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°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.     

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.     

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.     

Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium

Anaerobic ammonium-oxidizing (anammox) bacteria oxidize ammonium with nitrite and produce N(2). They reside in many natural ecosystems and contribute significantly to the cycling of marine nitrogen. Anammox bacteria generally live under ammonium limitation, and it was assumed that in nature anammox bacteria depend on other biochemical processes for ammonium. In this study we investigated the possibility of dissimilatory nitrate reduction to ammonium by anammox bacteria. Physically purified Kuenenia stuttgartiensis cells reduced (15)NO(3) (-) to (15)NH(4) (+) via (15)NO(2) (-) as the intermediate. This was followed by the anaerobic oxidation of the produced ammonium and nitrite. The overall end-product of this metabolism of anammox bacteria was (15)N(15)N dinitrogen gas. The nitrate reduction to nitrite proceeds at a rate of 0.3 +/- 0.02 fmol cell(-1) day(-1) (10% of the 'normal' anammox rate). A calcium-dependent cytochrome c protein with a high (305 mumol min(-1) mg protein(-1)) rate of nitrite reduction to ammonium was partially purified. We present evidence that dissimilatory nitrate reduction to ammonium occurs in Benguela upwelling system at the same site where anammox bacteria were previously detected. This indicates that anammox bacteria could be mediating dissimilatory nitrate reduction to ammonium in natural ecosystems.     

Application of anaerobic ammonium-oxidizing consortium to achieve completely autotrophic ammonium and sulfate removal

The simultaneous ammonium and sulfate removal was detected in an anammox reactor, consisted of ammonium oxidization with sulfate deoxidization, and subsequently traditional anammox process, in via of middle medium nitrite with solid sulfur and N2 as the terminal products. The pure anammox bacteria offered a great biotechnological potential for the completely autotrophic reaction indicated by batch tests. Denaturing gradient gel electrophoresis (DGGE) analysis further revealed that a new organism belonging to Planctomycetales was strongly enriched in the defined niche: the redox of ammonium and sulfate. The new species "Anammoxoglobussulfate" was so considered as holding a critical role in the ammonium oxidization with sulfate deoxidization to nitrite. Afterwards, the Planctomyces existing in the bacteria community performed the anammox process together to achieve the complete nitrogen and sulfate removal. The potential use of sulfate as electron acceptor for ammonium oxidizing widens the usage of anammox bacteria.     

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.     

Coupling between anammox and autotrophic denitrification for simultaneous removal of ammonium and sulfide by enriched marine sediments

In the present study, the capacity of enrichments derived from marine sediments collected from different sites of the Mexican littoral to perform anaerobic ammonium oxidation (anammox) coupled to sulfide-dependent denitrification for simultaneous removal of ammonium and sulfide linked to nitrite reduction was evaluated. Sulfide-dependent denitrification out-competed anammox during the simultaneous oxidation of sulfide and ammonium. Significant accumulation of elemental sulfur (ca. 14–30 % of added sulfide) occurred during the coupling between the two respiratory processes, while ammonium was partly oxidized (31–47 %) due to nitrite limitation imposed in sediment incubations. Nevertheless, mass balances revealed up to 38 % more oxidation of the electron donors available (ammonium and sulfide) than that expected from stoichiometry. Recycling of nitrite, from nitrate produced through anammox, is proposed to contribute to extra oxidation of sulfide, while additional ammonium oxidation is suggested by sulfate-reducing anammox (SR-anammox). The complex interaction between nitrogenous and sulfurous compounds occurring through the concomitant presence of autotrophic denitrification, conventional anammox and SR-anammox may significantly drive the nitrogen and sulfur fluxes in marine environments.     

高含氮稻田深层土壤的氨氧化古菌和厌氧氨氧化菌共存及对氮循环影响

随着海洋生态系统中的厌氧氨氧化反应和氨氧化古菌的发现,自然生态系统的氮循环过程被重新认识,但是目前尚无在陆地深层的相关报道。结合同位素示踪与分子生物学技术探索了稻田深层土壤中anammox与AOA的存在及特性。结果表明,在沼渣处理废水浇灌的高含氮稻田深层土壤中,anammox与AOA共存。通过构建克隆文库发现,此土壤中厌氧氨氧化菌的生物多样性相对较低,35个克隆序列只分为4个独立操作单元(OTU),代表序列与Genebank数据库中已探明的厌氧氨氧化菌Candidatus 'Kuenenia stuttgartiensis’的同源性超过95%;对氨氧化古菌的分析发现,20个克隆子共得到5个OTU,其与基因库中土壤/沉积物进化分支关系最近,序列的同源性部分超过98%。同位素示踪的初步结果表明,anammox产生的氮气占此土壤总氮气生成量的24.1%-29.8%。AOA与anammox的共存为anammox反应的广泛存在与发生提供了新思路。     

Anammox organism KSU-1 expresses a NirK-type copper-containing nitrite reductase instead of a NirS-type with cytochrome cd1

Anaerobic ammonium oxidation (anammox) and denitrification are two distinct microbial reactions relevant to the global nitrogen cycle. The proposed initial step of the anammox reactions, reduction of nitrite to nitric oxide, has been postulated to be identical to that in denitrification catalyzed by the dissimilatory nitrite reductase of the cytochrome cd(1)-type. Here, we characterized the copper-containing nitrite reductase homolog encoded by nirK detected in the genome of an anammox bacterium strain KSU-1. We hypothesize that this NirK-type nitrite reductase, rather than a nitrite reductase of the cytochrome cd(1)-type (NirS), is likely to catalyze nitrite reduction in anammox organism KSU-1.     

厌氧铵氧化过程中关键酶及相关分子标记在生态学研究中的应用进展

厌氧铵氧化是由微生物介导的氮素循环过程中的重要途径之一。近20年来,通过对厌氧铵氧化细菌生态学、基因组学和生理代谢特性的探索,人们对其微生物学机制已经有了较多的认识:厌氧铵氧化细菌通过亚硝酸盐还原酶将亚硝酸根离子还原为一氧化氮,进而与铵离子结合在联氨合成酶的作用下生成联氨,最后通过联氨氧化酶的催化产生终产物氮气。同时,对参与这些过程的关键酶及其功能基因的认识有助于选择新的分子标记,从而为研究厌氧铵氧化细菌的多样性和分子生态学特征提供新的工具,以弥补16S rRNA基因特异性相对较低且难以与生态功能关联等方面的不足。对目前已知的参与厌氧铵氧化过程的3种关键酶的研究历程和现状进行了评述,并总结了利用3种功能基因进行厌氧铵氧化细菌生态学研究的最新进展。     

Effect of sublethal concentrations of four chemicals on susceptibility of juvenile rainbow trout (Oncorhynchus mykiss) to saprolegniosis

The effects of sublethal concentrations of a variety of chemicals on the susceptibility of rainbow trout (Oncorhynchus mykiss) juveniles to Saprolegnia parasitica infection was examined. Sublethal concentrations of un-ionized ammonia (0.05 mg/liter) and nitrite (0.12 mg/liter) increased fish susceptibility after 10 days of exposure to the toxin, this increase being higher for ammonia (75% and 20% morbidity) than for nitrite (20% and 0% morbidity, respectively) with inoculum doses of 1.4 x 10(6) and 9.5 x 10(5) zoospores per liter, respectively. Sublethal concentrations of copper (0.05 mg/liter) or cyanide (0.05 mg/liter) did not show enhancement of infection by S. parasitica, even though the toxin exposure was for 21 days and the inoculum doses were higher than those for the experiments with the nitrogen compounds (4 x 10(6) and 3.2 x 10(6) zoospores per liter. However, infections began to appear in control animals.     

Effect of sublethal concentrations of four chemicals on susceptibility of juvenile rainbow trout (Oncorhynchus mykiss) to saprolegniosis.

The effects of sublethal concentrations of a variety of chemicals on the susceptibility of rainbow trout (Oncorhynchus mykiss) juveniles to Saprolegnia parasitica infection was examined. Sublethal concentrations of un-ionized ammonia (0.05 mg/liter) and nitrite (0.12 mg/liter) increased fish susceptibility after 10 days of exposure to the toxin, this increase being higher for ammonia (75% and 20% morbidity) than for nitrite (20% and 0% morbidity, respectively) with inoculum doses of 1.4 x 10(6) and 9.5 x 10(5) zoospores per liter, respectively. Sublethal concentrations of copper (0.05 mg/liter) or cyanide (0.05 mg/liter) did not show enhancement of infection by S. parasitica, even though the toxin exposure was for 21 days and the inoculum doses were higher than those for the experiments with the nitrogen compounds (4 x 10(6) and 3.2 x 10(6) zoospores per liter. However, infections began to appear in control animals.     

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.     

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     

Anammox bacteria enrichment in upflow anaerobic sludge blanket (UASB) reactor

We investigated the anaerobic ammonium oxidation (anammox) reaction in a labscale upflow anaerobic sludge blanket (UASB) reactor. Our aim was to detect and enrich the organisms responsible for the anammox reaction using a synthetic medium that contained low concentrations of substrates (ammonium and nitrite). The reactor was inoculated with granular sludge collected from a full-scale anaerobic digestor used for treating brewery wastewater. The experiment was performed during 260 days under conditions of constant ammonium concentration (50 mg NH₄/⁺-N/L) and different nitrite concentrations (50∼150 mg NO₂-N/L). After 200 days, anammox activity was observed in the system. The microorganisms involved in this anammox reaction were identified as CandidatusB. Anammoxidans andK. Stuttgartiensis using fluorescencein situ hybridization (FISH) method.     

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.     

Hydrazine synthase, a unique phylomarker with which to study the presence and biodiversity of anammox bacteria

Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the biogeochemical cycling of nitrogen. They derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. Several methods have been used to detect the presence and activity of anammox bacteria in the environment, including 16S rRNA gene-based approaches. The use of the 16S rRNA gene to study biodiversity has the disadvantage that it is not directly related to the physiology of the target organism and that current primers do not completely capture the anammox diversity. Here we report the development of PCR primer sets targeting a subunit of the hydrazine synthase (hzsA), which represents a unique phylogenetic marker for anammox bacteria. The tested primers were able to retrieve hzsA gene sequences from anammox enrichment cultures, full-scale anammox wastewater treatment systems, and a variety of freshwater and marine environmental samples, covering all known anammox genera.     

Candidatus "Anammoxoglobus propionicus" a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria

The bacteria that mediate the anaerobic oxidation of ammonium (anammox) are detected worldwide in natural and man-made ecosystems, and contribute up to 50% to the loss of inorganic nitrogen in the oceans. Two different anammox species rarely live in a single habitat, suggesting that each species has a defined but yet unknown niche. Here we describe a new anaerobic ammonium oxidizing bacterium with a defined niche: the co-oxidation of propionate and ammonium. The new anammox species was enriched in a laboratory scale bioreactor in the presence of ammonium and propionate. Interestingly, this particular anammox species could out-compete other anammox bacteria and heterotrophic denitrifiers for the oxidation of propionate in the presence of ammonium, nitrite and nitrate. We provisionally named the new species Candidatus "Anammoxoglobus propionicus".     

Global variations and controlling factors of anammox rates

Soil anammox is an environmentally friendly way to eliminate reactive nitrogen (N) without generating nitrous oxide. Nevertheless, the current earth system models have not incorporated the anammox due to the lack of parameters in anammox rates on a global scale, limiting the accurate projection for N cycling. A global synthesis with 1212 observations from 89 peer-reviewed papers showed that the average anammox rate was 1.60 ± 0.17 nmol N g⁻¹ h⁻¹ in terrestrial ecosystems, with significant variations across different ecosystems. Wetlands exhibited the highest rate (2.17 ± 0.31 nmol N g⁻¹ h⁻¹), followed by croplands at 1.02 ± 0.09 nmol N g⁻¹ h⁻¹. The lowest anammox rates were observed in forests and grasslands. The anammox rates were positively correlated with the mean annual temperature, mean annual precipitation, soil moisture, organic carbon (C), total N, as well as nitrite and ammonium concentrations, but negatively with the soil C:N ratio. Structural equation models revealed that the geographical variations in anammox rates were primarily influenced by the N contents (such as nitrite and ammonium) and abundance of anammox bacteria, which collectively accounted for 42% of the observed variance. Furthermore, the abundance of anammox bacteria was well simulated by the mean annual precipitation, soil moisture, and ammonium concentrations, and 51% variance of the anammox bacteria was accounted for. The key controlling factors for soil anammox rates differed from ecosystem type, for example, organic C, total N, and ammonium contents in croplands, versus soil C:N ratio and nitrite concentrations in wetlands. The controlling factors in soil anammox rate identified by this study are useful to construct an accurate anammox module for N cycling in earth system models.     

Simultaneous energy recovery and autotrophic nitrogen removal from sewage at moderately low temperatures

This study assessed the technical feasibility of treating sewage with a combination of direct anaerobic treatment and autotrophic nitrogen removal, while simultaneously achieving energy recovery and nitrogen removal under moderately low temperatures. The concentrations of ammonia, nitrite, and COD in effluent were below 1, 0.1, and 30 mg/L, respectively. In the up-flow, anaerobic sludge fixed-bed, there was no obvious change observed in the total methane production at temperatures of 35?±?1 °C, 28?±?1 °C, 24?±?3 °C, and 17?±?3 °C, with the accumulation of volatile fatty acids occurring with decreasing temperatures. The control strategy employed in this study achieved a stable effluent with equimolar concentrations of nitrite and ammonium, coupled with high nitrite accumulation (>97 %) in the partial nitrification sequencing batch reactor system at moderately low temperatures. In the anaerobic ammonium oxidation (anammox) reactor, a short hydraulic retention time of 0.96 h, with a nitrogen removal rate of 0.83 kgN/(m³/day) was achieved at 12–15 °C. At low temperatures, the corresponding fluorescence in situ hybridization image revealed a high amount of anammox bacteria. This study demonstrates that efficient nitrogen removal and energy recovery from sewage at moderately low temperatures can be achieved by utilizing a combined system. Additionally, this system has the potential to become energy-neutral or even energy-producing.