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.     

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".     

Detection of anaerobic processes and microorganisms in immobilized activated sludge of a wastewater treatment plant with intense aeration

Attached activated sludge from the Krasnaya Polyana (Sochi) wastewater treatment plant was studied after the reconstruction by increased aeration and water recycle, as well as by the installation of a bristle carrier for activated sludge immobilization. The activated sludge biofilms developing under conditions of intense aeration were shown to contain both aerobic and anaerobic microorganisms. Activity of a strictly anaerobic methanogenic community was revealed, which degraded organic compounds to methane, further oxidized by aerobic methanotrophs. Volatile fatty acids, the intermediates of anaerobic degradation of complex organic compounds, were used by both aerobic and anaerobic microorganisms. Anaerobic oxidation of ammonium with nitrite (anammox) and the presence of obligate anammox bacteria were revealed in attached activated sludge biofilms. Simultaneous aerobic and anaerobic degradation of organic contaminants by attached activated sludge provides for high rates of water treatment, stability of the activated sludge under variable environmental conditions, and decreased excess sludge formation.     

Genome analysis and heterologous expression of acetate-activating enzymes in the anammox bacterium Kuenenia stuttgartiensis

Anaerobic ammonium-oxidizing bacteria were recently shown to use short-chain organic acids as additional energy source. The AMP-forming acetyl-CoA synthetase gene (acs) of Kuenenia stuttgartiensis, encoding an important enzyme involved in the conversion of these organic acids, was identified and heterologously expressed in Escherichia coli to investigate the activation of several substrates, that is, acetate, propionate and butyrate. The heterologously expressed ACS enzyme could complement an E. coli triple mutant deficient in all pathways of acetate activation. Activity was observed toward several short-chain organic acids, but was highest with acetate. These properties are in line with a mixotrophic growth of anammox bacteria. In addition to acs, the genome of K. stuttgartiensis contained the essential genes of an acetyl-CoA synthase/CO dehydrogenase complex and genes putatively encoding two isoenzymes of archaeal-like ADP-forming acetyl-CoA synthetase underlining the importance of acetyl-CoA as intermediate in the carbon assimilation metabolism of anammox bacteria.     

厌氧氨氧化菌的物种多样性与生态分布

厌氧氨氧化是微生物和环境领域的重大发现,厌氧氨氧化可同时去除氨氮和亚硝氮,在环境工程上具有重大开发价值.由于厌氧氨氧化菌生长极慢,倍增时间长达11 d以上,严重制约了该反应的开发进程,因此,对厌氧氨氧化菌的研究具有重要意义.厌氧氨氧化菌种类丰富,除了人们最早认识的浮霉状菌外,还有硝化细菌和反硝化细菌,这些菌群生态分布广泛,为开辟新的厌氧氨氧化菌种资源创造了条件.硝化细菌和反硝化细菌兼有厌氧氨氧化能力,其代谢多样性为加速厌氧氨氧化反应器的启动提供了依据.厌氧消化污泥可呈现硫酸盐型厌氧氨氧化活性,可为新型生物脱氮工艺的研发奠定基础.探明厌氧氨氧化菌种资源及其生态分布,将有利于厌氧氨氧化的开发应用.     

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.     

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.     

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.     

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.     

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.     

Metagenomic evidence of a novel family of anammox bacteria in a subsea environment

Bacteria in the order ‘Candidatus Brocadiales’ within the phylum Planctomycetes (Planctomycetota) have the remarkable ability to perform anaerobic ammonium oxidation (anammox). Two families of anammox bacteria with different biogeographical distributions have been reported, marine Ca. Scalinduaceae and freshwater Ca. Brocadiaceae. Here we report evidence of three new species within a novel genus and family of anammox bacteria, which were discovered in biofilms of a subsea road tunnel under a fjord in Norway. In this particular ecosystem, the nitrogen cycle is likely fuelled by ammonia from organic matter degradation in the fjord sediments and the rock mass above the tunnel, resulting in the growth of biofilms where anammox bacteria can thrive under oxygen limitation. We resolved several metagenome-assembled genomes (MAGs) of anammox bacteria, including three Ca. Brocadiales MAGs that could not be classified at the family level. MAGs of this novel family had all the diagnostic genes for a full anaerobic ammonium oxidation pathway in which nitrite was probably reduced by a NirK-like reductase. A survey of published molecular data indicated that this new family of anammox bacteria occurs in many marine sediments, where its members presumably would contribute to nitrogen loss.     

Mimicking the oxygen minimum zones: stimulating interaction of aerobic archaeal and anaerobic bacterial ammonia oxidizers in a laboratory‐scale model system

In marine oxygen minimum zones (OMZs), ammonia‐oxidizing archaea (AOA) rather than marine ammonia‐oxidizing bacteria (AOB) may provide nitrite to anaerobic ammonium‐oxidizing (anammox) bacteria. Here we demonstrate the cooperation between marine anammox bacteria and nitrifiers in a laboratory‐scale model system under oxygen limitation. A bioreactor containing ‘Candidatus Scalindua profunda’ marine anammox bacteria was supplemented with AOA (Nitrosopumilus maritimus strain SCM1) cells and limited amounts of oxygen. In this way a stable mixed culture of AOA, and anammox bacteria was established within 200 days while also a substantial amount of endogenous AOB were enriched. ‘Ca. Scalindua profunda’ and putative AOB and AOA morphologies were visualized by transmission electron microscopy and a C₁₈ anammox [3]‐ladderane fatty acid was highly abundant in the oxygen‐limited culture. The rapid oxygen consumption by AOA and AOB ensured that anammox activity was not affected. High expression of AOA, AOB and anammox genes encoding for ammonium transport proteins was observed, likely caused by the increased competition for ammonium. The competition between AOA and AOB was found to be strongly related to the residual ammonium concentration based on amoA gene copy numbers. The abundance of archaeal amoA copy numbers increased markedly when the ammonium concentration was below 30 μM finally resulting in almost equal abundance of AOA and AOB amoA copy numbers. Massive parallel sequencing of mRNA and activity analyses further corroborated equal abundance of AOA and AOB. PTIO addition, inhibiting AOA activity, was employed to determine the relative contribution of AOB versus AOA to ammonium oxidation. The present study provides the first direct evidence for cooperation of archaeal ammonia oxidation with anammox bacteria by provision of nitrite and consumption of oxygen.     

Enrichment and characterization of an anammox bacterium from a rotating biological contactor treating ammonium-rich leachate

Anaerobic ammonium oxidation with nitrite to N2 (anammox) is a recently discovered microbial reaction with interesting potential for nitrogen removal from wastewater. We enriched an anammox culture from a rotating disk contactor (near K?lliken, Switzerland) that was used to treat ammonium-rich leachate with low organic carbon content. This enrichment led to a relative population size of 88% anammox bacteria. The microorganism carrying out the anammox reaction was identified by analysis of the 16S rDNA sequence and by fluorescence in situ hybridization (FISH) with 16S-rRNA-targeting probes. The percentage sequence identity between the 16S rDNA sequences of the K?lliken anammox organism and the archetype anammox strain Candidatus Brocadia anammoxidans was 90.9%, but between 98.5 and 98.9% with Candidatus Kuenenia stuttgartiensis, an organism identified in biofilms by molecular methods. The K?lliken culture catalyzed the anaerobic oxidation of ammonium with nitrite in a manner seemingly identical to that of Candidatus B. anammoxidans, but exhibited higher tolerance to phosphate (up to 20 mM) and to nitrite (up to 13 mM) and was active at lower cell densities. Anammox activity was observed only between pH 6.5 and 9, with an optimum at pH 8 and a temperature optimum at 37 degrees C. Hydroxylamine and hydrazine, which are intermediates of the anammox reaction of Candidatus B. anammoxidans, were utilized by the K?lliken organisms, and approximately 15% of the nitrite utilized during autotrophic growth was converted to nitrate. Electron microscopy showed a protein-rich region in the center of the cells surrounded by a doughnut-shaped region containing ribosomes and DNA. This doughnut-shape region was observed with FISH as having a higher fluorescence intensity. Similar to Candidatus B. anammoxidans, the K?lliken anammox organism typically formed homogenous clusters containing up to several hundred cells within an extracellular matrix.     

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     

PVA-alginate immobilized cells for anaerobic ammonium oxidation (anammox) process

The feasibility of an anaerobic ammonium oxidation (anammox) process combined with a cell-immobilization technique for autotrophic nitrogen removal was investigated. Anammox biomass was cultivated from local activated sludge and achieved significant anammox activity in 6 months. The development of a mature anammox biomass was confirmed by fluorescence in situ hybridization (FISH) analysis and off-line activity measurements. The abundance fraction of the anammox bacteria determined by FISH analysis was estimated by software. The anaerobic ammonia oxidizers occupied almost half of the total cells. Additionally, the anammox biomass was granulated as spherical gel beads of 3-4 mm in diameter by using a cell-immobilization technique. The nitrogen removal activity was proved to be successfully retained in the beads, with about 80% of nitrogenous compounds (NH(4) (+), NO(2) (- )and total nitrogen) removed after 48 h. These results offer a promising technique for the preservation of anammox microorganisms, the protection of them against the unfavorable surroundings, and the prevention of biomass washout towards the implementation of sustainable nitrogen elimination biotechnology. This is the first report on the immobilization of anammox biomass as gel beads.     

Alterations in anaerobic ammonium oxidation of paddy soil following organic carbon treatment estimated using 13C-DNA stable isotope probing

In this study, soil samples from the typical rice-wheat cropping system in Jiangsu Province, China, subjected to different fertilizer application treatments―no carbon (CK), urea (UR), straw (SR), pig manure (PM), starch (ST), and glucose (GL)―were used to determine potential anaerobic ammonium oxidation (anammox) rate and its association with bacterial abundance, diversity, and activity by using DNA stable isotope probing combined with ¹⁵N isotope tracing and molecular techniques. The effects of different organic carbon sources on anammox were significant, in the following order: GL > ST, SR > UR > PM; anammox activity differed significantly across treatments; however, the ¹³C active anammox bacteria were only closely related to Ca. Brocadia. The anammox hydrazine synthase β subunit functional gene sequences were highly associated with the Candidatus genus Brocadia in PM and CK treatments. The different organic carbon sources had different inhibitory effects with anammox rate, which dropped from 3.19 to 1.04 nmol dinitrogen gas g⁻¹ dry soil h⁻¹ among treatments. About 4.2–22.3% of dinitrogen gas emissions were attributed to anammox and indicated that a specific population of anammox bacteria was present and varied with the addition of exogenous organic compounds in paddy soils, although a small part of dinitrogen gas was emitted from the soil via anammox.

     

自然生态系统中的厌氧氨氧化

厌氧氨氧化（anaerobic ammonium oxidation,anammox）是由anammox菌在缺氧条件下以氨为电子供体、以亚硝酸为电子受体的生物反应,反应产物为氮气,该反应的发现为全球氮素循环增添了新的内容。参与anammox反应的微生物是anammox菌,anammox菌是一群分支很深的浮霉状菌,目前已发现的anammox菌有5个属8个种。催化anammox反应的是一特殊的细胞结构-厌氧氨氧化体,每种已发现的anammox菌中都存在该特殊结构。有关anammox反应的生化机理目前普遍认为,NO和联氨（N2H4）是anammox反应的重要中间体,NO可将NH4 直接氧化,形成N2H4,N2H4在联氨氧化酶的作用下最终转化为氮气。Anammox最初发现于人工脱氮系统,已发现的8种anammox菌中7种来自于人工系统。但越来越多的证据表明,anammox菌广泛分布于自然界的海洋、淡水和陆地生态系统中,在区域氮素循环中起着不同程度的作用。影响自然生态系统中anammox反应的主要环境因子包括有机质含量、NO3-浓度和盐度等,但在不同的生态系统,anammox反应的主导影响因子存在较明显差异。本文综述了anammox菌的类群和生化反应机理,总结了anammox菌在各种自然生态系统中的分布与生态多样性,并论述了anammox反应在全球氮素循环中的重要性以及影响此过程发挥的主要环境因子。     

Community Structures and Distribution of Anaerobic Ammonium Oxidizing and nirS-Encoding Nitrite-Reducing Bacteria in Surface Sediments of the South China Sea

Anaerobic ammonium oxidation (anammox) and denitrification are two important processes responsible for nitrogen loss; monitoring of microbial communities carrying out these two processes offers a unique opportunity to understand the microbial nitrogen cycle. The aim of the current study was to characterize community structures and distribution of anammox and nirS-encoding nitrite-reducing bacteria in surface sediments of the northern South China Sea (SCS). The consistent phylogenetic results of three biomarkers of anammox bacteria, including 16S rRNA, hzo, and Scalindua-nirS genes, showed that Scalindua-like bacteria were the only anammox group presenting in surface sediments of the SCS. However, a relatively high micro-diversity was found within this group, including several SCS habitat-specific phylotypes, Candidatus “Scalindua zhenghei”. Comparing to 16S rRNA gene, hzo and Scalindua-nirS genes provided a relatively higher resolution to elucidate anammox bacteria. For the nirS-encoding nitrite-reducing bacteria, the detected nirS gene sequences were closely related to various marine nirS denitrifiers, especially those which originated from coastal and estuarine sediments with a much higher diversity than anammox bacteria. Anammox bacterial communities shifted along with the seawater depth, while nirS-encoding nitrite-reducing bacteria did not. Although nirS-encoding nitrite-reducing bacteria have a much higher abundance and diversity than anammox bacteria, they showed similar abundance variation patterns in research sites, suggesting the two microbial groups might be affected by the similar environmental factors. The significant correlations among the abundance of the two microbial groups with the molar ratio of NH₄ ⁺ to (NO₂ ^??+?NO₃ ^?), pH, and organic matters of sediments strongly supported this hypothesis.     

Effects of inorganic carbon limitation on anaerobic ammonium oxidation (anammox) activity

Anammox bacteria are chemoautotrophic bacteria that oxidize ammonium with nitrite as the electron acceptor and with CO₂ as the main carbon source. The effects of inorganic carbon (IC) limitation on anammox bacteria were investigated using continuous feeding tests. In this study, a gel carrier with entrapped anammox sludge was used. It was clearly shown that the anammox activity deteriorated with a decrease in the influent IC concentration. The relationship between the influent IC concentration and the anammox activity was analyzed using Michaelis-Menten kinetics, and the apparent K_m was determined to be 1.2 mg-C/L. The activity could be recovered by adding IC to the influent. The consumption ratio of IC to ammonium was not constant and mainly depended on the influent ratio of the IC to ammonium concentrations (inf.IC/inf.NH₄-N). The results indicated that an inf.IC/inf.NH₄-N ratio of 0.2 in the anammox reactor was ideal for the anammox process using gel cubes.     

Contrasting spatiotemporal patterns and environmental drivers of diversity and community structure of ammonia oxidizers,denitrifiers, and anammox bacteria in sediments of estuarine tidal flats

The spatial and temporal patterns of diversity, community structure, and their drivers are fundamental issues in microbial ecology. This study aimed to investigate the relative importance of spatial and seasonal controls on the distribution of nitrogen cycling microbes in sediments of estuarine tidal flats, and to test the hypothesis that metals impact the distribution of nitrogen-cycling microbes in the coastal system. Two layers of sediment samples were collected from three estuarine tidal flats of Laizhou Bay in 2010 winter and 2011 summer. The alpha diversities (Shannon and Simpson indices) and community structure of ammonia oxidizing bacteria (AOB) and archaea (AOA), denitrifier and anammox bacteria (AMB) were revealed using denaturing gradient gel electrophoresis and clone library analysis of amoA, nosZ and 16S rRNA gene markers. We found that both AOB and AMB exhibited distinct seasonal patterns in either alpha diversity or community turnover; AOA had different alpha diversities in two layers, but neither spatial nor seasonal patterns were found for their community turnover. However, no distinct spatiotemporal pattern was observed for either diversity or community composition of nosZ-type denitrifiers. For correlations between alpha diversities and environmental factors, significant correlations were found between AOB and ammonium, temperature and As, between denitrifiers and nitrite, salinity and Pb, and between AMB and Pb, ratio of organic carbon to nitrogen, ammonium, pH and dissolved oxygen. Salinity and sediment grain size were the most important factors shaping AOB and AOA communities, respectively; whereas AMB community structure was mostly determined by temperature, dissolved oxygen, pH and heavy metals As and Cd. These results stress that ammonia oxidizers, denitrifiers and anammox bacteria have generally different distributional patterns across time and space, and heavy metals might have contributed to their differentiated distributions in coastal sediments.