox activity, reactor design, and final polishing.

     

Isolation and characterization of a prokaryotic cell organelle from the anammox bacterium Kuenenia stuttgartiensis

Anaerobic ammonium oxidizing (anammox) bacteria oxidize ammonium with nitrite to nitrogen gas in the absence of oxygen. These microorganisms form a significant sink for fixed nitrogen in the oceans and the anammox process is applied as a cost‐effective and environment‐friendly nitrogen removal system from wastewater. Anammox bacteria have a compartmentalized cell plan that consists of three separate compartments. Here we report the fractionation of the anammox bacterium Kuenenia stuttgartiensis in order to isolate and analyze the innermost cell compartment called the anammoxosome. The subcellular fractions were microscopically characterized and all membranes in the anammox cell were shown to contain ladderane lipids which are unique for anammox bacteria. Proteome analyses and activity assays with the isolated anammoxosomes showed that these organelles harbor the energy metabolism in anammox cells. Together the experimental data provide the first thorough characterization of a respiratory cell organelle from a bacterium and demonstrate the essential role of the anammoxosome in the production of a major portion of the nitrogen gas in our atmosphere.     

厌氧氨氧化菌的中心代谢研究进展

摘要: 厌氧氨氧化是以NH +4为电子供体,以NO－2为电子受体产生N2的生物反应。厌氧氨氧化菌是厌氧氨氧化过程的执行者,在废水生物脱氮和地球氮素循环中扮演着重要角色。研究厌氧氨氧化菌的代谢特性,将有助于理解厌氧氨氧化过程,开发厌氧氨氧化工艺。厌氧氨氧化菌是化能自养型细菌,以CO2或HCO－3为碳源,并通过偶联NH+4氧化和NO －2还原的生物反应获得能量。在NH+4/NO－2的生物氧化还原反应过程中,检出了中间产物N2H4,但未检出其他中间产物(如NH2OH、NO)。此外,由基因组信息推断,厌氧氨氧化菌     

Biochemistry and molecular biology of anammox bacteria

Anaerobic ammonium-oxidizing (anammox) bacteria are one of the latest additions to the biogeochemical nitrogen cycle. These bacteria derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. These slowly growing microorganisms belong to the order Brocadiales and are affiliated to the Planctomycetes. Anammox bacteria are characterized by a compartmentalized cell architecture featuring a central cell compartment, the “anammoxosome”. Thus far unique “ladderane” lipid molecules have been identified as part of their membrane systems surrounding the different cellular compartments. Nitrogen formation seems to involve the intermediary formation of hydrazine, a very reactive and toxic compound. The genome of the anammox bacterium Kuenenia stuttgartiensis was assembled from a complex microbial community grown in a sequencing batch reactor (74% enriched in this bacterium) using a metagenomics approach. The assembled genome allowed the in silico reconstruction of the anammox metabolism and identification of genes most likely involved in the process. The present anammox pathway is the only one consistent with the available experimental data, thermodynamically and biochemically feasible, and consistent with Ockham’s razor: it invokes minimum biochemical novelty and requires the fewest number of biochemical reactions. The worldwide presence of anammox bacteria has now been established in many oxygen-limited marine and freshwater systems, including oceans, seas, estuaries, marshes, rivers and large lakes. In the marine environment over 50% of the N₂ gas released may be produced by anammox bacteria. Application of the anammox process offers an attractive alternative to current wastewater treatment systems for the removal of ammonia-nitrogen. Currently, at least five full scale reactor systems are operational.     

厌氧氨氧化反应器启动过程的影响因素及微生物群落变化研究进展

自厌氧氨氧化反应发现以来,由于其具有低能耗、无需外加碳源等优点,已成为人们在污水生物脱氮研究与应用中的最新关注点。然而,由于极低的生长速率、极长的倍增时间以及严格的代谢条件等特点,限制了厌氧氨氧化菌的应用。综述了厌氧氨氧化菌富集培养过程中的影响因素,介绍了不同污泥来源的厌氧氨氧化优势菌属、分子鉴定方法,提供了部分用于厌氧氨氧化菌鉴定使用的引物序列和厌氧氨氧化菌最新发现的属与种。最后,对未来的研究方向提出一些建议思考,以期为厌氧氨氧化工艺在污水处理中的应用提供参考。     

Nitrogen removal performance and operation strategy of anammox process under temperature shock

Sequencing batch reactors were used to study anaerobic ammonium oxidation (anammox) process under temperature shock. Both long-term (15–35 °C) and short-term (10–50 °C) temperature effects on nitrogen removal performance were performed. In reactor operation test, the results indicated that ammonium removal rate decreased from 0.35 kg/(m³ day) gradually to 0.059 kg/(m³ day) when temperature dropped from 35 to 15 °C. Although bacteria morphology was not modified, sludge settling velocity decreased with decreasing temperature. In batch test, apparent activation energy (E_a) increased with decreasing temperature, which suggested the activity decrease of anaerobic ammonium oxidizing bacteria (AAOB). Low temperature inhibited AAOB and weakened nitrogen removal performance. The cardinal temperature model with inflection was first used to describe temperature effect on anammox process. Simulated results revealed that anammox reaction could occur at 10.52–50.15 °C with maximum specific anammox activity of 0.50 kg/(kg day) at 36.72 °C. The cold acclimatization of AAOB could be achieved and glycine betaine could slightly improve nitrogen removal performance at low temperature.     

Ecological characteristics of anaerobic ammonia oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is the microbial conversion of ammonium and nitrite to dinitrogen gas. The functional microbes of anammox reaction are anammox bacteria, which were discovered in a wastewater treatment system for nitrogen removal. Anammox bacteria are prevalent in anoxic ecosystems and play an important role in both biological nitrogen cycle and nitrogen pollution control. In this paper, we reviewed the investigation on ecological characteristics of anammox bacteria, and tried to figure out their complicated intraspecies and interspecies relationships. As for intraspecies relationship, we focused on the quorum sensing system, a cell density-dependent phenomenon. As for interspecies relationship, we focused on the synergism and competition of anammox bacteria with other microorganisms for substrate and space. Finally, we discussed the great influence of environmental factors (e.g., dissolved oxygen, organic matters) on the constitution, structure and function of anammox bacteria community.     

Biological treatment of ammonium-rich wastewater by partial nitritation and subsequent anaerobic ammonium oxidation (anammox) in a pilot plant

In wastewater treatment plants with anaerobic sludge digestion, 15-20% of the nitrogen load is recirculated to the main stream with the return liquors from dewatering. Separate treatment of this ammonium-rich digester supernatant would significantly reduce the nitrogen load of the activated sludge system. Some years ago, a novel biological process was discovered in which ammonium is converted to nitrogen gas under anoxic conditions with nitrite as the electron acceptor (anaerobic ammonium oxidation, anammox). Compared to conventional nitrification and denitrification, the aeration and carbon-source demand is reduced by over 50 and 100%, respectively. The combination of partial nitritation to produce nitrite in a first step and subsequent anaerobic ammonium oxidation in a second reactor was successfully tested on a pilot scale (3.6 m(3)) for over half a year. This report focuses on the feasibility of nitrogen removal from digester effluents from two different wastewater treatment plants (WWTPs) with the combined partial nitritation/anammox process. Nitritation was performed in a continuously stirred tank reactor (V=2.0 m(3)) without sludge retention. Some 58% of the ammonium in the supernatant was converted to nitrite. At 30 degrees C the maximum dilution rate D(x) was 0.85 d(-1), resulting in nitrite production of 0.35 kg NO(2)-N m(-3)(reactor) d(-1). The nitrate production was marginal. The anaerobic ammonium oxidation was carried out in a sequencing batch reactor (SBR, V=1.6 m(3)) with a nitrogen elimination rate of 2.4 kg N m(-3)(reactor) d(-1) during the nitrite-containing periods of the SBR cycle. Over 90% of the inlet nitrogen load to the anammox reactor was removed and the sludge production was negligible. The nitritation efficiency of the first reactor limited the overall maximum rate of nitrogen elimination.     

Effects of vegetation,limestone and aeration on nitritation,anammox and denitrification in wetland treatment systems

Integration of partial nitrification (nitritation) and anaerobic ammonium oxidation (anammox) in constructed wetlands creates a sustainable design for nitrogen removal. Three wetland treatment systems were operated with synthetic wastewater (60 mg NH₃–N L^?1) in a batch mode of fill – 1-week reaction – drain. Each treatment system had a surface flow wetland (unplanted, planted, and planted plus aerated, respectively) with a rooting substrate of sandy loam and limestone pellets, followed by an unplanted subsurface flow wetland. Meanwhile, three surface flow wetlands with a substrate of sandy loam and pavestone were operated in parallel to the former surface flow wetlands. Influent and effluent were monitored weekly for five cycles. Aeration reduced nitrogen removal due to hindered nitrate reduction. Vegetation maintained pH near neutral and moderate dissolved oxygen, significantly improved ammonia removal by anammox, and had higher TN removal due to coexistence of anammox and denitrification in anaerobic biofilm layers. Nitrite production was at a peak at the residence time of 4–5 d. Relative to pavestone, limestone increased the nitrite mass production peak by 97%. The subsurface flow wetlands removed nitrogen via nitritation and anammox, having an anammox activity of up to 2.4 g N m^?3 d^?1 over a startup operation of two months.     

The anammoxosome: an intracytoplasmic compartment in anammox bacteria

Anammox bacteria belong to the phylum Planctomycetes and perform anaerobic ammonium oxidation (anammox); they oxidize ammonium with nitrite as the electron acceptor to yield dinitrogen gas. The anammox reaction takes place inside the anammoxosome: an intracytoplasmic compartment bounded by a single ladderane lipid-containing membrane. The anammox bacteria, first found in a wastewater treatment plant in The Netherlands, have the potential to remove ammonium from wastewater without the addition of organic carbon. Very recently anammox bacteria were also discovered in the Black Sea where they are responsible for 30-50% of the nitrogen consumption. This review will introduce different forms of intracytoplasmic membrane systems found in prokaryotes and discuss the compartmentalization in anammox bacteria and its possible functional relation to catabolism and energy transduction.     

铁矿物强化厌氧氨氧化效能及其微生物机制研究进展

自然界中的氮循环与铁循环相互交联,参与氮循环的厌氧氨氧化（anaerobic ammonium oxidation,anammox）菌的生长代谢及活性发挥也与铁元素紧密关联。自然界广泛存在的铁矿物因具有运行成本低廉、稳定性好、二次污染小等优势,在污水处理领域得到广泛应用。在厌氧氨氧化脱氮系统中引入适量铁矿物,不仅有助于促进anammox菌和铁还原菌的富集,提高功能基因丰度和相关酶活性,还可能通过影响污泥浓度、血红素c含量、胞外聚合物含量和颗粒化程度,改善污泥性能和提高厌氧氨氧化系统的稳定性。同时,铁矿物具有促进体系多种氮素转化途径（如anammox、铁自养反硝化、铁氨氧化、异化硝酸盐还原成铵和反硝化）相耦合的潜能,可以提高anammox污水处理系统的总氮去除率。本文基于铁矿物在促进污水生物脱氮方面的良好性能及其在anammox系统中的变化,从脱氮效能、污泥特性、微生物特征及酶活性等方面,系统综述了铁矿物对厌氧氨氧化系统的强化作用机制,并从anammox菌对铁矿物的利用及铁元素的摄取角度展望了后续的研究方向,以期为铁矿物强化厌氧氨氧化系统的实际应用提供理论和技术指导。     

Development of a fixed-bed anammox reactor with high treatment potential

A plug-flow type anaerobic ammonium oxidation (anammox) reactor was developed using malt ceramics (MC) produced from carbonized spent grains as the biomass carriers for anammox sludge. Partial nitrified effluent of the filtrate from the sludge dehydrator of a brewery company was used as influent to a 20 L anammox reactor using MC. An average volumetric nitrogen removal rate (VNR) of 8.78 kg-N/m³/day was maintained stably for 76 days with 1 h of HRT. In a larger anammox reactor (400 L), an average VNR of 4.84 kg-N/m³/day could be maintained for 86 days during the treatment of low strength synthetic inorganic wastewater. As a result of bacterial community analysis for the 20 L anammox reactor, Asahi BRW1, probably originating from the wastewater collected at Asahi Breweries, was detected as the dominant anammox bacterium. These anammox reactors were characterized by a high NH₄-N removal capacity for low strength wastewater with a short hydraulic retention time.     

Nitrogen removal from animal waste treatment water by anammox enrichment

The aim of this work was to examine the applicability of the anaerobic ammonium oxidation (anammox) process to three kinds of low BOD/N ratio wastewaters from animal waste treatment processes in batch mode. A rapid decrease of NO(2)(-) and NH(4)(+) was observed during incubation with wastewaters from AS and UASB/trickling filter and their corresponding control artificial wastewaters. This nitrogen removal resulted from the anammox reaction, because the ratio of removed NO(2)(-) and NH(4)(+) was close to the theoretical ratio of the anammox reaction. Comparison of the inorganic nitrogen removal rate of the actual wastewater and that of control artificial wastewater showed that these two kinds of wastewater were very suitable for anammox treatment. Incubation with wastewater from RW did not show a clear anammox reaction; however, diluting it by half enabled the reaction, suggesting the presence of an inhibitory factor. This study showed that the three kinds of wastewater from animal waste treatment processes were suitable for anammox treatment.