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.     

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.     

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。     

污水短程硝化体系氨氧化菌与亚硝酸盐氧化菌的生态学研究进展

短程硝化(partial nitrification, PN)是一种绿色低碳的生物脱氮创新技术,伴随厌氧氨氧化(anaerobic ammonia oxidation, Anammox)污水脱氮技术的进一步推广,短程硝化作为提供其电子受体的重要环节,已成为了污水脱氮领域的研究热点。氨氧化菌(ammonia-oxidizing bacteria,AOB)和亚硝酸盐氧化菌(nitrite-oxidizing bacteria, NOB)是该技术的核心竞争微生物,掌握这两类微生物的生态学特征,借助生态学理论和手段调控AOB淘汰NOB,提高种群的可预测性,对于实现稳定高效的短程硝化具有重要意义。本文基于生态学角度介绍了AOB和NOB基础分类、生理性能及生态位分离,重点综述了短程硝化系统中AOB和NOB的生长动力学、群落构建、环境因素和相互作用,最后对这两类微生物的未来研究重点和研究方法进行了展望,为短程硝化工艺的快速启动和稳定运行提供理论指导。     

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...     

Enhancing mainstream nitrogen removal by employing nitrate/nitrite-dependent anaerobic methane oxidation processes

Due to serious eutrophication in water bodies, nitrogen removal has become a critical stage for wastewater treatment plants (WWTPs) over past decades. Conventional biological nitrogen removal processes are based on nitrification and denitrification (N/DN), and are suffering from several major drawbacks, including substantial aeration consumption, high fugitive greenhouse gas emissions, a requirement for external carbon sources, excessive sludge production and low energy recovery efficiency, and thus unable to satisfy the escalating public needs. Recently, the discovery of anaerobic ammonium oxidation (anammox) bacteria has promoted an update of conventional N/DN-based processes to autotrophic nitrogen removal. However, the application of anammox to treat domestic wastewater has been hindered mainly by unsatisfactory effluent quality with nitrogen removal efficiency below 80%. The discovery of nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) during the last decade has provided new opportunities to remove this barrier and to achieve a robust system with high-level nitrogen removal from municipal wastewater, by utilizing methane as an alternative carbon source. In the present review, opportunities and challenges for nitrate/nitrite-dependent anaerobic methane oxidation are discussed. Particularly, the prospective technologies driven by the cooperation of anammox and n-DAMO microorganisms are put forward based on previous experimental and modeling studies. Finally, a novel WWTP system acting as an energy exporter is delineated.     

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.     

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.

     

Propionate Oxidation by and Methanol Inhibition of Anaerobic Ammonium-Oxidizing Bacteria

Anaerobic ammonium oxidation (anammox) is a recently discovered microbial pathway and a cost-effective way to remove ammonium from wastewater. Anammox bacteria have been described as obligate chemolithoautotrophs. However, many chemolithoautotrophs (i.e., nitrifiers) can use organic compounds as a supplementary carbon source. In this study, the effect of organic compounds on anammox bacteria was investigated. It was shown that alcohols inhibited anammox bacteria, while organic acids were converted by them. Methanol was the most potent inhibitor, leading to complete and irreversible loss of activity at concentrations as low as 0.5 mM. Of the organic acids acetate and propionate, propionate was consumed at a higher rate (0.8 nmol min⁻¹ mg of protein⁻¹) by Percoll-purified anammox cells. Glucose, formate, and alanine had no effect on the anammox process. It was shown that propionate was oxidized mainly to CO₂, with nitrate and/or nitrite as the electron acceptor. The anammox bacteria carried out propionate oxidation simultaneously with anaerobic ammonium oxidation. In an anammox enrichment culture fed with propionate for 150 days, the relative amounts of anammox cells and denitrifiers did not change significantly over time, indicating that anammox bacteria could compete successfully with heterotrophic denitrifiers for propionate. In conclusion, this study shows that anammox bacteria have a more versatile metabolism than previously assumed.     

Research of Iron Reduction and the Iron Reductase Localization of Anammox Bacteria

The iron-reducing capability of anammox bacteria was examined in this study using Percoll purified anammox bacteria. Anammox bacteria could reduce Fe(III) to Fe(II) with organic matters as the electron donor. The activity of anammox iron-reducing process was dependent on different electron donor, acceptor and pH. The highest iron-reducing activity of anammox bacteria was achieved with Fe(III)-NTA (nitrilotriacetic acid) as electron acceptor and formate as the electron donor at pH7. Similar to other iron reducers, 80 % of the iron reductase in anammox bacteria was located in the membrane fraction. Due to the chemical oxidant of NO₂ ^? and the NO₃ ^? dependent ferrous iron oxidation by anammox bacteria, the iron-reducing activity of anammox bacteria could be severely inhibited when iron-reducing pathway and the anammox process were coupled. However, the total nitrogen removal efficiency was not significantly affected in the presence of Fe(III). The iron-reducing capability of anammox bacteria could influence both N and Fe cycle on earth, and it is a potential way for wastewater treatment.     

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.     

Applicability of one-stage partial nitritation and anammox in MBBR for anaerobically pre-treated municipal wastewater

Energy consumption of municipal wastewater treatment plants can be reduced by the anaerobic pre-treatment of the main wastewater stream. After this pre-treatment, nitrogen can potentially be removed by partial nitritation and anammox (PN/A). Currently, the application of PN/A is limited to nitrogen-rich streams (>500 mg L^?1) and temperatures 25–35 °C. But, anaerobically pretreated municipal wastewater is characterized by much lower nitrogen concentrations (20–100 mg L^?1) and lower temperatures (10–25 °C). We operated PN/A under similar conditions: total ammonium nitrogen concentration 50 mg L^?1 and lab temperature (22 °C). PN/A was operated for 342 days in a 4 L moving bed biofilm reactor (MBBR). At 0.4 mg O₂ L^?1, nitrogen removal rate 33 g N m^?3 day^?1 and 80 % total nitrogen removal efficiency was achieved. The capacity of the reactor was limited by low AOB activity. We observed significant anammox activity (40 g N m^?3 day^?1) even at 12 °C, improving the applicability of PN/A for municipal wastewater treatment.     

厌氧氨氧化颗粒污泥聚集机制研究进展

厌氧氨氧化(anaerobic ammonium oxidation,anammox)工艺被认为是当前污水生物脱氮领域最经济的处理工艺,有利于实现污水处理厂的能源自给。厌氧氨氧化菌是该工艺的核心功能微生物。以厌氧氨氧化菌为主导微生物形成的厌氧氨氧化颗粒污泥具有沉速大、污泥持留能力强及对不利环境抵抗能力强等突出优势,是实现厌氧氨氧化工艺最有前景的污泥形态。本论文围绕厌氧氨氧化颗粒,介绍了厌氧氨氧化菌的特性、种类及代谢途径,综述了厌氧氨氧化颗粒污泥的形成假说及与厌氧氨氧化颗粒污泥聚集密切相关的胞外聚合物(extracellular polymeric substance,EPS)和群体感应研究现状,并对今后厌氧氨氧化颗粒的研究进行了展望,以期为后续厌氧氨氧化颗粒的研究及厌氧氨氧化颗粒工艺的优化提供参考。     

Denitrification and anammox in tropical aquaculture settlement ponds: an isotope tracer approach for evaluating n(2) production

Settlement ponds are used to treat aquaculture discharge water by removing nutrients through physical (settling) and biological (microbial transformation) processes. Nutrient removal through settling has been quantified, however, the occurrence of, and potential for microbial nitrogen (N) removal is largely unknown in these systems. Therefore, isotope tracer techniques were used to measure potential rates of denitrification and anaerobic ammonium oxidation (anammox) in the sediment of settlement ponds in tropical aquaculture systems. Dinitrogen gas (N₂) was produced in all ponds, although potential rates were low (0–7.07 nmol N cm⁻³ h⁻¹) relative to other aquatic systems. Denitrification was the main driver of N₂ production, with anammox only detected in two of the four ponds. No correlations were detected between the measured sediment variables (total organic carbon, total nitrogen, iron, manganese, sulphur and phosphorous) and denitrification or anammox. Furthermore, denitrification was not carbon limited as the addition of particulate organic matter (paired t-Test; P = 0.350, n = 3) or methanol (paired t-Test; P = 0.744, n = 3) did not stimulate production of N₂. A simple mass balance model showed that only 2.5% of added fixed N was removed in the studied settlement ponds through the denitrification and anammox processes. It is recommended that settlement ponds be used in conjunction with additional technologies (i.e. constructed wetlands or biological reactors) to enhance N₂ production and N removal from aquaculture wastewater.     

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.     

厌氧氨氧化菌的研究进展

近年来,有关厌氧氨氧化过程这一特殊的生化机制以及微生物类群的研究引起了人们的极大关注,尤其是这类微生物的生态生境可能比人们预想的范围更加广泛,因而在自然界N循环中可能具有重要意义。对这类菌结构特征、系统发育地位以及厌氧氨氧化小体和厌氧氨氧化机制的更深入认识将大大促进它们在污水处理工程中的应用。综述了近年来有关厌氧氨氧化菌的生理特性、生化机制、结构特点、生态生境以及工程应用等方面的最新进展。     

Application, eco-physiology and biodiversity of anaerobic ammonium-oxidizing bacteria

The demand for new and sustainable systems for nitrogen removal has increased dramatically in the last decade. It is clear that the conventional systems cannot deal with the increasing nitrogen loads in a cost effective way. As an alternative, the implementation of the anammox (anaerobic ammonium oxidation) process in the treatment of wastewater with high ammonium concentrations has been started. The compact anammox reactors can sustain high nitrogen loads without any problems. The highest observed anammox capacity is 8.9 kg N removed m^-3 reactor day^-1. The first 75 m³ anammox reactor is operating in Rotterdam, the Netherlands, combined with the partial nitrification process Single reaction system for High Ammonium Removal Over Nitrite (SHARON). Partial nitrification and anammox can also be combined in one reactor systems like Completely Autotrophic Nitrogen removal Over Nitrite (CANON) or Oxygen Limited Ammonium removal via Nitrification Denitrification (OLAND) where aerobic ammonium-oxidizing bacteria (AOB) and anammox bacteria cooperate under oxygen-limitation. These systems remove about 1.5 kg N m^-3 reactor day^-1. In addition to ammonium, urea can also be converted in the CANON system after a two-week adaptation period. The ecophysiological properties of the anammox bacteria make them very well suited to convert ammonium and nitrite. The K_s values for ammonium and nitrite are below 5 M. However, nitrite above 10 mM is detrimental for the anammox process, and oxygen reversibly inhibits the process at concentrations as low as 1 M. Acetate and propionate can be used by the anammox bacteria to convert nitrite and nitrate, whereas methanol and ethanol severely inhibit the anammox reaction. The enzyme hydroxylamine/hydrazine oxidoreductase (HAO), one of the key enzymes, is located in the anammoxosome, which is a membrane bound organelle. The membranes of the anammox bacteria contain unique ladderane lipids and hopanoids. The bacteria responsible for the anammox reaction are related to the Planctomycetes. The first anammox bacteria were isolated via Percoll centrifugation and characterized as Candidatus Brocadia anammoxidans. Survey of different wastewater treatment plants using anammox specific 16S rRNA gene primers and anammox specific oligonucleotide probes has revealed the presence of at least three other anammox bacteria, which have been tentatively named Candidatus Kuenenia stuttgartiensis, Candidatus Scalindua wagneri and Candidatus Scalindua brodae. A close relative of the latter, Candidatus Scalindua sorokinii was found to be responsible for about 50% of the nitrogen conversion in the anoxic zone of the Black Sea, making the anammox bacteria an important player in the oceanic nitrogen cycle.     

Treatment of Biogas Produced in Anaerobic Reactors for Domestic Wastewater: Odor Control and Energy/Resource Recovery

Anaerobic municipal wastewater treatment in developing countries has important potential applications considering their huge lack of sanitation infrastructure and their advantageous climatic conditions. At present, among the obstacles that this technology encounters, odor control and biogas utilization or disposal should be properly addressed. In fact, in most of small and medium size anaerobic municipal treatment plants, biogas is just vented, transferring pollution from water to the atmosphere, contributing to the greenhouse gas inventory. Anaerobic municipal sewage treatment should not be considered as an energy producer, unless a significant wastewater flow is treated. In these cases, more than half of the methane produced is dissolved and lost in the effluent so yield values will be between 0.08 and 0.18 N m³ CH₄/kg COD removed. Diverse technologies for odor control and biogas cleaning are currently available. High pollutant concentrations may be treated with physical-chemical methods, while biological processes are used mainly for odor control to prevent negative impacts on the treatment facilities or nearby areas. In general terms, biogas treatment is accomplished by physico-chemical methods, scrubbing being extensively used for H₂S and CO₂ removal. However, dilution (venting) has been an extensive disposal method in some small- and medium-size anaerobic plants treating municipal wastewaters. Simple technologies, such as biofilters, should be developed in order to avoid this practice, matching with the simplicity of anaerobic wastewater treatment processes. In any case, design and specification of biogas handling system should consider safety standards. Resource recovery can be added to anaerobic sewage treatment if methane is used as electron donor for denitrification and nitrogen control purposes. This would result in a reduction of operational cost and in an additional advantage for the application of anaerobic sewage treatment. In developing countries, biogas conversion to energy may apply for the clean development mechanism (CDM) of the Kyoto Protocol. This would increase the economic feasibility of the project through the marketing of certified emission reductions (CERs).     

Correlation between Anammox Activity and Microscale Distribution of Nitrite in a Subtropical Mangrove Sediment

The distribution of anaerobic ammonium oxidation (anammox) in nature has been addressed by only a few environmental studies, and our understanding of how anammox bacteria compete for substrates in natural environments is therefore limited. In this study, we measure the potential anammox rates in sediment from four locations in a subtropical tidal river system. Porewater profiles of NO_x⁻ (NO₂⁻ plus NO₃⁻) and NO₂⁻ were measured with microscale biosensors, and the availability of NO₂⁻ was compared with the potential for anammox activity. The potential rate of anammox increased with increasing distance from the mouth of the river and correlated strongly with the production of nitrite in the sediment and with the average concentration or total pool of nitrite in the suboxic sediment layer. Nitrite accumulated both from nitrification and from NO_x⁻ reduction, though NO_x⁻ reduction was shown to have the greatest impact on the availability of nitrite in the suboxic sediment layer. This finding suggests that denitrification, though using NO₂⁻ as a substrate, also provides a substrate for the anammox process, which has been suggested in previous studies where microscale NO₂⁻ profiles were not measured.     

Effects of Specific Inhibitors on Anammox and Denitrification in Marine Sediments

The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N₂ production were investigated by using short anoxic incubations of marine sediment with a ¹⁵N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10 ± 0.02 μM⁻¹, and there was 95% inhibition at ~30 μM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 μM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049 ± 0.004 μM⁻¹; 95% inhibition at ~70 μM). Methanol specifically inhibited anammox with a decay constant of 0.79 ± 0.12 mM⁻¹, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by ~50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N₂ production in marine sediments and the use of ¹⁵N isotope methods for their quantification.