Microbial Composition and Structure of a Rotating Biological Contactor Biofilm Treating Ammonium-Rich Wastewater without Organic Carbon

High nitrogen losses were observed in a rotating biological contactor (RBC) treating ammonium-rich (up to 500 mg NH4(+)-N/L) but organic-carbon-poor leachate from a hazardous waste landfill in K?lliken, Switzerland. The composition and spatial structure of the microbial community in the biofilm on the RBC was analyzed with specific attention for the presence of aerobic ammonium and nitrite oxidizing bacteria and anaerobic ammonium oxidizers. Anaerobic ammonium oxidation (anammox) involves the oxidation of ammonium with nitrite to N2. First the diversity of the biofilm community was determined from sequencing cloned PCR-amplified 16S rDNA fragments. This revealed the presence of a number of very unusual 16S rDNA sequences, but very few sequences related to known ammonium or nitrite oxidizing bacteria. From analysis of biofilm samples by fluorescence in situ hybridization with known phylogenetic probes and by dot-blot hybridization of the same probes to total RNA purified from biofilm samples, the main groups of microorganisms constituting the biofilm were found to be ammonium-oxidizing bacteria from the Nitrosomonas europaea/eutropha group, anaerobic ammonium-oxidizing bacteria of the "Candidatus Kuenenia stuttgartiensis" type, filamentous bacteria from the phylum Bacteroidetes, and nitrite-oxidizing bacteria from the genus Nitrospira. Aerobic and anaerobic ammonium-oxidizing bacteria were present in similar amounts of around 20 to 30% of the biomass, whereas members of the CFB phylum were present at around 7%. Nitrite oxidizing bacteria were only present in relatively low amounts (less than 5% determined with fluorescence in situ hybridization). Data from 16S rRNA dot-blot and in situ hybridization were not in all cases congruent. FISH analysis of thin-sliced and fixed biofilm samples clearly showed that the aerobic nitrifiers were located at the top of the biofilm in an extremely high density and in alternating clusters. Anammox bacteria were exclusively present in the lower half of the biofilm, whereas CFB-type filamentous bacteria were present throughout the biofilm. The structure and composition of these biofilms correlated very nicely with the proposed physiological functional separations in ammonium conversion.     

16S rRNA gene and lipid biomarker evidence for anaerobic ammonium-oxidizing bacteria (anammox) in California and Nevada hot springs

Anammox, the oxidation of ammonium with nitrite to dinitrogen gas under anoxic conditions, is an important process in mesophilic environments such as wastewaters, oceans and freshwater systems, but little is known of this process at elevated temperatures. In this study, we investigated anammox in microbial mats and sediments obtained from several hot springs in California and Nevada, using geochemical and molecular microbiological methods. Anammox bacteria-specific ladderane core lipids with concentrations ranging between 0.3 and 52 ng g⁻¹ sediment were detected in five hot springs analyzed with temperatures up to 65 °C. In addition, 16S rRNA gene analysis showed the presence of genes phylogenetically related to the known anammox bacteria Candidatus Brocadia fulgida, Candidatus Brocadia anammoxidans and Candidatus Kuenenia stuttgartiensis (96.5–99.8% sequence identity) in three hot springs with temperatures up to 52 °C. Our data indicate that anammox bacteria may be able to thrive at thermophilic temperatures and thus may play a significant role in the nitrogen cycle of hot spring environments.     

Inhibitory effects of free ammonia on Anammox bacteria

Anammox bacteria can effectively treat high ammonia and nitrite concentrations under anoxic environments. However, the presence of high ammonia and nitrite concentrations may cause free ammonia and nitrous acid inhibition at high pH and temperature environments. In this study, the inhibitory effect of free ammonia on Anammox bacteria was investigated in a lab-scale upflow fixed-bed reactor with Kaldnes biofilm carriers. Results of continuous operation showed that inhibition was not observed in the Anammox reactor when the free ammonia concentration gradually increased up to 150?mg/L. However, Anammox activity suddenly dropped to 10 % when the free ammonia concentration reached to 190?mg/L. Nevertheless, high influent ammonia and nitrite concentrations up to 1,500 and 500?mg/L, respectively, did not noticeably inhibit the Anammox activity. Gradually decreasing Anammox activity was also supported by fluorescent in situ hybridization (FISH) analysis. FISH and 16S rRNA gene analysis results revealed that main Anammox organisms were phylogenetically related to Candidatus Kuenenia stuttgartiensis, Candidatus Jettenia asiatica and Candidatus Brocadia anammoxidans.     

Stable carbon isotopic fractionations associated with inorganic carbon fixation by anaerobic ammonium-oxidizing bacteria

Isotopic analyses of Candidatus "Brocadia anammoxidans," a chemolithoautotrophic bacterium that anaerobically oxidizes ammonium (anammox), show that it strongly fractionates against (13)C; i.e., lipids are depleted by up to 47 per thousand versus CO(2). Similar results were obtained for the anammox bacterium Candidatus "Scalindua sorokinii," which thrives in the anoxic water column of the Black Sea, suggesting that different anammox bacteria use identical carbon fixation pathways, which may be either the Calvin cycle or the acetyl coenzyme A pathway.     

Microbiology and application of the anaerobic ammonium oxidation ('anammox') process

Ten years ago, an anaerobic ammonium oxidation ('anammox') process was discovered in a denitrifying pilot plant reactor. From this system, a highly enriched microbial community was obtained, dominated by a single deep-branching planctomycete, Candidatus Brocadia anammoxidans. Phylogenetic inventories of different wastewater treatment plants with anammox activity have suggested that at least two genera in Planctomycetales can catalyse the anammox process. Electron microscopy of the ultrastructure of B. anammoxidans has shown that several membrane-bounded compartments are present inside the cytoplasm. Hydroxylamine oxidoreductase, a key anammox enzyme, is found exclusively inside one of these compartments, tentatively named the 'anammoxosome'.     

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.     

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。     

单级自养脱氮系统中厌氧氨氧化菌的分子生物学鉴定

对具有厌氧氨氧化作用的细菌进行更深入的分析有助于了解该菌在生物脱氮过程的应用。对稳定运行、氨氮转化率及总氮去除率分别达到90%及80%左右的单级自养脱氮系统的底部取活性污泥,采用分子生物学方法提取活性污泥细菌总DNA,利用特异引物Pla46rc/Amx820对单级自养脱氮系统中的厌氧氨氧化菌16S rDNA基因进行PCR扩增。扩增产物经克隆、测序及BLAST分析,结果表明该单级自养脱氮系统中存在的厌氧氨氧化菌与Candidatus Kueneniastuttgartiensis和Candidatus Brocadia anammoxidans的16S rDNA序列同源性达99%,进化分析证明与Candidatus Kuenenia stuttgartiensis进化上较为接近。     

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".     

Anaerobic ammonia oxidation in the presence of nitrogen oxides (NO(x)) by two different lithotrophs

The anaerobic ammonia-oxidizing activity of the planctomycete Candidatus "Brocadia anammoxidans" was not inhibited by NO concentrations up to 600 ppm and NO2 concentrations up to 100 ppm. B. anammoxidans was able to convert (detoxify) NO, which might explain the high NO tolerance of this organism. In the presence of NO2, the specific ammonia oxidation activity of B. anammoxidans increased, and Nitrosomonas-like microorganisms recovered an NO2-dependent anaerobic ammonia oxidation activity. Addition of NO2 to a mixed population of B. anammoxidans and Nitrosomonas induced simultaneous specific anaerobic ammonia oxidation activities of up to 5.5 mmol of NH4+ g of protein(-1) h(-1) by B. anammoxidans and up to 1.5 mmol of NH4+ g of protein(-1) h(-1) by Nitrosomonas. The stoichiometry of the converted N compounds (NO2-/NH3 ratio) and the microbial community structure were strongly influenced by NO2. The combined activity of B. anammoxidans and Nitrosomonas-like ammonia oxidizers might be of relevance in natural environments and for technical applications.     

Induced cooperation between marine nitrifiers and anaerobic ammonium-oxidizing bacteria by incremental exposure to oxygen

In oxygen-limited marine ecosystems cooperation between marine nitrifiers and anaerobic ammonium-oxidizing (anammox) bacteria is of importance to nitrogen cycling. Strong evidence for cooperation between anammox bacteria and nitrifiers has been provided by environmental studies but little is known about the development of such communities, the effects of environmental parameters and the physiological traits of their constituents. In this study, a marine laboratory model system was developed. Cooperation between marine nitrifiers and anammox bacteria was induced by incremental exposure of a marine anammox community dominated by Scalindua species to oxygen in a bioreactor set-up under high ammonium (40 mM influent) conditions. Changes in the activities of the relevant functional groups (anammox bacteria, aerobic ammonia oxidizers and nitrite oxidizers) were monitored by batch tests. Changes in community composition were followed by Fluorescence in situ Hybridization (FISH) and by amplification and sequencing of 16S rRNA and amoA genes. A co-culture of Scalindua sp., an aerobic ammonia-oxidizing Nitrosomonas-like species, and an aerobic (most likely Nitrospira sp.) nitrite oxidizer was obtained. Aerobic ammonia oxidizers became active immediately upon exposure to oxygen and their numbers increased 60-fold. Crenarchaea closely related to the ammonia-oxidizer Candidatus 'Nitrosopumilus maritimus' were detected in very low numbers and their contribution to nitrification was assumed negligible. Activity of anammox bacteria was not inhibited by the increased oxygen availability. The developed marine model system proved an effective tool to study the interactions between marine anammox bacteria and nitrifiers and their responses to changes in environmentally relevant conditions.     

Candidatus "Scalindua brodae", sp. nov., Candidatus "Scalindua wagneri", sp. nov., two new species of anaerobic ammonium oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world's oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 +/- 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candidatus "Scalindua brodae" and "Scalindua wagneri" considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus "Scalindua sorokinii", was detected in the water column of the Black Sea, making this genus the most widespread of all anammox bacteria described so far.     

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.     

Candidatus 'Brocadia fulgida': an autofluorescent anaerobic ammonium oxidizing bacterium

Anaerobic ammonium oxidizing (anammox) bacteria are detected in many natural ecosystems and wastewater treatment plants worldwide. This study describes the enrichment of anammox bacteria in the presence of acetate. The results obtained extend the concept that the anammox bacteria can be enriched to high densities in the presence of substrates for heterotrophic growth. Batch experiments showed that among the tested biomass, the biomass from the Candidatus 'Brocadia fulgida' enrichment culture oxidizes acetate at the highest rate. Continuous cultivation experiments showed that in the presence of acetate, ammonium, nitrite and nitrate, Candidatus 'Brocadia fulgida' out-competed other anammox bacteria. The results indicated that Candidatus 'Brocadia fulgida' did not incorporate acetate directly into their biomass. Candidatus 'Brocadia fulgida' exhibited the common characteristics of anammox bacteria: the presence of an anammoxosome and ladderane lipids and the production of hydrazine in the presence of hydroxylamine. Interestingly, the biofilm aggregates of this species showed strong autofluorescence. It is the only known anammox species exhibiting this feature. The autofluorescent extracellular polymeric substance had two excitation (352 and 442 nm) and two emission (464 and 521 nm) maxima.     

Anaerobic ammonium oxidation in a tropical freshwater system (Lake Tanganyika)

Here we provide the first direct evidence for the anammox process (anaerobic ammonium oxidation) in a lacustrine system, Lake Tanganyika, the second largest lake in the world. Incubations with (15)N labelled nitrate showed that anammox occurred in the suboxic water layer at 100-110 m water depth. Anammox rates up to 10 nM N(2) h(-1) are comparable to those reported for the marine water column. Up to approximately 13% of produced N(2) could be attributed to the anammox process whereas the remainder was related to denitrification. Typical lipid biomarkers characteristic of anammox bacteria were found in filtered water from the depths where anammox occurred, thus supporting the presence of anammox bacteria. Further evidence is provided by fluorescence in situ hybridization (FISH), revealing up to 13 000 anammox bacteria cells per ml or 1.4% of all DAPI (4'-6-Diamidino-2-phenylindole)-stained cells. Phylogenetic analyses of partial 16S rRNA genes indicated the presence of sequences most closely related to the known anammox bacterium Candidatus "Scalindua brodae" (95.7% similarity). Using the incubation results, a total loss of 0.2 Tg N(2) per year linked to anammox was estimated for the Northern basin of Lake Tanganyika.     

16S-23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium-oxidizing bacteria: implications for phylogeny and in situ detection

Recently, anaerobic ammonium-oxidizing bacteria (AAOB) were identified by comparative 16S rDNA sequence analysis as a novel, deep-branching lineage within the Planctomycetales . This lineage consists currently of only two, not yet culturable bacteria which have been provisionally described as Candidatus 'Brocadia anammoxidans' and Candidatus 'Kuenenia stuttgartiensis'. In this study, a large fragment of the rDNA operon, including the 16S rDNA, the intergenic spacer region (ISR) and approximately 2 000 bases of the 23S rDNA, was polymerase chain reaction (PCR) amplified, cloned and sequenced from both AAOB. The retrieved 16S rDNA sequences of both species contain an insertion at helix 9 with a previously overlooked pronounced secondary structure (new subhelices 9a and 9b). This insertion, which is absent in all other known prokaryotes, is detectable by fluorescence in situ hybridization (FISH) and thus present in the mature 16S rRNA. In contrast with the genera Pirellula , Planctomyces and Gemmata that possess unlinked 16S and 23S rRNA genes, both AAOB have the respective genes linked together by an ISR of approximately 450 bp in length. Phylogenetic analysis of the obtained 23S rRNA-genes confirmed the deep branching of the AAOB within the Planctomycetales and allowed the design of additional specific FISH probes. Remarkably, the ISR of the AAOB also could be successfully detected by FISH via simultaneous application of four monolabelled oligonucleotide probes. Quantitative FISH experiments with cells of Candidatus 'Brocadia anammoxidans' that were inhibited by exposure to oxygen for different time periods demonstrated that the concentration of transcribed ISR reflected the activity of the cells more accurately than the 16S or 23S rRNA concentration. Thus the developed ISR probes might become useful tools for in situ monitoring of the activity of AAOB in their natural environment.     

The metagenomic basis of anammox metabolism in Candidatus 'Brocadia fulgida'

Anammox (anaerobic ammonium oxidation) coupled to nitrite reduction is an important step in the nitrogen cycle and has been recognized as an important sink for fixed nitrogen in the ocean. Still little is known about the genomic blueprint of different anammox species. In the present article, we discuss the important genes of anammox metabolism in Candidatus 'Brocadia fulgida' that were retrieved via a metagenomic approach.     

A microdiversity study of anammox bacteria reveals a novel Candidatus Scalindua phylotype in marine oxygen minimum zones

The anaerobic oxidation of ammonium (anammox) contributes significantly to the global loss of fixed nitrogen and is carried out by a deep branching monophyletic group of bacteria within the phylum Planctomycetes. Various studies have implicated anammox to be the most important process responsible for the nitrogen loss in the marine oxygen minimum zones (OMZs) with a low diversity of marine anammox bacteria. This comprehensive study investigated the anammox bacteria in the suboxic zone of the Black Sea and in three major OMZs (off Namibia, Peru and in the Arabian Sea). The diversity and population composition of anammox bacteria were investigated by both, the 16S rRNA gene sequences and the 16S-23S rRNA internal transcribed spacer (ITS). Our results showed that the anammox bacterial sequences of the investigated samples were all closely related to the Candidatus Scalindua genus. However, a greater microdiversity of marine anammox bacteria than previously assumed was observed. Both phylogenetic markers supported the classification of all sequences in two distinct anammox bacterial phylotypes: Candidatus Scalindua clades 1 and 2. Scalindua 1 could be further divided into four distinct clusters, all comprised of sequences from either the Namibian or the Peruvian OMZ. Scalindua 2 consisted of sequences from the Arabian Sea and the Peruvian OMZ and included one previously published 16S rRNA gene sequence from Lake Tanganyika and one from South China Sea sediment (97.9-99.4% sequence identity). This cluster showed only 相似文献